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Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells
Azizah Ugusman, Zaiton Zakaria, Chua Kien Hui, Nor Anita Megat Mohd Nordin
Department of Physiology, Universiti Kebangsaan, Malaysia Medical Center - Kuala Lumpur/Malaysia
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="cesec10" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle60">INTRODUCTION</span><p id="para10" class="elsevierStylePara elsevierViewall">Nitric oxide &#40;NO&#41; in the endothelium is synthesized by endothelial nitric oxide synthase &#40;eNOS&#41; via the conversion of L-arginine to L-citrulline&#46; The reaction requires the presence of the following cofactors&#58; nicotinamide adenine dinucleotide phosphate &#40;NADPH&#41;&#44; calcium&#47;calmodulin &#40;CaM&#41;&#44; flavin adenine dinucleotide &#40;FAD&#41;&#44; flavin mononucleotide &#40;FMN&#41; and tetrahydrobiopterin &#40;BH<span class="elsevierStyleInf">4</span>&#41;&#46;<a class="elsevierStyleCrossRefs" href="#bib1">1&#44;2</a></p><p id="para20" class="elsevierStylePara elsevierViewall">Nitric oxide has been recognized as a major anti-atherogenic factor because of its vasoprotective activity&#46;<a class="elsevierStyleCrossRef" href="#bib3">3</a> Nitric oxide induces vasorelaxation by activating soluble guanylate cyclase&#59; thus&#44; NO plays an important role in regulating the vascular tone&#46; Nitric oxide has also been shown to inhibit oxidation of low-density lipoprotein &#40;LDL&#41; and antagonize platelet aggregation by inhibiting platelet activation&#46;<a class="elsevierStyleCrossRef" href="#bib3">3</a> Moreover&#44; NO inhibits nuclear factor-&#954;B-dependent expression of adhesion molecules that mediate recruitment of leukocytes to the endothelium in the early phase of atherosclerosis&#46; Nitric oxide has been shown to suppress abnormal proliferation of vascular smooth muscle cells&#44; which contribute to the narrowing of atherosclerotic vessel walls&#46;<a class="elsevierStyleCrossRef" href="#bib3">3</a> Based on these anti-atherosclerotic properties&#44; the enhancement of endothelial NO production may play an important role in the prophylaxis or treatment of cardiovascular diseases&#46;<a class="elsevierStyleCrossRef" href="#bib4">4</a></p><p id="para30" class="elsevierStylePara elsevierViewall">Oxidative stress plays an important role in the pathogenesis of atherosclerosis and cardiovascular diseases by promoting endothelial dysfunction&#44; inflammation and lipid&#47;lipoprotein peroxidation as well as by reducing NO bioavailability&#46;<a class="elsevierStyleCrossRef" href="#bib5">5</a> Loss of normal NO production from the endothelium is a cardinal feature of endothelial dysfunction&#46;<a class="elsevierStyleCrossRef" href="#bib6">6</a> Endothelial dysfunction is characterized by a reduction in the bioavailability of NO&#44; which is followed by increased levels of endothelium-derived vasoconstrictors such as endothelin-1&#46;<a class="elsevierStyleCrossRef" href="#bib7">7</a> This imbalance leads to impairment of endothelium-derived relaxation &#40;EDR&#41;&#46; Arteries may thereby be predisposed to increased vascular tone and vasospasm&#46; Impairment of EDR was found in atherosclerotic vessels even before vascular structural changes had ensued&#46;<a class="elsevierStyleCrossRef" href="#bib8">8</a></p><p id="para40" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Piper sarmentosum</span> &#40;PS&#41; is a creeping terrestrial herbaceous plant that belongs to the <span class="elsevierStyleItalic">Piperaceae</span> family&#46; It is commonly found in the tropical and subtropical regions of the world&#44; such as the Asian region&#46; The leaves and roots of this plant have been used for the treatment of toothache&#44; fungoid dermatitis on the feet&#44; cough&#44; asthma and pleurisy&#46; <a class="elsevierStyleCrossRef" href="#bib9">9</a> Chloroform extracts of <span class="elsevierStyleItalic">Piper sarmentosum</span> have the ability to act as an anti-malarial agent against <span class="elsevierStyleItalic">Plasmodium falciparum</span> and <span class="elsevierStyleItalic">Plasmodium berghei</span>&#46;<a class="elsevierStyleCrossRef" href="#bib10">10</a> The aqueous extract of the entire plant has been reported to have hypoglycemic effects in experimental rats&#46;<a class="elsevierStyleCrossRef" href="#bib11">11</a> Furthermore&#44; the ethanolic extract of PS exerted anti-carcinogenic effects through an intrinsic apoptosis pathway in HepG2 cells&#46;<a class="elsevierStyleCrossRef" href="#bib13">13</a> The aqueous extract of PS also exhibited anti-nociceptive and anti-inflammatory activities <span class="elsevierStyleItalic">in vivo</span>&#46;<a class="elsevierStyleCrossRef" href="#bib14">14</a> Recent research has demonstrated that various extracts prepared from PS leaves have antioxidant and anti-tuberculous activities&#46;<a class="elsevierStyleCrossRef" href="#bib12">12</a> To date&#44; however&#44; there is no direct evidence linking PS to the eNOS system&#46; Based on the properties of PS extracts mentioned above&#44; the present study was designed to investigate the effects of PS on the eNOS system and NO synthesis in human umbilical vein endothelial cells &#40;HUVECs&#41;&#46; The results of the present study may help in the prevention and treatment of atherosclerosis&#44; which is linked to various cardiovascular diseases&#46;</p></span><span id="cesec20" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle70">MATERIALS AND METHODS</span><span id="cesec30" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle80">Preparation of aqueous extract of <span class="elsevierStyleItalic">Piper sarmentosum</span></span><p id="para50" class="elsevierStylePara elsevierViewall">Leaves of PS were collected in Sungai Buloh&#44; Malaysia&#44; and identified by a plant taxonomist from the Forest Research Institute of Malaysia &#40;voucher specimen&#58; FRI 45870&#41;&#46; The leaves were washed with tap water&#44; cut into small pieces&#44; sun-dried and ground into powder form&#46; A 10&#37; aqueous extract of <span class="elsevierStyleItalic">Piper sarmentosum</span> &#40;AEPS&#41; was prepared by soaking 100 g of the powdered leaves in 900 mL of purified water followed by incubation in a high-speed mixer at 80 &#176;C for 3 hours&#46; After cooling&#44; the extract was filtered using mesh and further concentrated&#46; The aqueous extract was then freeze-dried to powdered form and kept at 4 &#176;C until the experiments&#46;</p></span><span id="cesec40" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle90">Cell culture and treatment protocols</span><p id="para60" class="elsevierStylePara elsevierViewall">Human umbilical vein endothelial cells were obtained from umbilical cord veins using 0&#46;1 &#37; type I collagenase &#40;Gibco-Invitrogen Corp&#46;&#44; Grand Island&#44; N&#46;Y&#46;&#41; digestion&#46; Cells were grown in medium-200 &#40;Cascade Biologics&#44; USA&#41; supplemented with LSGS &#40;low-serum growth supplement&#59; Cascade Biologics&#44; USA&#41; at 37 &#176;C in a humidified atmosphere of 5&#37; CO<span class="elsevierStyleInf">2</span> and 95&#37; air&#46; Human umbilical vein endothelial cells were identified by the typical endothelial cell cobblestone morphology and positive expression of vonWillebrand factor and CD31 in immunocytochemistry&#46; The culture medium was changed every other day until the cells reached confluence&#46; Human umbilical vein endothelial cells from passage 3 at 80&#37; confluency were used for the experiments&#46; The cells were divided into four groups&#58; control &#40;CTRL&#41;&#44; treatment with 180 &#956;M hydrogen peroxide &#40;H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#41; to induce oxidative stress&#59; treatment with 150 &#956;g&#47;mL AEPS&#44; and concomitant treatment with 150 &#956;g&#47;mL AEPS and 180 &#956;M H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#46; All treatments were given for 24 hours&#46;</p></span><span id="cesec50" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle100">Quantitative reverse transcription polymerase chain reaction &#40;qPCR&#41; for analysis of eNOS mRNA expression</span><p id="para70" class="elsevierStylePara elsevierViewall">After treatment for 24 hours&#44; total ribonucleic acid &#40;RNA&#41; from HUVECs was extracted using TRI Reagent &#40;Molecular Research Center&#44; Cincinnati&#44; USA&#41; according to a previously published protocol&#46;<a class="elsevierStyleCrossRef" href="#bib15">15</a> Polyacryl carrier &#40;Molecular Research Center&#44; Cincinnati&#44; USA&#41; was added to precipitate the total RNA&#46; The extracted RNA pellet was then washed with 75&#37; ethanol and dried prior to being dissolving in RNase- and DNase-free water &#40;Invitrogen&#44; Carlsbad&#44; USA&#41;&#46; The extracted total RNA was assessed for its purity and quantity using a Nanodrop ND-100 spectrophotometer &#40;Wilmington DE&#44; USA&#41; and stored at &#8722;80 &#176;C&#46; Complimentary DNA &#40;cDNA&#41; was synthesized using SuperScript III First-Strand Synthesis SuperMix &#40;Invitrogen&#44; Carlsbad&#44; USA&#41;&#46; A total reaction volume of 20 &#956;l&#44; which consisted of 10 &#956;l of 2X RT Reaction Mix&#44; 2 &#956;l of RT enzyme&#44; 5 &#956;l of total RNA and 3 &#956;l of DEPC-treated water&#44; was incubated at 25 &#176;C for 10 minutes for primer annealing&#46; The reaction was then incubated at 50 &#176;C for 30 minutes for reverse transcription&#46; Finally&#44; the reaction was terminated at 85 &#176;C for 5 minutes and chilled on ice for 1 minute&#44; after which 1 &#956;l of <span class="elsevierStyleItalic">E&#46; coli</span> RNase H was added to the mixture&#46; The cDNA was further incubated at 37 &#176;C for 20 minutes and stored at &#8722;20 &#176;C&#46; Subsequently&#44; qPCR was carried out to determine the mRNA expression level of eNOS&#46; Glyceraldehyde-3-phosphate dehydrogenase &#40;GAPDH&#41; was used as the reference gene&#46; Primer 3 software &#40;<a href="http://frodo:wi.mit.edu/cgi-bin/primer3/primer3-www.cgi">http&#58;&#47;&#47;frodo&#58;wi&#46;mit&#46;edu&#47;cgi-bin&#47;primer3&#47;primer3-www&#46;cgi</a>&#41; was used to design the primers from the NIH GenBank database&#46; The following sequences were used as primers for eNOS &#91;GenBank&#58; NM&#95;000603&#93; and GAPDH &#91;GenBank&#58; BC020308&#93;&#58; CTCCAGCCCCGGTACTACTC &#40;forward&#41; and TTAGCCACGTGGAGCAGACT &#40;reverse&#41;&#44; and TCCCTGAGCTGAACGGGAAG &#40;forward&#41; and GGAGGAGTGGGTGTCGCTGT &#40;reverse&#41;&#44; respectively&#46; The qPCR reaction was performed in a BioRad iCycler &#40;Bio-Rad&#44; USA&#41; with 1 &#956;l of cDNA&#44; 5 &#956;M of each forward and reverse primer and 12&#46;5 &#956;l of IQ SYBR Green Supermix &#40;Bio-Rad&#44; USA&#41;&#46; The reaction profile consisted of 40 cycles using the following parameters&#58; 95&#176;C &#40;10 seconds&#41; and 61&#176;C &#40;30 seconds&#41;&#46; The reaction kinetics of each primer set and protocol were verified with the melting profile&#44; and product size was further confirmed with 2&#37; agarose gel electrophoresis stained with ethidium bromide &#40;Sigma&#44; St Louis&#44; USA&#41;&#46; The threshold cycle &#40;C<span class="elsevierStyleInf">T</span>&#41; value was determined&#44; and the relative mRNA expression of eNOS was calculated with the following equation&#58; 2<span class="elsevierStyleSup">&#916;&#916;CT</span>&#44; where &#916;&#916;C<span class="elsevierStyleInf">T</span> &#61; C<span class="elsevierStyleInf">T</span> GAPDH - C<span class="elsevierStyleInf">T</span> eNOS&#46;</p></span><span id="cesec60" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle110">Enzyme-linked immunosorbent assay &#40;ELISA&#41; for eNOS protein analyses</span><p id="para80" class="elsevierStylePara elsevierViewall">The eNOS protein level of the cultured HUVECs was determined using the Quantikine human eNOS ELISA kit &#40;R&#38;D Systems&#41;&#46; Human umbilical vein endothelial cells were washed twice with phosphate-buffered saline &#40;PBS&#41;&#44; manually scraped from the culture flask and lysed with 400 &#956;L of lysis buffer&#46; The assay was performed using 100 &#956;L of the cell lysate&#46; The cell lysate was pipetted into the 96-well plate so that any eNOS that was present would be bound to the immobilized antibody in the plate&#46; After washing away any unbound substances&#44; eNOS conjugate was added to the wells&#46; This was followed by the addition of substrate solution and stop solution&#46; The optical density of each well was determined at 450 nm using an ELISA microplate reader&#46;</p></span><span id="cesec70" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle120">Determination of eNOS Activity</span><p id="para90" class="elsevierStylePara elsevierViewall">The eNOS activity was determined using a commercially available kit &#40;Nitric Oxide Synthase Colorimetric Assay&#44; Calbiochem&#44; USA&#41;&#46; The principle of this assay was based on the measurement of nitrite produced by eNOS in the sample in a timed reaction&#46; Human umbilical vein endothelial cells were scraped from the culture flask&#44; homogenized in PBS and centrifuged at 10&#44;000 g for 20 minutes&#46; Then&#44; the cell lysate in the supernatant solution was filtered through a 0&#46;45 &#956;m filter prior to ultracentrifugation at 100&#44;000 g for 15 minutes&#46; A total of 40 &#956;L of the cell lysate was diluted with 20 &#956;L of assay buffer&#46; Next&#44; the samples were mixed with NADPH&#44; nitrate reductase&#44; cofactor preparation solution and lactate dehydrogenase &#40;LDH&#41;&#46; Total nitrite was measured at 540 nm with Griess reagents &#40;sulfanilamide and naphthalene&#8211;ethylene diamine dihydrochloride&#41;&#46; The concentration of nitrite in the sample was calculated using a standard curve&#46; The eNOS activity was expressed as nmol of nitrite&#47;min per mL of sample&#46;</p></span><span id="cesec80" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle130">Determination of endothelial nitric oxide production</span><p id="para100" class="elsevierStylePara elsevierViewall">Production of NO by HUVECs was measured via its stable oxidation product&#44; nitrite&#44; using a commercial kit &#40;BIOXYTECH Nitric Oxide Colorimetric Assay&#44; OXIS Research&#44; USA&#41;&#46; Briefly&#44; 50 &#956;L of the culture medium was diluted with 35 &#956;L of assay buffer and mixed with 10 &#956;L of nitrate reductase and 10 &#956;L NADH&#46; After 20 minutes of incubation to convert nitrate to nitrite&#44; total nitrite was measured at 540 nm with Griess reagents &#40;sulfanilamide and naphthalene&#8211;ethylene diamine dihydrochloride&#41;&#46;</p></span><span id="cesec90" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle140">Statistical analysis</span><p id="para110" class="elsevierStylePara elsevierViewall">Data were tested for normality using the Kolmogorov-Smirnov test&#44; and all variables were normally distributed&#46; Data were expressed as mean &#177; SEM&#46; Statistical analyses between two groups were performed with Student&#8217;s paired <span class="elsevierStyleItalic">t-</span>test using SPSS version 16&#46;0 software&#46; Values of p &#60; 0&#46;05 were considered statistically significant&#46;</p></span></span><span id="cesec100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle150">RESULTS</span><span id="cesec110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle160">eNOS mRNA expression in HUVECs</span><p id="para120" class="elsevierStylePara elsevierViewall">Treatment of HUVECs with AEPS significantly enhanced eNOS mRNA expression by 2&#46;3-fold &#40;4&#46;405 &#177; 0&#46;892 x 10<span class="elsevierStyleSup">&#8722;3</span>&#41; compared with the control group &#40;1&#46;915 &#177; 0&#46;428 x 10<span class="elsevierStyleSup">&#8722;3</span>&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig1">Figure 1</a>&#41;&#46; In the oxidative stress-induced group&#44; HUVECs treated with H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> also showed a significantly higher &#40;2&#46;2-fold&#41; level of eNOS mRNA expression &#40;4&#46;280 &#177; 0&#46;760 x 10<span class="elsevierStyleSup">&#8722;3</span>&#41; compared with the control group&#46; However&#44; concomitant treatment of HUVECs with both AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> did not result in a significant increase in eNOS mRNA expression &#40;2&#46;95 &#177; 0&#46;697 x 10<span class="elsevierStyleSup">&#8722;3</span>&#41; compared to the control group&#46;</p><elsevierMultimedia ident="fig1"></elsevierMultimedia></span><span id="cesec120" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle170">eNOS protein level in HUVECs</span><p id="para130" class="elsevierStylePara elsevierViewall">The aqueous extract of PS significantly increased the eNOS protein level by 1&#46;4-fold &#40;1&#46;706 &#177; 0&#46;154 x 10<span class="elsevierStyleSup">3</span> pg&#47;mL&#41; compared with the control group &#40;1&#46;235 &#177; 0&#46;170 x 10<span class="elsevierStyleSup">3</span> pg&#47;mL&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig2">Figure 2</a>&#41;&#44; which was consistent with the changes in eNOS mRNA&#46; Treatment of HUVECs with H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> alone also resulted in a significantly higher &#40;1&#46;3-fold&#41; level of eNOS protein &#40;1&#46;669 &#177; 0&#46;137 x 10<span class="elsevierStyleSup">3</span> pg&#47;mL&#41; compared with the control group&#46; Concomitant treatment of HUVECs with both AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> did not result in a significant increase in eNOS protein level &#40;1&#46;549 &#177; 0&#46;096 x 10<span class="elsevierStyleSup">3</span> pg&#47;mL&#41; compared with the control group&#46;</p><elsevierMultimedia ident="fig2"></elsevierMultimedia></span><span id="cesec130" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle180">eNOS activity in HUVECs</span><p id="para140" class="elsevierStylePara elsevierViewall">Treatment of HUVECs with AEPS significantly promoted eNOS enzyme activity &#40;5&#46;237 &#177; 0&#46;55x10<span class="elsevierStyleSup">&#8722;2</span> nmoles&#47;mL&#47;min&#41; compared with the control group &#40;4&#46;393 &#177; 0&#46;74 x 10<span class="elsevierStyleSup">&#8722;2</span> nmoles&#47;mL&#47;min&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig3">Figure 3</a>&#41;&#46; In the oxidative stress-induced group&#44; HUVECs treated with H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> also showed a significantly higher level of eNOS enzyme activity &#40;5&#46;863 &#177; 0&#46;57 x 10<span class="elsevierStyleSup">&#8722;2</span> nmoles&#47;mL&#47;min&#41; compared with the control group&#46; Concomitant treatment of HUVECs with both AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> did not result in a significant increase in eNOS enzyme activity &#40;5&#46;228 &#177; 1&#46;473 x 10<span class="elsevierStyleSup">&#8722;2</span> nmoles&#47;mL&#47;min&#41; compared with the control group&#46;</p><elsevierMultimedia ident="fig3"></elsevierMultimedia></span><span id="cesec140" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle190">Nitric oxide production in HUVECs</span><p id="para150" class="elsevierStylePara elsevierViewall">The aqueous extract of PS significantly promoted NO production in HUVECs by 6&#46;3-fold &#40;15&#46;446 &#177; 3&#46;879 &#956;M&#41; compared with the control group &#40;2&#46;454 &#177; 0&#46;799 &#956;M&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig4">Figure 4</a>&#41;&#46; In the oxidative stress-induced group&#44; HUVECs treated with H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> also showed a significantly higher &#40;1&#46;7-fold increase&#41; level of NO production &#40;4&#46;175 &#177; 0&#46;966 &#956;M&#41; compared with the control group&#46; The greatest increase in NO production &#40;17&#46;536 &#177; 3&#46;55 &#956;M&#41; was observed in HUVECs treated with both AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#59; the level of NO production was significantly higher than that in the control and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> groups&#46;</p><elsevierMultimedia ident="fig4"></elsevierMultimedia></span></span><span id="cesec150" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle200">DISCUSSION</span><p id="para160" class="elsevierStylePara elsevierViewall">Oxidative stress can contribute to the development and progression of atherosclerosis by promoting endothelial dysfunction&#44; inflammation and lipid peroxidation&#44; as well as by reducing NO bioavailability&#46;<a class="elsevierStyleCrossRef" href="#bib16">16</a> Based on the results of a previous study&#44; we used 180 &#956;M H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> to induce oxidative stress in HUVECs&#46;<a class="elsevierStyleCrossRef" href="#bib17">17</a> This concentration of H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> increased eNOS mRNA expression&#44; eNOS protein level and eNOS activity &#40;<a class="elsevierStyleCrossRef" href="#fig1">Figure 1</a>&#44; <a class="elsevierStyleCrossRef" href="#fig2">2</a>&#44; <a class="elsevierStyleCrossRef" href="#fig3">3</a>&#41;&#46; In another study&#44; incubation of bovine aortic endothelial cells with 150 &#956;M H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> for 24 hours also caused an increase in eNOS mRNA&#44; eNOS protein and eNOS enzyme activity&#46;<a class="elsevierStyleCrossRef" href="#bib18">18</a></p><p id="para170" class="elsevierStylePara elsevierViewall">The NO level was higher in the H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>-treated group compared to the control group&#46; This may have been due to induction of NO production by H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> as part of the self-protective mechanism of the cells&#46; The dose of H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> used in this study was not lethal to HUVECs&#59; therefore&#44; the cells were still able to increase their endogenous NO production after an H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> challenge&#46; However&#44; H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> also caused oxidative destruction of the synthesized NO&#44; which explains why the increase in NO in the H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2-</span>treated group was not as high as in the other groups &#40;i&#46;e&#46;&#44; the AEPS and the combined AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> groups&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig4">Figure 4</a>&#41;&#46;</p><p id="para180" class="elsevierStylePara elsevierViewall">In this study&#44; the responses to H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> are in agreement with earlier reports&#46;<a class="elsevierStyleCrossRef" href="#bib5">5</a> The expression of eNOS in endothelial cells is regulated by NO through a negative feedback mechanism at both the transcriptional and translational levels&#46; Hydrogen peroxide-derived upregulation of eNOS was mediated by diminished NO availability and a consequent reduction in the negative feedback regulatory action of NO on eNOS expression&#46; This represents a compensatory protective mechanism of the cells to a reduction in NO availability induced by acute exposure to H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#46;<a class="elsevierStyleCrossRef" href="#bib5">5</a> Hydrogen peroxide has been shown to increase eNOS activity by inducing changes in the phosphorylation status of the enzyme&#46; This response represents an attempt by the endothelial cells to maintain NO bioactivity under conditions of increased oxidative stress&#46;<a class="elsevierStyleCrossRef" href="#bib19">19</a></p><p id="para190" class="elsevierStylePara elsevierViewall">It is well recognized that NO produced by eNOS plays a protective role against the development of atherosclerosis and endothelial dysfuntion&#46;<a class="elsevierStyleCrossRefs" href="#bib20">20&#44;21</a> The results of this study show that AEPS &#40;150 &#956;g&#47;mL&#41; significantly increased NO production in HUVECs &#40;<a class="elsevierStyleCrossRef" href="#fig4">Figure 4</a>&#41;&#46; AEPS also induced increases in eNOS mRNA&#44; protein and activity &#40;<a class="elsevierStyleCrossRef" href="#fig1">Figure 1</a>&#44; <a class="elsevierStyleCrossRef" href="#fig2">2</a>&#44; <a class="elsevierStyleCrossRef" href="#fig3">3</a>&#41;&#46; The higher amount of eNOS protein caused a higher level of eNOS activity&#46; This resulted in an increase in NO production by HUVECs&#46; The results of the present study suggest that AEPS may improve endothelial function by augmenting NO production in human endothelial cells&#46; Thus&#44; AEPS could reduce the risk of atherosclerosis&#46;</p><p id="para200" class="elsevierStylePara elsevierViewall">Antioxidants are known to enhance the biological actions of NO by protecting NO against oxidative destruction by reactive oxygen species&#46;<a class="elsevierStyleCrossRef" href="#bib21">21</a> AEPS has been shown to exhibit antioxidant properties&#46;<a class="elsevierStyleCrossRef" href="#bib12">12</a> Thus&#44; AEPS can directly protect NO from oxidative destruction by H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> &#40;<a class="elsevierStyleCrossRef" href="#fig4">Figure 4</a>&#41;&#46; The aqueous extract of PS also promoted NO production from HUVECs by increasing eNOS protein synthesis and enzyme activity &#40;<a class="elsevierStyleCrossRef" href="#fig2">Figure 2</a>&#44; <a class="elsevierStyleCrossRef" href="#fig3">3</a>&#41;&#46; Therefore&#44; both protection of NO from oxidative destruction and enhancement of eNOS activity by AEPS caused an increase in NO level&#46;</p><p id="para210" class="elsevierStylePara elsevierViewall">We observed the largest increase in NO production in the group that received AEPS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#46; Since AEPS can directly protect NO from oxidative destruction by H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#44; NO is available in the cells at a higher level&#46; A higher level of NO in the cells reduced the mRNA expression and protein synthesis of eNOS via a negative feedback mechanism&#46;<a class="elsevierStyleCrossRef" href="#bib5">5</a></p><p id="para220" class="elsevierStylePara elsevierViewall">Previous phytochemical screening of PS revealed the presence of a variety of natural products&#44; such as amides&#44; polyphenols and flavonoids&#46;<a class="elsevierStyleCrossRef" href="#bib12">12</a> Myricetin&#44; apigenin and quercetin are examples of flavonoids identified in PS leaves&#46;<a class="elsevierStyleCrossRef" href="#bib22">22</a> Quercetin improved endothelial dysfunction by increasing NO synthesis in HUVECs&#46; The increase in NO synthesis was attributed to an enhancement of eNOS activity via increased calcium concentration&#46;<a class="elsevierStyleCrossRef" href="#bib23">23</a> Quercetin has also been reported to exert endothelium-dependent vasodilatation of porcine aortic rings&#46;<a class="elsevierStyleCrossRef" href="#bib24">24</a> Therefore&#44; in the present study&#44; the flavonoids appear to be the active constituents of AEPS responsible for enhancing NO production in HUVECs&#46;</p></span><span id="cesec160" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle210">STUDY LIMITATIONS</span><p id="para230" class="elsevierStylePara elsevierViewall">This experiment was an <span class="elsevierStyleItalic">in vitro</span> study that investigated some fundamental biomolecular and cellular activities&#46; The data suggest that AEPS could reduce the risk of atherosclerosis by increasing the bioavailability of NO to defend against oxidative stress&#46; A clinical placebo-controlled study may be needed before employing AEPS as an effective supplement&#46; If this study were performed in a clinical setting&#44; we could attempt to determine dosage&#44; pharmacokinetics and pharmacodynamics of AEPS&#46;</p></span><span id="cesec170" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle220">CONCLUSION</span><p id="para240" class="elsevierStylePara elsevierViewall">In summary&#44; the present study demonstrated that AEPS increased eNOS mRNA expression&#44; protein synthesis&#44; eNOS activity and NO production that could protect HUVECs from from oxidative stress&#46; Based on the vasoprotective and anti-atherosclerotic effects of endothelial NO&#44; AEPS has the ability to reduce the risk of atherosclerosis&#46; Further studies are needed to corroborate these findings&#46;</p></span></span>"
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              "titulo" => "Preparation of aqueous extract of Piper sarmentosum"
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              "titulo" => "Quantitative reverse transcription polymerase chain reaction &#40;qPCR&#41; for analysis of eNOS mRNA expression"
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              "titulo" => "Enzyme-linked immunosorbent assay &#40;ELISA&#41; for eNOS protein analyses"
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              "titulo" => "Determination of eNOS Activity"
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              "titulo" => "Determination of endothelial nitric oxide production"
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              "titulo" => "eNOS mRNA expression in HUVECs"
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              "titulo" => "eNOS protein level in HUVECs"
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              "titulo" => "eNOS activity in HUVECs"
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              "titulo" => "Nitric oxide production in HUVECs"
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            0 => "<span class="elsevierStyleItalic">Piper sarmentosum</span>"
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        "resumen" => "<span id="ceabs10" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle10">OBJECTIVE&#58;</span><p id="spara50" class="elsevierStyleSimplePara elsevierViewall">Nitric oxide produced by endothelial nitric oxide synthase &#40;eNOS&#41; possesses multiple anti-atherosclerotic properties&#46; Hence&#44; enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis&#46; <span class="elsevierStyleItalic">Piper sarmentosum</span> is a tropical plant with antioxidant and anti-inflammatory activities&#46; This study aimed to investigate the effects of <span class="elsevierStyleItalic">Piper sarmentosum</span> on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells &#40;HUVECs&#41;&#46;</p></span> <span id="ceabs20" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle20">METHODS&#58;</span><p id="spara60" class="elsevierStyleSimplePara elsevierViewall">HUVECs were divided into four groups&#58; control&#44; treatment with 180 &#956;M hydrogen peroxide &#40;H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span>&#41;&#44; treatment with 150 &#956;g&#47;mL aqueous extract of <span class="elsevierStyleItalic">Piper sarmentosum</span>&#44; and concomitant treatment with aqueous extract of PS and H<span class="elsevierStyleInf">2</span>O<span class="elsevierStyleInf">2</span> for 24 hours&#46; Subsequently&#44; HUVECs were harvested and eNOS mRNA expression was determined using qPCR&#46; The eNOS protein level was measured using ELISA&#44; and the eNOS activity and Nitric oxide level were determined by the Griess reaction&#46;</p></span> <span id="ceabs30" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle30">RESULTS&#58;</span><p id="spara70" class="elsevierStyleSimplePara elsevierViewall">Human umbilical vein endothelial cells treated with aqueous extract of <span class="elsevierStyleItalic">Piper sarmentosum</span> showed a marked induction of Nitric oxide&#46; Treatment with PS also resulted in increased eNOS mRNA expression&#44; eNOS protein level and eNOS activity in HUVECs&#46;</p></span> <span id="ceabs40" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle40">CONCLUSION&#58;</span><p id="spara80" class="elsevierStyleSimplePara elsevierViewall">Aqueous extract of <span class="elsevierStyleItalic">Piper sarmentosum</span> may improve endothelial function by promoting NO production in HUVECs&#46;</p></span>"
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