Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation

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(A) Different siRNAs (#25, #33 and #34) targeting frizzled-2 mRNA at different regions and non-targeting siRNA serving as negative control (NC) were transfected at final doses of 25, 50 and 100nM for 24h in BRL-3 cells. Relative expression levels of frizzled-2 were analyzed qRT-PCR and normalized to β-actin. * denoted the group with the frizzled-2 siRNA (#34) at 50nM achieved maximally silencing efficiency among groups. (B) Transfection of frizzled-2 siRNA (#34) and negative control siRNA (NC) was performed at 50nM in BRL-3 cells for 48h. Cell group was set up as mock control. Frizzled-2 protein level was analyzed by Western blot. Relative expression of Frizzled-2 was normalized to GAPDH. Data were presented as means±SEM from three independent experiments. ##p<0.001, Cell or NC groups vs. siRNA group." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Xiang Hu, Chenjie Zhou, Guolin He, Yuan Cheng, Mingxin Pan, Yi Gao" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Xiang" "apellidos" => "Hu" ] 1 => array:2 [ "nombre" => "Chenjie" "apellidos" => "Zhou" ] 2 => array:2 [ "nombre" => "Guolin" "apellidos" => "He" ] 3 => array:2 [ "nombre" => "Yuan" "apellidos" => "Cheng" ] 4 => array:2 [ "nombre" => "Mingxin" "apellidos" => "Pan" ] 5 => array:2 [ "nombre" => "Yi" "apellidos" => "Gao" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "en" => array:9 [ "pii" => "S0210570519300986" "doi" => "10.1016/j.gastrohep.2019.02.006" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0210570519300986?idApp=UINPBA00004N" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2444382420300171?idApp=UINPBA00004N" "url" => "/24443824/0000004300000003/v1_202004030631/S2444382420300171/v1_202004030631/en/main.assets" ] ] "itemSiguiente" => array:19 [ "pii" => "S0210570519302535" "issn" => "02105705" "doi" => "10.1016/j.gastrohep.2019.09.009" "estado" => "S300" "fechaPublicacion" => "2020-03-01" "aid" => "1460" "copyright" => "Elsevier España, S.L.U." 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McNicholl, Ana Garre, Laura Llorca, Luis Bujanda, Javier Molina-Infante, Merce Barenys, Julia Perez, Maria D. Guerrero-Torres, Esther Tamayo, Milagrosa Montes, Raul Prados-Manzano, Ariadna Sanchez-Garcia, Mercedes Ramas, Veronica B. Valdez Blanco, Miguel Montoro, Xavier Calvet, Ariadna Figuerola, Sergio Lario, Eia Quilez, Angel Lanas, Pia Silva-Pomarino, Angeles Perez-Aisa, Maria G. Donday, Blanca Belloc, Antonia Montserrat-Torres, Nuria Fernandez-Moreno, María José Ramírez, Teresa Alarcon, Javier P. Gisbert" "autores" => array:29 [ 0 => array:2 [ "nombre" => "Adrian G." 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"apellidos" => "Gisbert" ] ] ] ] ] "idiomaDefecto" => "en" "Traduccion" => array:1 [ "en" => array:9 [ "pii" => "S2444382420300183" "doi" => "10.1016/j.gastre.2019.09.009" "estado" => "S300" "subdocumento" => "" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S2444382420300183?idApp=UINPBA00004N" ] ] "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0210570519302535?idApp=UINPBA00004N" "url" => "/02105705/0000004300000003/v2_202010310705/S0210570519302535/v2_202010310705/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "107" "paginaFinal" => "116" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Xiang Hu, Chenjie Zhou, Guolin He, Yuan Cheng, Mingxin Pan, Yi Gao" "autores" => array:6 [ 0 => array:3 [ "nombre" => "Xiang" "apellidos" => "Hu" "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] ] ] 1 => array:3 [ "nombre" => "Chenjie" "apellidos" => "Zhou" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "Guolin" "apellidos" => "He" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "Yuan" "apellidos" => "Cheng" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "Mingxin" "apellidos" => "Pan" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 5 => array:4 [ "nombre" => "Yi" "apellidos" => "Gao" "email" => array:1 [ 0 => "drgaoy@126.com" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Department of Hepatobiliary Surgery II, State Key Laboratory of Organ Failure Research, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, Guangdong Province, China" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "General Surgery Department, The Second Hospital of Shenzhen Baoan People's Hospital Group, Shenzhen 518108, Guangdong Province, China" "etiqueta" => "b" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "La inhibición de Frizzled-2 por ARN interferente pequeño protege a los hepatocitos BRL-3A de rata frente a la citotoxicidad y la apoptosis inducidas por hipoxia/reoxigenación" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 3435 "Ancho" => 2500 "Tamanyo" => 489627 ] ] "descripcion" => array:1 [ "en" => "Transfection of frizzled-2 siRNA inhibited the H/R induced apoptosis in BRL-3A cells by Flow Cytometry using Annexin-V-FITC method. (A) BRL-3A cells alone; (B) BRL-3A cells with H/R treatment (Cell-H/R); (C) H/R treatment of BRL-3A cells with transfection of negative control siRNA (NC-H/R); (D) H/R treatment of BRL-3A cells with transfection of frizzled-2 siRNA (Si-H/R); (E) quantitative analysis of the early cell apoptosis; (F) quantitative analysis of the late cell apoptosis; (G) quantitative analysis of the early cell apoptosis. Data were presented as means±SEM from three independent experiments. *p<0.05 and **p<0.001 when comparing to Cell group, and #p<0.05 and ##p<0.001 when comparing to Si-H/R group." ] ] ] "textoCompleto" => "IntroductionHepatic ischemia–reperfusion injury (HIRI) is a major cause of morbidity and mortality in liver resection and transplantation surgery.<a class="elsevierStyleCrossRefs" href="#bib0115">1,2</a> HIRI of the liver results in microvascular changes and acute inflammation, ultimately leading to cell death.<a class="elsevierStyleCrossRef" href="#bib0120">2</a> HIRI induced cell death includes apoptosis, autophagy, necrosis and necroptosis in hepatocytes and non-parenchymal cells.<a class="elsevierStyleCrossRefs" href="#bib0125">3,4</a> Restoration of blood flow is necessary to restore cellular function, but paradoxically reperfusion can initiate a cascade of pathways that cause further cellular injury after prolonged ischemia. Understanding the molecular events of liver ischemia–reperfusion injury helps develop strategies to counterattack this injury and reduce acute complications in hepatic resection and transplantation.Wnt signaling pathway is selectively activated in hepatic development, regeneration and hepatocellular carcinoma (HCC), in a canonical or a non-canonical pathway.<a class="elsevierStyleCrossRefs" href="#bib0135">5–9</a> Wnt signaling is mediated by its receptors, Frizzled family proteins, which contain seven trans-membranes in humans and mice.<a class="elsevierStyleCrossRef" href="#bib0155">9</a> Canonical Wnt signaling is mediated by Frizzled-1, and regulates the nuclear accumulation of β-catenin.<a class="elsevierStyleCrossRef" href="#bib0160">10</a> Non-canonical Wnt signaling is β-catenin-independent, and includes pathways, such as mitogen-activated protein kinase,<a class="elsevierStyleCrossRef" href="#bib0165">11</a> NFκB and c-Jun N terminal kinase (JNK),<a class="elsevierStyleCrossRef" href="#bib0170">12</a> ROCK/Rho kinase,<a class="elsevierStyleCrossRef" href="#bib0175">13</a> protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII).<a class="elsevierStyleCrossRef" href="#bib0180">14</a> Frizzled-2 binds Wnt5a to activate the non-canonical Wnt pathway.<a class="elsevierStyleCrossRef" href="#bib0185">15</a> Activation of Wnt5a/Frizzled-2 signaling has been shown to turn off β-catenin during hepatic development and liver regeneration after partial hepatectomy.<a class="elsevierStyleCrossRef" href="#bib0190">16</a> Wnt5a loss leads to enhanced β-catenin signaling in hepatocellular carcinoma.<a class="elsevierStyleCrossRefs" href="#bib0145">7,17</a> Wnt5a/Frizzled-2 signaling pathway was activated in response to H/R in H9C2 cells,<a class="elsevierStyleCrossRef" href="#bib0200">18</a> indicating its important role in in vivo myocardial cells post reperfusion. However, the Frizzled-2 signaling pathway has not been understood yet in the hepatic development under pathophysiological HIRI conditions.In this study, we examined the effects of Frizzled-2 on the H/R treated rat normal liver BRL-3A cells, an in vitro cell model established to mimic the pathophysiological HIRI. The Wnt5a/Frizzled-2 signaling was suppressed by silencing frizzled-2 gene expression using the siRNA approach. Inhibition of Frizzled-2 significantly attenuated the H/R induced cytotoxicity, apoptosis and intracellular Ca2+ in BRL-3A cells. Our data suggest the inhibition of Wnt5a/Frizzled-2 signaling pathway may represent a novel and promising approach to protect liver cells against HIRI-related disorders.Materials and methodsCell culture and Hypoxia/Reoxygenation (H/R) modelThis project was approved by the Ethical committee guide of our hospital, Zhujiang Hospital, and Southern Medical University for the care and the use of laboratory animals (No. ZJYY-2016-GDEK-001). BRL-3A cell (derived from rat liver cells) was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and maintained in HyClone™ Dulbecco's modified Eagle's medium (DMEM) with 4.5g/L glucose supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin (v/v) in a humidified air atmosphere with a 5% CO2 at 37°C. All cell culture reagents were purchased from Thermo Scientific (Waltham, MA, USA). To develop cells under Hypoxia/Reoxygenation (H/R), BRL-3A cells in culture medium with low serum media (0.5% FBS) were exposed to hypoxia for 2h in a humidified incubation chamber at 37°C and flushed with a gas mixture of 95% N2 and 5% CO2, followed by incubation in fresh medium with 95% air and 5% CO2 for reoxygenation. The control cells were kept in normoxic conditions. After reoxygenation for16h, cells were harvested for RNA or protein analysis.Transfection with small interfering RNA (siRNA) targeting frizzled-2 geneFor transfection, BRL-3A cells were plated in 6-well plate at a density of 105 cells per well or in 100mm dishes with 106 cells in growth medium without antibiotics. After cell growth reached 50–60% confluence, transfection was performed using Lipofectamine 2000 from Invitrogen (ThermoFisher Scientific, MA, USA). Cells were incubated in Opti-MEM I Reduced Serum Medium from Invitrogen with or without frizzled-2 siRNA–Lipofectamine 2000 complexes (25, 50, or 100nM) for 6h and then replaced with fresh DMEM for 48h. The transfection efficiency of siRNA was monitored by observing the fluorescence of cells transfected with FAM labeled siRNA negative control (siRNA-NC) after 6h. Then cells were subjected to H/R treatment and then harvested for other experiments. Three different non-overlapping siRNA duplexes and FAM labeled siRNA negative control were designed and purchased from Sigma-Aldrich (Guangzhou, China). The sequences were as follows: firizzled-2 siRNA #25: 5′-GUGCUGUGCUGCGCUUCUAdTdT-3′; firizzled-2 siRNA #33: 5′-CUAUCUCAGCUAUAAGUUUdTdT-3′; firizzled-2 siRNA #34: 5′-GCUGUACUAUACUCUUCAUdTdT-3′ and negative control: 5′-UUCUCCGAACGUGUCACGUdTdT-3′.RNA isolation and quantitative real-time PCRTotal RNA was isolated from BRL-3A cells s using Trizol from Invitrogen (ThermoFisher Scientific, MA, USA) according to the manufacturer's protocols. Quantitative Real-Time PCR (qRT-PCR) was carried out using an ABI 7500 Real-time PCR System (Applied Biosystems, Foster, CA, USA). SYBR Green PCR SuperMix was used as a double-stranded DNA-specific dye according to the manufacturer's instructions (ThermoFisher Scientific, MA USA). Primers were designed for a single qRT-PCR thermal profile (95°C for 1min, and 40 cycles of 95°C for 15s and 55°C for 1min for frizzled-2 and 95°C for 1min, and 40 cycles of 95°C for 15s and 60°C for 1min for Wnt5a and β-actin). The expression levels of examined transcripts were normalized to β-actin. Primer sequences for frizzled-2, Wnt5a and β-actin were listed below: frizzled-2-forward: 5′-TCGTTTTGCCCGTCTCT-3′, frizzled-2-reverse: 5′-TAGCGGAATCGCTGCAT-3′; Wnt5a-forward: 5′-CGTGGCTATGACCAGTTTAAG-3′; Wnt5a-reverse: 5′-CCACAATCTCCGTGCACTT-3′; β-actin-forward: 5′-AGGGAAATCGTGCGTGACAT-3′; β-actin-reverse: 5′-GAACCGCTCATTGCCGATAG-3′.Protein lysate preparation and Western blotCultured BRL-3A cells were lysed in ice-cold Radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (Beyotime Biotechnology, Shanghai, China) and total protein samples were prepared in extraction buffer (10mM Tris, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1mM NaF, 20mM Na4P2O7, 2mM Na3VO4, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-100, 10% glycerol and 1mM PMSF) containing 1× Cocktail of Proteinase inhibitors and Phosphatase inhibitors (ThermoFisher Scientific, MA, USA). Protein concentration was measured using BCA protein assay kit (Kaiji Biotechnology, Nanjing, China). A 20μg of protein for each sample was loaded into each well and resolved by SDS-10% PAGE and transferred onto PVDF membranes via electroblotting (Millipore, Billerica, MA, USA). After blocking 1h in 1× TBST containing 5% bovine serum albumin (BSA), membranes were washed three times in 1× TBST and probed with the following primary antibodies at 4°C overnight: rabbit polyclonal to Frizzled-2 (1:1000; Abcam, Cambridge, MA, USA), rabbit polyclonal to Wnt5a (1:2000; Abcam, Cambridge, MA, USA) and goat polyclonal antibody to human GAPDH (1:1000; Santa Cruz, California, CA, USA). The blots were then incubated with goat anti-rabbit IgG peroxidase-conjugated secondary antibody (1:6000; Calbiochem, Merck KgaA, Darmstadt, Germany) and visualized by enhanced SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, USA). GAPDH was used as the internal loading control. For quantification, Image-Pro Plus 6.0 software (Media Cybernetics Inc., Silver Spring, MD, USA) was used to measure the integrated optical density (IOD) of the bands. The relative level of protein expression was expressed as a ratio to GAPDH. Experiments were repeated three times for each experimental condition.LDH cytotoxicity assayThe cytotoxicity of BRL-3A cells was determined by Lactate Dehydrogenase (LDH) released from the damaged cells. Cells were seeded in triplicate in 6-well plates and incubated overnight. Then cells were subject to transfection of siRNA of frizzled-2 or control siRNA for 24h, respectively then followed by H/R treatment. Cell lysates were harvested for LDH assay according to the manufactory protocol from Pierce Biotechnology (Rockford, IL, USA). The absorbance was measured at 490nm and 680nm. To determine LDH activity, subtract the 680nm absorbance value as background from the 490nm absorbance before calculation of % cytotoxicity. Experiments were repeated three times independently.Apoptosis assay by flow cytometryTo assess cell apoptosis, an Annexin V-fluorescein isothiocyanate (V-FITC) staining assay from Invitrogen (ThermoFisher Scientific, MA, USA) was performed according to the manufacturer's instructions. Briefly, the BRL-3A cells were detached by 0.25% Trypsin and collected for staining with Annexin V-FITC and PI for 15min at room temperature (18–24°C). The cells were then washed twice with PBS, and the fluorescence was analyzed using a FACSCalibur flow cytometry system and CellQuest software (BD Biosciences, New Jersey, USA).Fluorescence of Ca2+ and laser scanning confocal microscopyFor calcium detection, cells grown in the 6-well plates were incubated with the intracellular Ca2+ indicator Fluo 8-AM at 37°C for 30min according to the manufacturer's protocol from Invitrogen (ThermoFisher, MA, USA). The fluorescence intensity was monitored at 490nm/515nm (Ex/Em). Fluorescent images were acquired by a LSM510 confocal laser scanning microscope (Carl-Zeiss, Jena, Germany) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). The average fluorescence density of cell preparations from each group was compared among the groups. Values of integrated optical densities were expressed as the mean±SEM of at least three different experiments.In vivo animal study and surgeries for hepatic ischemia and reperfusion injury (IRI)Total 12 male C57BL/6 mice at 8- to 10-week old were bred and housed at State Key Laboratory of Biological Artificial Liver in Zhujiang Hospital. For surgery for hepatic ischemia and reperfusion injury (IRI), 6 mice were anesthetized with isoflurane and a small midline was vertically incised on the abdominal wall to access the liver and portal triad. The blood supply to left and central hepatic lobes was occluded via vascular clips on the hepatic artery and portal vein for 90min, followed by a 6h period of reperfusion before sacrifice. For IRI sham operations, 6 mice were also anesthetized and the abdominal wall was vertically incised at midline and left open but covered with saline-soaked gauze for 30min before the incision was closed. Mice were placed on a warming pad during and after surgery to maintain a body temperature of 37°C. At sacrifice, liver tissues were collected and stored at −80°C for further analysis. All procedures were approved by the Institutional Animal Care and Use Committee and followed at guide of our hospital, Zhujiang Hospital, and Southern Medical University for the care and the use of laboratory animals.Statistical analysisAll data are presented as the mean±standard error of the mean (SEM). Student's t-test and one-way analysis of variance (ANOVA) were used to examine the overall statistical differences using SPSS software (Version 24.0, Chicago, IL, USA). Differences between the groups were considered statistically significant at a p<0.05.ResultsInhibition of frizzled-2 gene by siRNA in rat normal hepatic cell line BRL-3A cellsTo validate the frizzled-2 siRNA silencing efficiency, the frizzled-2 gene expression was detected in BRL-3A cells using qRT-PCR after siRNA transfection for 24h. The frizzled-2 mRNA levels were inhibited by 30.44%, 69.22% and 74.79% by transfection of siRNA-#25, -#33 and -#34 at 50nM, respectively, compared to the negative control siRNA (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A). A significant decrease in Frizzle protein expression was also shown in the group with transfection of siRNA-#34 at 50nM by Western blot compared to mock control Cell group or negative control (NC) group (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B). With the highest efficiency (74.79%) in silencing frizzled-2 expression in BRL-3A cells, transfection of siRNA-#34 at 50nM was chosen to knockdown frizzled-2 gene expression in the subsequent experiments.<elsevierMultimedia ident="fig0005"></elsevierMultimedia>H/R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cellsTo determine whether Frizzled-2/Wnt5a pathway was responded to the H/R treatment in BRL-3A cells, the expression of Frizzled-2 and Wnt5a were examined in BRL-3A cells with or without H/R (Cell-H/R or Cell) by qRT-PCR and Western blot. Significant increases in the expression of both Frizzled-2 and Wnt5a were detected at mRNA (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A and B) and protein levels in BRL-3A cells after H/R treatment (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>D–E). The H/R induced frizzled-2 mRNA as well as its protein levels were diminished by frizzled-2 siRNA transfection in BRL-3A cells (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A, C and D), whereas Wnt5a gene expression was induced (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>B, C and E).<elsevierMultimedia ident="fig0010"></elsevierMultimedia>Frizzled-2 siRNA inhibited the H/R-induced cytotoxicity in BRL-3A cellsTo examine the effect of H/R treatment on cell cytotoxicity in BRL-3A cells, the activity of LDH released from the dead cells was measured. LDH assay showed transfection of frizzled-2 siRNA significantly inhibited the LDH activity by reducing the OD value from 3.27±0.09 to 2.87±0.05 (p<0.05) (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>A and B), suggesting silencing frizzled-2 gene expression enables to protect hepatic cells injury caused by H/R.<elsevierMultimedia ident="fig0015"></elsevierMultimedia>Frizzled-2 siRNA inhibited the H/R-induced apoptosis in BRL-3A cellsAs a critical protein involved in cell apoptotic process, Caspase-3 protein level was used to monitor cell apoptosis during H/R. Western blot analysis showed Caspase-3 level was significantly increased in BRL-3A cells after H/R treatment (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>A) and this increase was suppressed by the frizzled-2 siRNA transfection. The fold change of Caspase-3 protein level was further presented by the bar graph (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>B). The early apoptosis rates were further determined by Annexin-V-FITC using flow cytometry assay. Total cell apoptosis was significantly induced by the H/R treatment in BRL-3A cells (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>A and B). Compared to the negative control group, transfection of frizzled-2 siRNA decreased early apoptosis in BRL-3A cells during H/R (0.74±0.09 vs. 6.06±0.59, p<0.001; <a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>C–E). Similar levels of cell late apoptosis rates (3.12±0.70 vs. 3.34±0.40, p>0.05; <a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>C, D and F) but a significant decrease in total cell apoptosis (3.86±0.64 vs. 9.41±0.56, p<0.001; <a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>C, D and G) were shown in cell group with the transfection of frizzled-2 siRNA during H/R treatment compared to the negative control group.<elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia>Frizzled-2 siRNA inhibited the H/R-induced intracellular Ca2+ in BRL-3A cellsAs Ca2+ overload is usually involved in the HIRI process, we detected the intracellular Ca2+ concentration in BRL-3A cells with H/R treatment through the mean fluorescence intensity (MFI) of Furo 8-AM using confocal microscopy. The intracellular Ca2+ level was clearly increased in the BRL-3A cells with H/R treatment compared to the normal cells (MFI: 55.34±3.42 vs. 46.49±6.3, p<0.5, <a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>A, D and E). To test whether inhibition of Wnt5a/Frizzled-2 pathway regulates the intracellular Ca2+ level, transfection of frizzled-2 siRNA was performed to examine the intracellular Ca2+ level. The Ca2+ concentration was significantly suppressed in the H/R treated BRL-3A cells with transfection of frizzled-2 siRNA compared to the normal cells (MFI 23.71±7.68 vs. 46.49±6.30, p<0.05; <a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>C–E).<elsevierMultimedia ident="fig0030"></elsevierMultimedia>Up-regulation of Frizzled-2/Wnt 5 pathway in liver tissues from mice treated with ischemia and reperfusion injury (IRI)To validate the role of Frizzled-2/Wnt5 pathway mediating H/R in a hepatocyte-like cell line, in vivo animal study was further performed in a clinically relevant setting of hepatic IRI in mice (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 7</a>). The expression of Wnt5a, Frizzled-2, and cleaved Caspase-3 at 17kDa and 19kDa was individually up-regulated significantly accompanied by a significant decrease in β-catenin in liver tissues from mice with IRI compared to the control group (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 7</a>A–F).<elsevierMultimedia ident="fig0035"></elsevierMultimedia>DiscussionAs a clinical condition, HIRI may lead to cellular injury and organ dysfunction and is mediated mainly through different mechanisms, which include Wnt/β-catenin signaling pathway and Ca2+ overload. To address the potential hepatocyte functionality in response to HIRI, we first established the in vitro H/R hepatic cell model using rat normal liver BRL-3A cells to mimic in vivo hepatic cells under HIRI condition, which allows for in vitro cellular functional and mechanistic studies. In this study, we investigated the effects of H/R treatment on the expression of Frizzled-2/Wnt5a pathway. Frizzled-2 and Wnt5a gene and protein expression were significantly increased in BRL-3A cells by H/R treatment. We further demonstrated H/R treatment induced cell cytotoxicity, apoptosis and the intracellular Ca2+ level in BRL-3A cells. This induction could be suppressed by transfection of frizzled-2 siRNA, suggesting suppression of Frizzled-2/Wnt5a signaling played important roles in protection of hepatic cells against HIRI.The ischemic injury could be mediated through various possible mechanisms. The Wnt signaling pathway is well established in liver cell biology and plays a critical role as a molecular regulator in hepatic development, regeneration and carcinogenesis.<a class="elsevierStyleCrossRef" href="#bib0150">8</a> Wnt5a/Frizzled-2 has been shown to inhibit liver regeneration process by suppressing β-catenin signaling in hepatocytes.<a class="elsevierStyleCrossRef" href="#bib0190">16</a> Frizzled proteins can stimulate different Wnts at the cell surface and discriminate among different Wnt ligands,<a class="elsevierStyleCrossRefs" href="#bib0120">2,19</a> which determines the specific downstream pathway activated in the cells. Pharmacologic activation of canonical β-catenin dependent Wnt signaling protects against hepatic ischemia/reperfusion injury through inducing proliferation and reducing apoptosis.<a class="elsevierStyleCrossRef" href="#bib0210">20</a> Noncanonical Wnt5a strongly inhibited canonical Wnt signaling.<a class="elsevierStyleCrossRef" href="#bib0145">7</a> In hepatocellular carcinoma, high levels of Frizzled-2 expression induced EMT and enhanced cell migration and invasiveness, while transfection of frizzled-2 siRNA resulted in a significant reduction in cell migration and invasion but not proliferation in HCC cell lines.<a class="elsevierStyleCrossRef" href="#bib0215">21</a> Canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.<a class="elsevierStyleCrossRef" href="#bib0145">7</a> In the present study, siRNA silencing the frizzled-2 gene expression led to a decrease in cell cytotoxicity and apoptosis, indicating the activation of Wnt5a/Frizzled-2 might enhance the cell death after HIRI whereas inhibition of Frizzled-2 may protect liver cell against injury and help to maintain hepatic cell integrity.During reperfusion injury, both apoptosis and necrosis pathways are present in cell death.<a class="elsevierStyleCrossRef" href="#bib0125">3</a> Several proapoptotic proteins are activated during the reperfusion phase, including the proteases Caspase-3 and Caspase-8, which finally leads to the destruction of nuclear DNA, resulting in cell death.<a class="elsevierStyleCrossRef" href="#bib0115">1</a> Caspase-3 is one of the mediators in apoptotic pathway which can be enhanced by Ca2+ accumulation post-injury. In this study, our data demonstrated the H/R induced cell death and apoptosis in BRL-3A cells. In addition to morphological criteria, induction of Caspase-3 activity provided corroborative evidence for hepatocyte apoptosis. Transfection of frizzled-2 siRNA blocked the H/R-induced LDH and Caspase-3 levels, which emphasized the role of Frizzled-2 in apoptotic cell death in the hepatic H/R. The liver cell membrane structure, the endoplasmic reticulum and sarcoplasmic reticulum are damaged in response to HIRI, which may lead to the abnormal function of the calcium pump and subsequent elevation of the intracellular Ca2+ concentration. Activation of Wnt5a pathway has been shown to trigger the intracytoplasmic release of Ca2+ and the activation of subsequent Ca2+-related signaling.<a class="elsevierStyleCrossRef" href="#bib0220">22</a> HIRI-induced elevations in intracellular Ca2+ can also lead to the pathological activation of Ca2+/calmodulin-dependent protein kinases (CaMKs),<a class="elsevierStyleCrossRef" href="#bib0130">4</a> eventually contributing to cell death and organ dysfunction following ischemia. We demonstrated H/R treatment induced the intracellular Ca2+ level in BRL-3A cells and this induction was attenuated after siRNA silencing frizzled-2 gene expression, which therefore may rescue calcium level and efficiently mitigate the defects of HIRI.Using in vivo animal study, the expression of Wnt5a and Frizzled-2 was up-regulated significantly in liver tissues from mice with IRI accompanied by a significant decrease in β-catenin compared to the control mice with sham operations, indicating the Wnt5a/Frizzled-2 pathway is involved in mediating hepatic IRI. The increase of cleaved Caspase-3 in liver tissues suggested the involvement of Caspase-3 dependent apoptosis on hepatic IRI.In summary, we showed Wnt5a/Frizzled-2 pathway was involved in maintaining cell cytotoxicity and apoptosis in BRL-3A cells with H/R treatment as well as in liver tissues from mice with IRI. Frizzled-2 mediated Wnt/Ca2+ signaling pathway via Ca2+ overload during H/R. Our data provided an understanding of the role of Wnt5a/Frizzled-2 signaling in the hepatic cell growth and apoptosis under hepatic IRI, indicating Frizzled-2 may be a molecular therapeutic target in the management of a complex clinical physiopathological HIRI condition.Conflict of interestThe authors declare no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:11 [ 0 => array:3 [ "identificador" => "xres1406774" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1287073" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres1406775" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec1287074" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:9 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Cell culture and Hypoxia/Reoxygenation (H/R) model" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Transfection with small interfering RNA (siRNA) targeting frizzled-2 gene" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "RNA isolation and quantitative real-time PCR" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Protein lysate preparation and Western blot" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "LDH cytotoxicity assay" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Apoptosis assay by flow cytometry" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Fluorescence of Ca and laser scanning confocal microscopy" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "In vivo animal study and surgeries for hepatic ischemia and reperfusion injury (IRI)" ] 8 => array:2 [ "identificador" => "sec0055" "titulo" => "Statistical analysis" ] ] ] 6 => array:3 [ "identificador" => "sec0060" "titulo" => "Results" "secciones" => array:6 [ 0 => array:2 [ "identificador" => "sec0065" "titulo" => "Inhibition of frizzled-2 gene by siRNA in rat normal hepatic cell line BRL-3A cells" ] 1 => array:2 [ "identificador" => "sec0070" "titulo" => "H/R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cells" ] 2 => array:2 [ "identificador" => "sec0075" "titulo" => "Frizzled-2 siRNA inhibited the H/R-induced cytotoxicity in BRL-3A cells" ] 3 => array:2 [ "identificador" => "sec0080" "titulo" => "Frizzled-2 siRNA inhibited the H/R-induced apoptosis in BRL-3A cells" ] 4 => array:2 [ "identificador" => "sec0085" "titulo" => "Frizzled-2 siRNA inhibited the H/R-induced intracellular Ca in BRL-3A cells" ] 5 => array:2 [ "identificador" => "sec0090" "titulo" => "Up-regulation of Frizzled-2/Wnt 5 pathway in liver tissues from mice treated with ischemia and reperfusion injury (IRI)" ] ] ] 7 => array:2 [ "identificador" => "sec0095" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0100" "titulo" => "Conflict of interest" ] 9 => array:2 [ "identificador" => "xack489508" "titulo" => "Acknowledgements" ] 10 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2018-06-03" "fechaAceptado" => "2019-02-22" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1287073" "palabras" => array:7 [ 0 => "Frizzled-2" 1 => "siRNA" 2 => "Hypoxia/Reoxygenation (H/R)" 3 => "Apoptosis" 4 => "Intracellular Ca2+" 5 => "Hepatic BRL-3A cells" 6 => "In vivo mice study" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1287074" "palabras" => array:7 [ 0 => "Frizzled-2" 1 => "ARNip" 2 => "Hipoxia/reoxigenación" 3 => "Apoptosis" 4 => "Ca2+ intracelular" 5 => "Hepatocitos BRL-3A" 6 => "Estudio de ratones in vivo" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "Frizzled-2 desempeña un papel importante en el mantenimiento de la funcionalidad normal de los hepatocitos. Este estudio tiene como objetivo analizar el papel de la inhibición de Frizzled-2 en la protección de los hepatocitos BRL-3A de rata de la hipoxia/reoxigenación (H/R). El modelo de hepatocitos H/R in vitro se demostró con el cultivo de células BRL-3A en condiciones de H/R. El ARNip de Frizzled-2 se transinfectó en células BRL-3A para inhibir la señalización de Frizzled-2. Wnt5a y Frizzled-2 aumentaron considerablemente en las células BRL-3A tras el tratamiento con H/R. El tratamiento con H/R provocó citotoxicidad celular, una tasa de apoptosis temprana y el nivel de Ca2+ intracelular en células BRL-3A mientras que el gen frizzled-2 silenciado redujo la citotoxicidad celular inducida por H/R, la apoptosis y el nivel de Ca2+ intracelular. El estudio in vivo con ratones mostró, además, la regulación al alza de la vía de Frizzled-2/Wnt 5 y la expresión de caspasa 3 escindida en tejidos hepáticos con lesión por isquemia y reperfusión (LIR). En resumen, la inhibición de Frizzled-2 por su ARNip puede proteger a las células BRL-3A al atenuar la citotoxicidad celular y la apoptosis inducida por H/R." ] ] "multimedia" => array:7 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1008 "Ancho" => 2125 "Tamanyo" => 112979 ] ] "descripcion" => array:1 [ "en" => "Validation of siRNA silencing frizzled-2 gene expression. (A) Different siRNAs (#25, #33 and #34) targeting frizzled-2 mRNA at different regions and non-targeting siRNA serving as negative control (NC) were transfected at final doses of 25, 50 and 100nM for 24h in BRL-3 cells. Relative expression levels of frizzled-2 were analyzed qRT-PCR and normalized to β-actin. * denoted the group with the frizzled-2 siRNA (#34) at 50nM achieved maximally silencing efficiency among groups. (B) Transfection of frizzled-2 siRNA (#34) and negative control siRNA (NC) was performed at 50nM in BRL-3 cells for 48h. Cell group was set up as mock control. Frizzled-2 protein level was analyzed by Western blot. Relative expression of Frizzled-2 was normalized to GAPDH. Data were presented as means±SEM from three independent experiments. ##p<0.001, Cell or NC groups vs. siRNA group." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2919 "Ancho" => 2500 "Tamanyo" => 266435 ] ] "descripcion" => array:1 [ "en" => "H/R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cells. (A) Relative Frizzled-2 gene expression. (B) Relative Wnt5a gene expression; (C) representative photographs of protein expression of Frizzled-2, Wnt5a and GAPDH; (D) relative protein level of Frizzled-2; (E) relative protein level of Wnt5a. The relative protein expression of Frizzled-2 and Wnt5a was normalized to GAPDH. Data were presented as means±SEM from three independent experiments. *p<0.05 and **p<0.001 when comparing to Cell group, and #p<0.05 and ##p<0.001 when comparing to Si-H/R group." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 891 "Ancho" => 2085 "Tamanyo" => 82109 ] ] "descripcion" => array:1 [ "en" => "The effect of transfection of frizzled-2 siRNA on cell cytotoxicity in BRL-3A cells. (A) LDH assay showed transfection of frizzled-2 siRNA significantly inhibited the LDH activity from 3.27±0.09 to 2.87±0.05 (p<0.05) at OD values; (B) LDH activities were shown as percentage of OD values. *p<0.05 Cell or NC groups vs. Si-H/R group." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 1832 "Ancho" => 1452 "Tamanyo" => 120860 ] ] "descripcion" => array:1 [ "en" => "Caspase-3 protein expression was induced by H/R and suppressed by transfection of frizzled-2 siRNA. (A) Representative photographs of protein expression of Caspase-3 and GAPDH; (B) relative Caspase-3 protein level was normalized to GAPDH; data were presented as means±SEM from three independent experiments. *p<0.05 when comparing to Cell group, and #p<0.05 when comparing to Si-H/R group." ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 3435 "Ancho" => 2500 "Tamanyo" => 489627 ] ] "descripcion" => array:1 [ "en" => "Transfection of frizzled-2 siRNA inhibited the H/R induced apoptosis in BRL-3A cells by Flow Cytometry using Annexin-V-FITC method. (A) BRL-3A cells alone; (B) BRL-3A cells with H/R treatment (Cell-H/R); (C) H/R treatment of BRL-3A cells with transfection of negative control siRNA (NC-H/R); (D) H/R treatment of BRL-3A cells with transfection of frizzled-2 siRNA (Si-H/R); (E) quantitative analysis of the early cell apoptosis; (F) quantitative analysis of the late cell apoptosis; (G) quantitative analysis of the early cell apoptosis. Data were presented as means±SEM from three independent experiments. *p<0.05 and **p<0.001 when comparing to Cell group, and #p<0.05 and ##p<0.001 when comparing to Si-H/R group." ] ] 5 => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2065 "Ancho" => 1500 "Tamanyo" => 127595 ] ] "descripcion" => array:1 [ "en" => "Transfection of frizzled-2 siRNA inhibited the H/R-induced intracellular Ca2+ concentration in BRL-3A cells. Fluorescence was shown in green color (528nm) to monitor the intracellular Ca2+ in BRL-3A cells with H/R treatment by Fluro 8-AM using confocal microscopy. (A) BRL-3A cells with H/R treatment (Cell-H/R); (B) H/R treatment of BRL-3A cells with transfection of negative control siRNA; (C) H/R treatment of BRL-3A cells with transfection of frizzled-2 siRNA; (D) control cells without H/R treatment; (E) bar graph showed the quantitative analysis of mean fluorescence intensity (MFI) of the intracellular Ca2+ concentration in different cell groups. Blue color denoted the nuclear stained with DAPI. Data were presented as means±SEM from three independent experiments. *p<0.05 and **p<0.001 when comparing to Cell group, and #p<0.05 and ##p<0.001 when comparing to Si-H/R group." ] ] 6 => array:7 [ "identificador" => "fig0035" "etiqueta" => "Figure 7" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr7.jpeg" "Alto" => 2370 "Ancho" => 2519 "Tamanyo" => 299553 ] ] "descripcion" => array:1 [ "en" => "The up-regulation of Wnt5a, Frizzled-2, cleaved Caspase-3 (cCaspase-3) accompanied by a decrease in β-catenin was shown in liver tissues from mice with IRI. (A) Representative photographs of protein expression of Frizzled-2, Wnt5a, cCaspase-3 (17kDa and 19kDa), β-catenin and GAPDH; (B) relative protein level of Wnt5a; (C) relative protein level of Frizzled-2; (D) relative cCaspase-3 (19kDa); (E) relative cCaspase-3 (17kDa); (F) relative β-catenin gene expression. Bar graphs showing the relative protein expression of Wnt5a, Frizzled-2, cCaspase-3 (17kDa and 19kDa) and β-catenin were individually normalized to GAPDH and presented as means±SEM from six mice from group IRI or control group with sham operations. *p<0.05 when comparing to control group." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0015" "bibliografiaReferencia" => array:22 [ 0 => array:3 [ "identificador" => "bib0115" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Protective strategies against ischemic injury of the liver" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:4 [ 0 => "N. Selzner" 1 => "H. Rudiger" 2 => "R. Graf" 3 => "P.A. 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Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation

La inhibición de Frizzled-2 por ARN interferente pequeño protege a los hepatocitos BRL-3A de rata frente a la citotoxicidad y la apoptosis inducidas por hipoxia/reoxigenación

Xiang Hua,b, Chenjie Zhoua, Guolin Hea, Yuan Chenga, Mingxin Pana, Yi Gaoa,

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drgaoy@126.com

Corresponding author.

a Department of Hepatobiliary Surgery II, State Key Laboratory of Organ Failure Research, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, Guangdong Province, China

b General Surgery Department, The Second Hospital of Shenzhen Baoan People's Hospital Group, Shenzhen 518108, Guangdong Province, China

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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Hepatic ischemia&#8211;reperfusion injury &#40;HIRI&#41; is a major cause of morbidity and mortality in liver resection and transplantation surgery&#46;<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">1&#44;2</span></a> HIRI of the liver results in microvascular changes and acute inflammation&#44; ultimately leading to cell death&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">2</span></a> HIRI induced cell death includes apoptosis&#44; autophagy&#44; necrosis and necroptosis in hepatocytes and non-parenchymal cells&#46;<a class="elsevierStyleCrossRefs" href="#bib0125"><span class="elsevierStyleSup">3&#44;4</span></a> Restoration of blood flow is necessary to restore cellular function&#44; but paradoxically reperfusion can initiate a cascade of pathways that cause further cellular injury after prolonged ischemia&#46; Understanding the molecular events of liver ischemia&#8211;reperfusion injury helps develop strategies to counterattack this injury and reduce acute complications in hepatic resection and transplantation&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Wnt signaling pathway is selectively activated in hepatic development&#44; regeneration and hepatocellular carcinoma &#40;HCC&#41;&#44; in a canonical or a non-canonical pathway&#46;<a class="elsevierStyleCrossRefs" href="#bib0135"><span class="elsevierStyleSup">5&#8211;9</span></a> Wnt signaling is mediated by its receptors&#44; Frizzled family proteins&#44; which contain seven trans-membranes in humans and mice&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">9</span></a> Canonical Wnt signaling is mediated by Frizzled-1&#44; and regulates the nuclear accumulation of &#946;-catenin&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">10</span></a> Non-canonical Wnt signaling is &#946;-catenin-independent&#44; and includes pathways&#44; such as mitogen-activated protein kinase&#44;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">11</span></a> NF&#954;B and c-Jun N terminal kinase &#40;JNK&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">12</span></a> ROCK&#47;Rho kinase&#44;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">13</span></a> protein kinase C &#40;PKC&#41; and Ca<span class="elsevierStyleSup">2&#43;</span>&#47;calmodulin-dependent protein kinase II &#40;CaMKII&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">14</span></a> Frizzled-2 binds Wnt5a to activate the non-canonical Wnt pathway&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">15</span></a> Activation of Wnt5a&#47;Frizzled-2 signaling has been shown to turn off &#946;-catenin during hepatic development and liver regeneration after partial hepatectomy&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">16</span></a> Wnt5a loss leads to enhanced &#946;-catenin signaling in hepatocellular carcinoma&#46;<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">7&#44;17</span></a> Wnt5a&#47;Frizzled-2 signaling pathway was activated in response to H&#47;R in H9C2 cells&#44;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">18</span></a> indicating its important role in <span class="elsevierStyleItalic">in vivo</span> myocardial cells post reperfusion&#46; However&#44; the Frizzled-2 signaling pathway has not been understood yet in the hepatic development under pathophysiological HIRI conditions&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">In this study&#44; we examined the effects of Frizzled-2 on the H&#47;R treated rat normal liver BRL-3A cells&#44; an <span class="elsevierStyleItalic">in vitro</span> cell model established to mimic the pathophysiological HIRI&#46; The Wnt5a&#47;Frizzled-2 signaling was suppressed by silencing <span class="elsevierStyleItalic">frizzled-2</span> gene expression using the siRNA approach&#46; Inhibition of Frizzled-2 significantly attenuated the H&#47;R induced cytotoxicity&#44; apoptosis and intracellular Ca<span class="elsevierStyleSup">2&#43;</span> in BRL-3A cells&#46; Our data suggest the inhibition of Wnt5a&#47;Frizzled-2 signaling pathway may represent a novel and promising approach to protect liver cells against HIRI-related disorders&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Cell culture and Hypoxia&#47;Reoxygenation &#40;H&#47;R&#41; 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BRL-3A cells in culture medium with low serum media &#40;0&#46;5&#37; FBS&#41; were exposed to hypoxia for 2<span class="elsevierStyleHsp" style=""></span>h in a humidified incubation chamber at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and flushed with a gas mixture of 95&#37; N<span class="elsevierStyleInf">2</span> and 5&#37; CO<span class="elsevierStyleInf">2</span>&#44; followed by incubation in fresh medium with 95&#37; air and 5&#37; CO<span class="elsevierStyleInf">2</span> for reoxygenation&#46; The control cells were kept in normoxic conditions&#46; After reoxygenation for16<span class="elsevierStyleHsp" style=""></span>h&#44; cells were harvested for RNA or protein analysis&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Transfection with small interfering RNA &#40;siRNA&#41; targeting <span class="elsevierStyleItalic">frizzled-2</span> gene</span><p id="par0025" class="elsevierStylePara elsevierViewall">For transfection&#44; BRL-3A cells were plated in 6-well plate at a density of 10<span class="elsevierStyleSup">5</span> cells per well or in 100<span class="elsevierStyleHsp" style=""></span>mm dishes with 10<span class="elsevierStyleSup">6</span> cells in growth medium without antibiotics&#46; After cell growth reached 50&#8211;60&#37; confluence&#44; transfection was performed using Lipofectamine 2000 from Invitrogen &#40;ThermoFisher Scientific&#44; MA&#44; USA&#41;&#46; Cells were incubated in Opti-MEM I Reduced Serum Medium from Invitrogen with or without <span class="elsevierStyleItalic">frizzled-2</span> siRNA&#8211;Lipofectamine 2000 complexes &#40;25&#44; 50&#44; or 100<span class="elsevierStyleHsp" style=""></span>nM&#41; for 6<span class="elsevierStyleHsp" style=""></span>h and then replaced with fresh DMEM for 48<span class="elsevierStyleHsp" style=""></span>h&#46; The transfection efficiency of siRNA was monitored by observing the fluorescence of cells transfected with FAM labeled siRNA negative control &#40;siRNA-NC&#41; after 6<span class="elsevierStyleHsp" style=""></span>h&#46; Then cells were subjected to H&#47;R treatment and then harvested for other experiments&#46; Three different non-overlapping siRNA duplexes and FAM labeled siRNA negative control were designed and purchased from Sigma-Aldrich &#40;Guangzhou&#44; China&#41;&#46; The sequences were as follows&#58; <span class="elsevierStyleItalic">firizzled-2</span> siRNA &#35;25&#58; 5&#8242;-GUGCUGUGCUGCGCUUCUAdTdT-3&#8242;&#59; <span class="elsevierStyleItalic">firizzled-2</span> siRNA &#35;33&#58; 5&#8242;-CUAUCUCAGCUAUAAGUUUdTdT-3&#8242;&#59; <span class="elsevierStyleItalic">firizzled-2</span> siRNA &#35;34&#58; 5&#8242;-GCUGUACUAUACUCUUCAUdTdT-3&#8242; and negative control&#58; 5&#8242;-UUCUCCGAACGUGUCACGUdTdT-3&#8242;&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">RNA isolation and quantitative real-time PCR</span><p id="par0030" class="elsevierStylePara elsevierViewall">Total RNA was isolated from BRL-3A cells s using Trizol from Invitrogen &#40;ThermoFisher Scientific&#44; MA&#44; USA&#41; according to the manufacturer&#39;s protocols&#46; Quantitative Real-Time PCR &#40;qRT-PCR&#41; was carried out using an ABI 7500 Real-time PCR System &#40;Applied Biosystems&#44; Foster&#44; CA&#44; USA&#41;&#46; SYBR Green PCR SuperMix was used as a double-stranded DNA-specific dye according to the manufacturer&#39;s instructions &#40;ThermoFisher Scientific&#44; MA USA&#41;&#46; Primers were designed for a single qRT-PCR thermal profile &#40;95<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>min&#44; and 40 cycles of 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 15<span class="elsevierStyleHsp" style=""></span>s and 55<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>min for <span class="elsevierStyleItalic">frizzled-2</span> and 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>min&#44; and 40 cycles of 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 15<span class="elsevierStyleHsp" style=""></span>s and 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>min for <span class="elsevierStyleItalic">Wnt5a</span> and <span class="elsevierStyleItalic">&#946;-actin</span>&#41;&#46; The expression levels of examined transcripts were normalized to &#946;-actin&#46; Primer sequences for <span class="elsevierStyleItalic">frizzled-2</span>&#44; <span class="elsevierStyleItalic">Wnt5a</span> and <span class="elsevierStyleItalic">&#946;-actin</span> were listed below&#58; <span class="elsevierStyleItalic">frizzled-2</span>-forward&#58; 5&#8242;-TCGTTTTGCCCGTCTCT-3&#8242;&#44; <span class="elsevierStyleItalic">frizzled-2</span>-reverse&#58; 5&#8242;-TAGCGGAATCGCTGCAT-3&#8242;&#59; <span class="elsevierStyleItalic">Wnt5a</span>-forward&#58; 5&#8242;-CGTGGCTATGACCAGTTTAAG-3&#8242;&#59; <span class="elsevierStyleItalic">Wnt5a</span>-reverse&#58; 5&#8242;-CCACAATCTCCGTGCACTT-3&#8242;&#59; <span class="elsevierStyleItalic">&#946;-actin</span>-forward&#58; 5&#8242;-AGGGAAATCGTGCGTGACAT-3&#8242;&#59; <span class="elsevierStyleItalic">&#946;-actin</span>-reverse&#58; 5&#8242;-GAACCGCTCATTGCCGATAG-3&#8242;&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Protein lysate preparation and Western blot</span><p id="par0035" class="elsevierStylePara elsevierViewall">Cultured BRL-3A cells were lysed in ice-cold Radioimmunoprecipitation assay &#40;RIPA&#41; Lysis and Extraction Buffer &#40;Beyotime Biotechnology&#44; Shanghai&#44; China&#41; and total protein samples were prepared in extraction buffer &#40;10<span class="elsevierStyleHsp" style=""></span>mM Tris&#44; 100<span class="elsevierStyleHsp" style=""></span>mM NaCl&#44; 1<span class="elsevierStyleHsp" style=""></span>mM EDTA&#44; 1<span class="elsevierStyleHsp" style=""></span>mM EGTA&#44; 1<span class="elsevierStyleHsp" style=""></span>mM NaF&#44; 20<span class="elsevierStyleHsp" style=""></span>mM Na<span class="elsevierStyleInf">4</span>P<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">7</span>&#44; 2<span class="elsevierStyleHsp" style=""></span>mM Na<span class="elsevierStyleInf">3</span>VO<span class="elsevierStyleInf">4</span>&#44; 0&#46;1&#37; SDS&#44; 0&#46;5&#37; sodium deoxycholate&#44; 1&#37; Triton-100&#44; 10&#37; glycerol and 1<span class="elsevierStyleHsp" style=""></span>mM PMSF&#41; containing 1&#215; Cocktail of Proteinase inhibitors and Phosphatase inhibitors &#40;ThermoFisher Scientific&#44; MA&#44; USA&#41;&#46; Protein concentration was measured using BCA protein assay kit &#40;Kaiji Biotechnology&#44; Nanjing&#44; China&#41;&#46; A 20<span class="elsevierStyleHsp" style=""></span>&#956;g of protein for each sample was loaded into each well and resolved by SDS-10&#37; PAGE and transferred onto PVDF membranes via electroblotting &#40;Millipore&#44; Billerica&#44; MA&#44; USA&#41;&#46; After blocking 1<span class="elsevierStyleHsp" style=""></span>h in 1&#215; TBST containing 5&#37; bovine serum albumin &#40;BSA&#41;&#44; membranes were washed three times in 1&#215; TBST and probed with the following primary antibodies at 4<span class="elsevierStyleHsp" style=""></span>&#176;C overnight&#58; rabbit polyclonal to Frizzled-2 &#40;1&#58;1000&#59; Abcam&#44; Cambridge&#44; MA&#44; USA&#41;&#44; rabbit polyclonal to Wnt5a &#40;1&#58;2000&#59; Abcam&#44; Cambridge&#44; MA&#44; USA&#41; and goat polyclonal antibody to human GAPDH &#40;1&#58;1000&#59; Santa Cruz&#44; California&#44; CA&#44; USA&#41;&#46; The blots were then incubated with goat anti-rabbit IgG peroxidase-conjugated secondary antibody &#40;1&#58;6000&#59; Calbiochem&#44; Merck KgaA&#44; Darmstadt&#44; Germany&#41; and visualized by enhanced SuperSignal West Pico Chemiluminescent Substrate &#40;Pierce Biotechnology&#44; Rockford&#44; USA&#41;&#46; GAPDH was used as the internal loading control&#46; For quantification&#44; Image-Pro Plus 6&#46;0 software &#40;Media Cybernetics Inc&#46;&#44; Silver Spring&#44; MD&#44; USA&#41; was used to measure the integrated optical density &#40;IOD&#41; of the bands&#46; The relative level of protein expression was expressed as a ratio to GAPDH&#46; Experiments were repeated three times for each experimental condition&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">LDH cytotoxicity assay</span><p id="par0040" class="elsevierStylePara elsevierViewall">The cytotoxicity of BRL-3A cells was determined by Lactate Dehydrogenase &#40;LDH&#41; released from the damaged cells&#46; Cells were seeded in triplicate in 6-well plates and incubated overnight&#46; Then cells were subject to transfection of siRNA of <span class="elsevierStyleItalic">frizzled-2</span> or control siRNA for 24<span class="elsevierStyleHsp" style=""></span>h&#44; respectively then followed by H&#47;R treatment&#46; Cell lysates were harvested for LDH assay according to the manufactory protocol from Pierce Biotechnology &#40;Rockford&#44; IL&#44; USA&#41;&#46; The absorbance was measured at 490<span class="elsevierStyleHsp" style=""></span>nm and 680<span class="elsevierStyleHsp" style=""></span>nm&#46; To determine LDH activity&#44; subtract the 680<span class="elsevierStyleHsp" style=""></span>nm absorbance value as background from the 490<span class="elsevierStyleHsp" style=""></span>nm absorbance before calculation of &#37; cytotoxicity&#46; Experiments were repeated three times independently&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Apoptosis assay by flow cytometry</span><p id="par0045" class="elsevierStylePara elsevierViewall">To assess cell apoptosis&#44; an Annexin V-fluorescein isothiocyanate &#40;V-FITC&#41; staining assay from Invitrogen &#40;ThermoFisher Scientific&#44; MA&#44; USA&#41; was performed according to the manufacturer&#39;s instructions&#46; Briefly&#44; the BRL-3A cells were detached by 0&#46;25&#37; Trypsin and collected for staining with Annexin V-FITC and PI for 15<span class="elsevierStyleHsp" style=""></span>min at room temperature &#40;18&#8211;24<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#46; The cells were then washed twice with PBS&#44; and the fluorescence was analyzed using a FACSCalibur flow cytometry system and CellQuest software &#40;BD Biosciences&#44; New Jersey&#44; USA&#41;&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Fluorescence of Ca<span class="elsevierStyleSup">2&#43;</span> and laser scanning confocal microscopy</span><p id="par0050" class="elsevierStylePara elsevierViewall">For calcium detection&#44; cells grown in the 6-well plates were incubated with the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> indicator Fluo 8-AM at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>min according to the manufacturer&#39;s protocol from Invitrogen &#40;ThermoFisher&#44; MA&#44; USA&#41;&#46; The fluorescence intensity was monitored at 490<span class="elsevierStyleHsp" style=""></span>nm&#47;515<span class="elsevierStyleHsp" style=""></span>nm &#40;Ex&#47;Em&#41;&#46; Fluorescent images were acquired by a LSM510 confocal laser scanning microscope &#40;Carl-Zeiss&#44; Jena&#44; Germany&#41; and analyzed using Image-Pro Plus 6&#46;0 software &#40;Media Cybernetics&#44; Silver Spring&#44; MD&#44; USA&#41;&#46; The average fluorescence density of cell preparations from each group was compared among the groups&#46; Values of integrated optical densities were expressed as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM of at least three different experiments&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070"><span class="elsevierStyleItalic">In vivo</span> animal study and surgeries for hepatic ischemia and reperfusion injury &#40;IRI&#41;</span><p id="par0055" class="elsevierStylePara elsevierViewall">Total 12 male C57BL&#47;6 mice at 8- to 10-week old were bred and housed at State Key Laboratory of Biological Artificial Liver in Zhujiang Hospital&#46; For surgery for hepatic ischemia and reperfusion injury &#40;IRI&#41;&#44; 6 mice were anesthetized with isoflurane and a small midline was vertically incised on the abdominal wall to access the liver and portal triad&#46; The blood supply to left and central hepatic lobes was occluded via vascular clips on the hepatic artery and portal vein for 90<span class="elsevierStyleHsp" style=""></span>min&#44; followed by a 6<span class="elsevierStyleHsp" style=""></span>h period of reperfusion before sacrifice&#46; For IRI sham operations&#44; 6 mice were also anesthetized and the abdominal wall was vertically incised at midline and left open but covered with saline-soaked gauze for 30<span class="elsevierStyleHsp" style=""></span>min before the incision was closed&#46; Mice were placed on a warming pad during and after surgery to maintain a body temperature of 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; At sacrifice&#44; liver tissues were collected and stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C for further analysis&#46; All procedures were approved by the Institutional Animal Care and Use Committee and followed at guide of our hospital&#44; Zhujiang Hospital&#44; and Southern Medical University for the care and the use of laboratory animals&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Statistical analysis</span><p id="par0060" class="elsevierStylePara elsevierViewall">All data are presented as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard error of the mean &#40;SEM&#41;&#46; Student&#39;s <span class="elsevierStyleItalic">t</span>-test and one-way analysis of variance &#40;ANOVA&#41; were used to examine the overall statistical differences using SPSS software &#40;Version 24&#46;0&#44; Chicago&#44; IL&#44; USA&#41;&#46; Differences between the groups were considered statistically significant at a <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#46;</p></span></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Results</span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Inhibition of <span class="elsevierStyleItalic">frizzled-2</span> gene by siRNA in rat normal hepatic cell line BRL-3A cells</span><p id="par0065" class="elsevierStylePara elsevierViewall">To validate the <span class="elsevierStyleItalic">frizzled-2</span> siRNA silencing efficiency&#44; the <span class="elsevierStyleItalic">frizzled-2</span> gene expression was detected in BRL-3A cells using qRT-PCR after siRNA transfection for 24<span class="elsevierStyleHsp" style=""></span>h&#46; The <span class="elsevierStyleItalic">frizzled-2</span> mRNA levels were inhibited by 30&#46;44&#37;&#44; 69&#46;22&#37; and 74&#46;79&#37; by transfection of siRNA-&#35;25&#44; -&#35;33 and -&#35;34 at 50<span class="elsevierStyleHsp" style=""></span>nM&#44; respectively&#44; compared to the negative control siRNA &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41;&#46; A significant decrease in Frizzle protein expression was also shown in the group with transfection of siRNA-&#35;34 at 50<span class="elsevierStyleHsp" style=""></span>nM by Western blot compared to mock control Cell group or negative control &#40;NC&#41; group &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B&#41;&#46; With the highest efficiency &#40;74&#46;79&#37;&#41; in silencing <span class="elsevierStyleItalic">frizzled-2</span> expression in BRL-3A cells&#44; transfection of siRNA-&#35;34 at 50<span class="elsevierStyleHsp" style=""></span>nM was chosen to knockdown <span class="elsevierStyleItalic">frizzled-2</span> gene expression in the subsequent experiments&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">H&#47;R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cells</span><p id="par0070" class="elsevierStylePara elsevierViewall">To determine whether Frizzled-2&#47;Wnt5a pathway was responded to the H&#47;R treatment in BRL-3A cells&#44; the expression of Frizzled-2 and Wnt5a were examined in BRL-3A cells with or without H&#47;R &#40;Cell-H&#47;R or Cell&#41; by qRT-PCR and Western blot&#46; Significant increases in the expression of both Frizzled-2 and Wnt5a were detected at mRNA &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A and B&#41; and protein levels in BRL-3A cells after H&#47;R treatment &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>D&#8211;E&#41;&#46; The H&#47;R induced <span class="elsevierStyleItalic">frizzled-2</span> mRNA as well as its protein levels were diminished by <span class="elsevierStyleItalic">frizzled-2</span> siRNA transfection in BRL-3A cells &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A&#44; C and D&#41;&#44; whereas <span class="elsevierStyleItalic">Wnt5a</span> gene expression was induced &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>B&#44; C and E&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Frizzled-2 siRNA inhibited the H&#47;R-induced cytotoxicity in BRL-3A cells</span><p id="par0075" class="elsevierStylePara elsevierViewall">To examine the effect of H&#47;R treatment on cell cytotoxicity in BRL-3A cells&#44; the activity of LDH released from the dead cells was measured&#46; LDH assay showed transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA significantly inhibited the LDH activity by reducing the OD value from 3&#46;27<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;09 to 2&#46;87<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;05 &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>A and B&#41;&#44; suggesting silencing <span class="elsevierStyleItalic">frizzled-2</span> gene expression enables to protect hepatic cells injury caused by H&#47;R&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Frizzled-2 siRNA inhibited the H&#47;R-induced apoptosis in BRL-3A cells</span><p id="par0080" class="elsevierStylePara elsevierViewall">As a critical protein involved in cell apoptotic process&#44; Caspase-3 protein level was used to monitor cell apoptosis during H&#47;R&#46; Western blot analysis showed Caspase-3 level was significantly increased in BRL-3A cells after H&#47;R treatment &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>A&#41; and this increase was suppressed by the <span class="elsevierStyleItalic">frizzled-2</span> siRNA transfection&#46; The fold change of Caspase-3 protein level was further presented by the bar graph &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>B&#41;&#46; The early apoptosis rates were further determined by Annexin-V-FITC using flow cytometry assay&#46; Total cell apoptosis was significantly induced by the H&#47;R treatment in BRL-3A cells &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>A and B&#41;&#46; Compared to the negative control group&#44; transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA decreased early apoptosis in BRL-3A cells during H&#47;R &#40;0&#46;74<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;09 vs&#46; 6&#46;06<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;59&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#59; <a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>C&#8211;E&#41;&#46; Similar levels of cell late apoptosis rates &#40;3&#46;12<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;70 vs&#46; 3&#46;34<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;40&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#59; <a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>C&#44; D and F&#41; but a significant decrease in total cell apoptosis &#40;3&#46;86<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;64 vs&#46; 9&#46;41<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;56&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#59; <a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>C&#44; D and G&#41; were shown in cell group with the transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA during H&#47;R treatment compared to the negative control group&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Frizzled-2 siRNA inhibited the H&#47;R-induced intracellular Ca<span class="elsevierStyleSup">2&#43;</span> in BRL-3A cells</span><p id="par0085" class="elsevierStylePara elsevierViewall">As Ca<span class="elsevierStyleSup">2&#43;</span> overload is usually involved in the HIRI process&#44; we detected the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> concentration in BRL-3A cells with H&#47;R treatment through the mean fluorescence intensity &#40;MFI&#41; of Furo 8-AM using confocal microscopy&#46; The intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level was clearly increased in the BRL-3A cells with H&#47;R treatment compared to the normal cells &#40;MFI&#58; 55&#46;34<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;42 vs&#46; 46&#46;49<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>6&#46;3&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;5&#44; <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>A&#44; D and E&#41;&#46; To test whether inhibition of Wnt5a&#47;Frizzled-2 pathway regulates the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level&#44; transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA was performed to examine the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level&#46; The Ca<span class="elsevierStyleSup">2&#43;</span> concentration was significantly suppressed in the H&#47;R treated BRL-3A cells with transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA compared to the normal cells &#40;MFI 23&#46;71<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>7&#46;68 vs&#46; 46&#46;49<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>6&#46;30&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#59; <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>C&#8211;E&#41;&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Up-regulation of Frizzled-2&#47;Wnt 5 pathway in liver tissues from mice treated with ischemia and reperfusion injury &#40;IRI&#41;</span><p id="par0090" class="elsevierStylePara elsevierViewall">To validate the role of Frizzled-2&#47;Wnt5 pathway mediating H&#47;R in a hepatocyte-like cell line&#44; <span class="elsevierStyleItalic">in vivo</span> animal study was further performed in a clinically relevant setting of hepatic IRI in mice &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 7</a>&#41;&#46; The expression of Wnt5a&#44; Frizzled-2&#44; and cleaved Caspase-3 at 17<span class="elsevierStyleHsp" style=""></span>kDa and 19<span class="elsevierStyleHsp" style=""></span>kDa was individually up-regulated significantly accompanied by a significant decrease in &#946;-catenin in liver tissues from mice with IRI compared to the control group &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 7</a>A&#8211;F&#41;&#46;</p><elsevierMultimedia ident="fig0035"></elsevierMultimedia></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Discussion</span><p id="par0095" class="elsevierStylePara elsevierViewall">As a clinical condition&#44; HIRI may lead to cellular injury and organ dysfunction and is mediated mainly through different mechanisms&#44; which include Wnt&#47;&#946;-catenin signaling pathway and Ca<span class="elsevierStyleSup">2&#43;</span> overload&#46; To address the potential hepatocyte functionality in response to HIRI&#44; we first established the <span class="elsevierStyleItalic">in vitro</span> H&#47;R hepatic cell model using rat normal liver BRL-3A cells to mimic <span class="elsevierStyleItalic">in vivo</span> hepatic cells under HIRI condition&#44; which allows for <span class="elsevierStyleItalic">in vitro</span> cellular functional and mechanistic studies&#46; In this study&#44; we investigated the effects of H&#47;R treatment on the expression of Frizzled-2&#47;Wnt5a pathway&#46; Frizzled-2 and Wnt5a gene and protein expression were significantly increased in BRL-3A cells by H&#47;R treatment&#46; We further demonstrated H&#47;R treatment induced cell cytotoxicity&#44; apoptosis and the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level in BRL-3A cells&#46; This induction could be suppressed by transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA&#44; suggesting suppression of Frizzled-2&#47;Wnt5a signaling played important roles in protection of hepatic cells against HIRI&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">The ischemic injury could be mediated through various possible mechanisms&#46; The Wnt signaling pathway is well established in liver cell biology and plays a critical role as a molecular regulator in hepatic development&#44; regeneration and carcinogenesis&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">8</span></a> Wnt5a&#47;Frizzled-2 has been shown to inhibit liver regeneration process by suppressing &#946;-catenin signaling in hepatocytes&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">16</span></a> Frizzled proteins can stimulate different Wnts at the cell surface and discriminate among different Wnt ligands&#44;<a class="elsevierStyleCrossRefs" href="#bib0120"><span class="elsevierStyleSup">2&#44;19</span></a> which determines the specific downstream pathway activated in the cells&#46; Pharmacologic activation of canonical &#946;-catenin dependent Wnt signaling protects against hepatic ischemia&#47;reperfusion injury through inducing proliferation and reducing apoptosis&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">20</span></a> Noncanonical Wnt5a strongly inhibited canonical Wnt signaling&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">7</span></a> In hepatocellular carcinoma&#44; high levels of Frizzled-2 expression induced EMT and enhanced cell migration and invasiveness&#44; while transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA resulted in a significant reduction in cell migration and invasion but not proliferation in HCC cell lines&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">21</span></a> Canonical and noncanonical Wnt pathways have complementary roles in HCC&#44; where the canonical signaling contributes to tumor initiation&#44; and noncanonical signaling to tumor progression&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">7</span></a> In the present study&#44; siRNA silencing the <span class="elsevierStyleItalic">frizzled-2</span> gene expression led to a decrease in cell cytotoxicity and apoptosis&#44; indicating the activation of Wnt5a&#47;Frizzled-2 might enhance the cell death after HIRI whereas inhibition of Frizzled-2 may protect liver cell against injury and help to maintain hepatic cell integrity&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">During reperfusion injury&#44; both apoptosis and necrosis pathways are present in cell death&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">3</span></a> Several proapoptotic proteins are activated during the reperfusion phase&#44; including the proteases Caspase-3 and Caspase-8&#44; which finally leads to the destruction of nuclear DNA&#44; resulting in cell death&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">1</span></a> Caspase-3 is one of the mediators in apoptotic pathway which can be enhanced by Ca<span class="elsevierStyleSup">2&#43;</span> accumulation post-injury&#46; In this study&#44; our data demonstrated the H&#47;R induced cell death and apoptosis in BRL-3A cells&#46; In addition to morphological criteria&#44; induction of Caspase-3 activity provided corroborative evidence for hepatocyte apoptosis&#46; Transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA blocked the H&#47;R-induced LDH and Caspase-3 levels&#44; which emphasized the role of Frizzled-2 in apoptotic cell death in the hepatic H&#47;R&#46; The liver cell membrane structure&#44; the endoplasmic reticulum and sarcoplasmic reticulum are damaged in response to HIRI&#44; which may lead to the abnormal function of the calcium pump and subsequent elevation of the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> concentration&#46; Activation of Wnt5a pathway has been shown to trigger the intracytoplasmic release of Ca<span class="elsevierStyleSup">2&#43;</span> and the activation of subsequent Ca<span class="elsevierStyleSup">2&#43;</span>-related signaling&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">22</span></a> HIRI-induced elevations in intracellular Ca<span class="elsevierStyleSup">2&#43;</span> can also lead to the pathological activation of Ca<span class="elsevierStyleSup">2&#43;</span>&#47;calmodulin-dependent protein kinases &#40;CaMKs&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">4</span></a> eventually contributing to cell death and organ dysfunction following ischemia&#46; We demonstrated H&#47;R treatment induced the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level in BRL-3A cells and this induction was attenuated after siRNA silencing <span class="elsevierStyleItalic">frizzled-2</span> gene expression&#44; which therefore may rescue calcium level and efficiently mitigate the defects of HIRI&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">Using <span class="elsevierStyleItalic">in vivo</span> animal study&#44; the expression of Wnt5a and Frizzled-2 was up-regulated significantly in liver tissues from mice with IRI accompanied by a significant decrease in &#946;-catenin compared to the control mice with sham operations&#44; indicating the Wnt5a&#47;Frizzled-2 pathway is involved in mediating hepatic IRI&#46; The increase of cleaved Caspase-3 in liver tissues suggested the involvement of Caspase-3 dependent apoptosis on hepatic IRI&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">In summary&#44; we showed Wnt5a&#47;Frizzled-2 pathway was involved in maintaining cell cytotoxicity and apoptosis in BRL-3A cells with H&#47;R treatment as well as in liver tissues from mice with IRI&#46; Frizzled-2 mediated Wnt&#47;Ca<span class="elsevierStyleSup">2&#43;</span> signaling pathway via Ca<span class="elsevierStyleSup">2&#43;</span> overload during H&#47;R&#46; Our data provided an understanding of the role of Wnt5a&#47;Frizzled-2 signaling in the hepatic cell growth and apoptosis under hepatic IRI&#44; indicating Frizzled-2 may be a molecular therapeutic target in the management of a complex clinical physiopathological HIRI condition&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Conflict of interest</span><p id="par0120" class="elsevierStylePara elsevierViewall">The authors declare no conflicts of interest&#46;</p></span></span>"
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          "identificador" => "xres1406774"
          "titulo" => "Abstract"
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          "titulo" => "Resumen"
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          "titulo" => "Palabras clave"
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        4 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
        ]
        5 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:9 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Cell culture and Hypoxia&#47;Reoxygenation &#40;H&#47;R&#41; model"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Transfection with small interfering RNA &#40;siRNA&#41; targeting frizzled-2 gene"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "RNA isolation and quantitative real-time PCR"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Protein lysate preparation and Western blot"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "LDH cytotoxicity assay"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Apoptosis assay by flow cytometry"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Fluorescence of Ca and laser scanning confocal microscopy"
            ]
            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "In vivo animal study and surgeries for hepatic ischemia and reperfusion injury &#40;IRI&#41;"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Statistical analysis"
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          ]
        ]
        6 => array:3 [
          "identificador" => "sec0060"
          "titulo" => "Results"
          "secciones" => array:6 [
            0 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Inhibition of frizzled-2 gene by siRNA in rat normal hepatic cell line BRL-3A cells"
            ]
            1 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "H&#47;R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cells"
            ]
            2 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Frizzled-2 siRNA inhibited the H&#47;R-induced cytotoxicity in BRL-3A cells"
            ]
            3 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Frizzled-2 siRNA inhibited the H&#47;R-induced apoptosis in BRL-3A cells"
            ]
            4 => array:2 [
              "identificador" => "sec0085"
              "titulo" => "Frizzled-2 siRNA inhibited the H&#47;R-induced intracellular Ca in BRL-3A cells"
            ]
            5 => array:2 [
              "identificador" => "sec0090"
              "titulo" => "Up-regulation of Frizzled-2&#47;Wnt 5 pathway in liver tissues from mice treated with ischemia and reperfusion injury &#40;IRI&#41;"
            ]
          ]
        ]
        7 => array:2 [
          "identificador" => "sec0095"
          "titulo" => "Discussion"
        ]
        8 => array:2 [
          "identificador" => "sec0100"
          "titulo" => "Conflict of interest"
        ]
        9 => array:2 [
          "identificador" => "xack489508"
          "titulo" => "Acknowledgements"
        ]
        10 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2018-06-03"
    "fechaAceptado" => "2019-02-22"
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          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1287073"
          "palabras" => array:7 [
            0 => "Frizzled-2"
            1 => "siRNA"
            2 => "Hypoxia&#47;Reoxygenation &#40;H&#47;R&#41;"
            3 => "Apoptosis"
            4 => "Intracellular Ca<span class="elsevierStyleSup">2&#43;</span>"
            5 => "Hepatic BRL-3A cells"
            6 => "<span class="elsevierStyleItalic">In vivo</span> mice study"
          ]
        ]
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        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Palabras clave"
          "identificador" => "xpalclavsec1287074"
          "palabras" => array:7 [
            0 => "Frizzled-2"
            1 => "ARNip"
            2 => "Hipoxia&#47;reoxigenaci&#243;n"
            3 => "Apoptosis"
            4 => "Ca<span class="elsevierStyleSup">2&#43;</span> intracelular"
            5 => "Hepatocitos BRL-3A"
            6 => "Estudio de ratones <span class="elsevierStyleItalic">in vivo</span>"
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Frizzled-2 plays an important role in maintaining normal hepatic cell functionality&#46; This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia&#47;Reoxygenation &#40;H&#47;R&#41;&#46; <span class="elsevierStyleItalic">In vitro</span> H&#47;R hepatic cell model was established by culturing BRL-3A cells under H&#47;R condition&#46; <span class="elsevierStyleItalic">Frizzled-2</span> siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling&#46; Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H&#47;R treatment&#46; H&#47;R treatment induced cell cytotoxicity&#44; the early apoptosis rate and the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level in BRL-3A cells while silencing <span class="elsevierStyleItalic">frizzled-2</span> gene decreased the H&#47;R induced cell cytotoxicity&#44; apoptosis and intracellular Ca<span class="elsevierStyleSup">2&#43;</span> level&#46; <span class="elsevierStyleItalic">In vivo</span> mice study further showed the up-regulation of Frizzled-2&#47;Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury &#40;IRI&#41;&#46; In summary&#44; inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H&#47;R induced cell cytotoxicity and apoptosis&#46;</p></span>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Frizzled-2 desempe&#241;a un papel importante en el mantenimiento de la funcionalidad normal de los hepatocitos&#46; Este estudio tiene como objetivo analizar el papel de la inhibici&#243;n de Frizzled-2 en la protecci&#243;n de los hepatocitos BRL-3A de rata de la hipoxia&#47;reoxigenaci&#243;n &#40;H&#47;R&#41;&#46; El modelo de hepatocitos H&#47;R <span class="elsevierStyleItalic">in vitro</span> se demostr&#243; con el cultivo de c&#233;lulas BRL-3A en condiciones de H&#47;R&#46; El ARNip de Frizzled-2 se transinfect&#243; en c&#233;lulas BRL-3A para inhibir la se&#241;alizaci&#243;n de Frizzled-2&#46; Wnt5a y Frizzled-2 aumentaron considerablemente en las c&#233;lulas BRL-3A tras el tratamiento con H&#47;R&#46; El tratamiento con H&#47;R provoc&#243; citotoxicidad celular&#44; una tasa de apoptosis temprana y el nivel de Ca<span class="elsevierStyleSup">2&#43;</span> intracelular en c&#233;lulas BRL-3A mientras que el gen <span class="elsevierStyleItalic">frizzled-2</span> silenciado redujo la citotoxicidad celular inducida por H&#47;R&#44; la apoptosis y el nivel de Ca<span class="elsevierStyleSup">2&#43;</span> intracelular&#46; El estudio <span class="elsevierStyleItalic">in vivo</span> con ratones mostr&#243;&#44; adem&#225;s&#44; la regulaci&#243;n al alza de la v&#237;a de Frizzled-2&#47;Wnt 5 y la expresi&#243;n de caspasa 3 escindida en tejidos hep&#225;ticos con lesi&#243;n por isquemia y reperfusi&#243;n &#40;LIR&#41;&#46; En resumen&#44; la inhibici&#243;n de Frizzled-2 por su ARNip puede proteger a las c&#233;lulas BRL-3A al atenuar la citotoxicidad celular y la apoptosis inducida por H&#47;R&#46;</p></span>"
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          "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Validation of siRNA silencing <span class="elsevierStyleItalic">frizzled-2</span> gene expression&#46; &#40;A&#41; Different siRNAs &#40;&#35;25&#44; &#35;33 and &#35;34&#41; targeting <span class="elsevierStyleItalic">frizzled-2</span> mRNA at different regions and non-targeting siRNA serving as negative control &#40;NC&#41; were transfected at final doses of 25&#44; 50 and 100<span class="elsevierStyleHsp" style=""></span>nM for 24<span class="elsevierStyleHsp" style=""></span>h in BRL-3 cells&#46; Relative expression levels of <span class="elsevierStyleItalic">frizzled-2</span> were analyzed qRT-PCR and normalized to &#946;-actin&#46; &#42; denoted the group with the <span class="elsevierStyleItalic">frizzled-2</span> siRNA &#40;&#35;34&#41; at 50<span class="elsevierStyleHsp" style=""></span>nM achieved maximally silencing efficiency among groups&#46; &#40;B&#41; Transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA &#40;&#35;34&#41; and negative control siRNA &#40;NC&#41; was performed at 50<span class="elsevierStyleHsp" style=""></span>nM in BRL-3 cells for 48<span class="elsevierStyleHsp" style=""></span>h&#46; Cell group was set up as mock control&#46; Frizzled-2 protein level was analyzed by Western blot&#46; Relative expression of Frizzled-2 was normalized to GAPDH&#46; Data were presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from three independent experiments&#46; <span class="elsevierStyleSup">&#35;&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#44; Cell or NC groups vs&#46; siRNA group&#46;</p>"
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          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">H&#47;R treatment induced the expression of Frizzled-2 and Wnt5a in BRL-3A cells&#46; &#40;A&#41; Relative <span class="elsevierStyleItalic">Frizzled-2</span> gene expression&#46; &#40;B&#41; Relative <span class="elsevierStyleItalic">Wnt5a</span> gene expression&#59; &#40;C&#41; representative photographs of protein expression of Frizzled-2&#44; Wnt5a and GAPDH&#59; &#40;D&#41; relative protein level of Frizzled-2&#59; &#40;E&#41; relative protein level of Wnt5a&#46; The relative protein expression of Frizzled-2 and Wnt5a was normalized to GAPDH&#46; Data were presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from three independent experiments&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Cell group&#44; and <span class="elsevierStyleSup">&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and <span class="elsevierStyleSup">&#35;&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Si-H&#47;R group&#46;</p>"
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          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">The effect of transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA on cell cytotoxicity in BRL-3A cells&#46; &#40;A&#41; LDH assay showed transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA significantly inhibited the LDH activity from 3&#46;27<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;09 to 2&#46;87<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;05 &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; at OD values&#59; &#40;B&#41; LDH activities were shown as percentage of OD values&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 Cell or NC groups vs&#46; Si-H&#47;R group&#46;</p>"
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          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Caspase-3 protein expression was induced by H&#47;R and suppressed by transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA&#46; &#40;A&#41; Representative photographs of protein expression of Caspase-3 and GAPDH&#59; &#40;B&#41; relative Caspase-3 protein level was normalized to GAPDH&#59; data were presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from three independent experiments&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 when comparing to Cell group&#44; and <span class="elsevierStyleSup">&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 when comparing to Si-H&#47;R group&#46;</p>"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA inhibited the H&#47;R induced apoptosis in BRL-3A cells by Flow Cytometry using Annexin-V-FITC method&#46; &#40;A&#41; BRL-3A cells alone&#59; &#40;B&#41; BRL-3A cells with H&#47;R treatment &#40;Cell-H&#47;R&#41;&#59; &#40;C&#41; H&#47;R treatment of BRL-3A cells with transfection of negative control siRNA &#40;NC-H&#47;R&#41;&#59; &#40;D&#41; H&#47;R treatment of BRL-3A cells with transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA &#40;Si-H&#47;R&#41;&#59; &#40;E&#41; quantitative analysis of the early cell apoptosis&#59; &#40;F&#41; quantitative analysis of the late cell apoptosis&#59; &#40;G&#41; quantitative analysis of the early cell apoptosis&#46; Data were presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from three independent experiments&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Cell group&#44; and <span class="elsevierStyleSup">&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and <span class="elsevierStyleSup">&#35;&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Si-H&#47;R group&#46;</p>"
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          "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA inhibited the H&#47;R-induced intracellular Ca<span class="elsevierStyleSup">2&#43;</span> concentration in BRL-3A cells&#46; Fluorescence was shown in green color &#40;528<span class="elsevierStyleHsp" style=""></span>nm&#41; to monitor the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> in BRL-3A cells with H&#47;R treatment by Fluro 8-AM using confocal microscopy&#46; &#40;A&#41; BRL-3A cells with H&#47;R treatment &#40;Cell-H&#47;R&#41;&#59; &#40;B&#41; H&#47;R treatment of BRL-3A cells with transfection of negative control siRNA&#59; &#40;C&#41; H&#47;R treatment of BRL-3A cells with transfection of <span class="elsevierStyleItalic">frizzled-2</span> siRNA&#59; &#40;D&#41; control cells without H&#47;R treatment&#59; &#40;E&#41; bar graph showed the quantitative analysis of mean fluorescence intensity &#40;MFI&#41; of the intracellular Ca<span class="elsevierStyleSup">2&#43;</span> concentration in different cell groups&#46; Blue color denoted the nuclear stained with DAPI&#46; Data were presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from three independent experiments&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and &#42;&#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Cell group&#44; and <span class="elsevierStyleSup">&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 and <span class="elsevierStyleSup">&#35;&#35;</span><span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001 when comparing to Si-H&#47;R group&#46;</p>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">The up-regulation of Wnt5a&#44; Frizzled-2&#44; cleaved Caspase-3 &#40;cCaspase-3&#41; accompanied by a decrease in &#946;-catenin was shown in liver tissues from mice with IRI&#46; &#40;A&#41; Representative photographs of protein expression of Frizzled-2&#44; Wnt5a&#44; cCaspase-3 &#40;17<span class="elsevierStyleHsp" style=""></span>kDa and 19<span class="elsevierStyleHsp" style=""></span>kDa&#41;&#44; &#946;-catenin and GAPDH&#59; &#40;B&#41; relative protein level of Wnt5a&#59; &#40;C&#41; relative protein level of Frizzled-2&#59; &#40;D&#41; relative cCaspase-3 &#40;19<span class="elsevierStyleHsp" style=""></span>kDa&#41;&#59; &#40;E&#41; relative cCaspase-3 &#40;17<span class="elsevierStyleHsp" style=""></span>kDa&#41;&#59; &#40;F&#41; relative &#946;-catenin gene expression&#46; Bar graphs showing the relative protein expression of Wnt5a&#44; Frizzled-2&#44; cCaspase-3 &#40;17<span class="elsevierStyleHsp" style=""></span>kDa and 19<span class="elsevierStyleHsp" style=""></span>kDa&#41; and &#946;-catenin were individually normalized to GAPDH and presented as means<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SEM from six mice from group IRI or control group with sham operations&#46; &#42;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 when comparing to control group&#46;</p>"
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Article information

ISSN: 02105705
Original language: English

DOI:10.1016/j.gastrohep.2019.02.006

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	2	0	2
2024 October	15	3	18
2024 September	19	7	26
2024 August	31	3	34
2024 July	19	5	24
2024 June	16	3	19
2024 May	24	3	27
2024 April	30	5	35
2024 March	51	3	54
2024 February	38	2	40
2024 January	32	2	34
2023 December	34	4	38
2023 November	34	1	35
2023 October	48	3	51
2023 September	45	1	46
2023 August	23	4	27
2023 July	12	1	13
2023 June	27	1	28
2023 May	25	1	26
2023 April	29	2	31
2023 March	25	1	26
2023 February	24	1	25
2023 January	8	6	14
2022 December	27	4	31
2022 November	39	13	52
2022 October	25	10	35
2022 September	25	10	35
2022 August	21	10	31
2022 July	20	14	34
2022 June	18	13	31
2022 May	18	5	23
2022 April	20	9	29
2022 March	17	6	23
2022 February	5	0	5
2022 January	3	0	3
2021 December	2	0	2
2020 December	1	2	3
2020 July	1	0	1
2020 June	1	0	1
2020 April	6	4	10
2020 March	10	4	14

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