郜攀，司良毅，徐強(qiáng)，王笑梅

白藜蘆醇對(duì)血管緊張素Ⅱ誘導(dǎo)的血管平滑肌細(xì)胞增殖的抑制作用及其機(jī)制觀察

郜攀，司良毅，徐強(qiáng)，王笑梅

目的探討白藜蘆醇對(duì)血管緊張素Ⅱ(AngⅡ)誘導(dǎo)的血管平滑肌細(xì)胞(VSMCs)增殖的影響及其可能機(jī)制。方法體外培養(yǎng)大鼠胸主動(dòng)脈血管平滑肌細(xì)胞(VSMCs)。在檢測(cè)白藜蘆醇影響AngⅡ誘導(dǎo)VSMCs增殖和活力的實(shí)驗(yàn)中，將細(xì)胞分為對(duì)照組、AngⅡ組(1μmol/L)、白藜蘆醇濃度梯度(10、30、100μmol/L)組及AngⅡ+白藜蘆醇濃度梯度組，各組細(xì)胞均反應(yīng)0、6、12、24h，采用細(xì)胞計(jì)數(shù)法檢測(cè)VSMCs增殖情況，MTT法檢測(cè)VSMCs活力。在檢測(cè)腺苷酸活化蛋白激酶(AMPK)抑制劑復(fù)合物C對(duì)VSMCs生物活性影響的實(shí)驗(yàn)中，將細(xì)胞分為對(duì)照組、AngⅡ組、白藜蘆醇+AngⅡ組及復(fù)合物C組+白藜蘆醇+AngⅡ組，各組細(xì)胞反應(yīng)24h后采用Western blotting檢測(cè)增殖細(xì)胞核抗原(PCNA)蛋白表達(dá)。結(jié)果與對(duì)照組比較，6、12、24h后AngⅡ組細(xì)胞活力和細(xì)胞數(shù)目均顯著增高(P＜0.05)；與AngⅡ組比較，不同濃度白藜蘆醇+AngⅡ組細(xì)胞活力和細(xì)胞數(shù)目均顯著降低(P＜0.05)。另一方面，與對(duì)照組比較，AngⅡ組PCNA蛋白表達(dá)顯著增高(P＜0.05)；與AngⅡ組比較，白藜蘆醇+AngⅡ組PCNA蛋白表達(dá)顯著降低(P＜0.05)；而與白藜蘆醇+AngⅡ組比較，復(fù)合物C+白藜蘆醇+AngⅡ組細(xì)胞數(shù)目、細(xì)胞活力及PCNA蛋白表達(dá)顯著增高(P＜0.05)。結(jié)論白藜蘆醇可抑制AngⅡ誘導(dǎo)的VSMCs增殖，其機(jī)制可能與AMPK的激活有關(guān)。

白藜蘆醇；肌細(xì)胞，平滑?。谎芫o張素Ⅱ；腺苷酸活化蛋白激酶

動(dòng)脈粥樣硬化是眾多心腦血管疾病發(fā)展的基礎(chǔ)。研究提示，炎癥因子刺激血管平滑肌細(xì)胞(vascular smooth muscle cells，VSMCs)表型轉(zhuǎn)換和異常增殖進(jìn)而導(dǎo)致血管重構(gòu)是動(dòng)脈粥樣硬化發(fā)生的重要病理基礎(chǔ)[1-2]。近年來(lái)，紅酒中的天然提取物白藜蘆醇(resveratrol)在心血管疾病中的保護(hù)作用逐漸受到關(guān)注，相關(guān)研究表明白藜蘆醇能夠抑制VSMCs增殖，但其作用機(jī)制尚未闡明[3-4]。本課題組前期研究發(fā)現(xiàn)，白藜蘆醇能激活內(nèi)皮細(xì)胞腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase，AMPK)，誘導(dǎo)一氧化氮釋放、改善內(nèi)皮功能，提示AMPK對(duì)細(xì)胞功能具有重要影響[5]。血管緊張素Ⅱ(angiotensin Ⅱ，AngⅡ)是腎素-血管緊張素-醛固酮系統(tǒng)(RAAS)中最重要的一種生物活性肽，除有收縮血管、促醛固酮分泌及心肌肥大[6]等作用外，還具有很強(qiáng)的促VSMCs增殖作用，同時(shí)AngⅡ是VSMCs分泌的一種重要炎癥介質(zhì)，除誘導(dǎo)血管平滑肌細(xì)胞增殖之外，還可促進(jìn)炎癥介質(zhì)釋放，導(dǎo)致動(dòng)脈粥樣硬化發(fā)生[7]。因此觀察白藜蘆醇與VSMCs內(nèi)AngⅡ的相互作用，進(jìn)而探討其相互作用機(jī)制對(duì)理解白藜蘆醇在心血管系統(tǒng)中的保護(hù)作用具有重要意義。

1 材料與方法

1.1 動(dòng)物及試劑 清潔級(jí)SD大鼠購(gòu)自第三軍醫(yī)大學(xué)動(dòng)物實(shí)驗(yàn)中心。白藜蘆醇、血管緊張素Ⅱ、復(fù)合物C(AMPK抑制劑)均購(gòu)自Sigma公司(美國(guó))；細(xì)胞計(jì)數(shù)試劑盒及MTT試劑盒購(gòu)買自北京鼎國(guó)重慶分公司；SM-actin蛋白、增殖細(xì)胞核抗原(proliferating cell nuclear antigen，PCNA)蛋白、β-actin蛋白一抗購(gòu)自Santa Cruz公司(美國(guó))；兔抗山羊二抗購(gòu)自北京中杉金橋生物公司。

1.2 原代VSMCs的培養(yǎng)和鑒定[8]組織貼塊法培養(yǎng)大鼠胸主動(dòng)脈VSMCs。倒置相差顯微鏡下觀察細(xì)胞形態(tài)及生長(zhǎng)狀況。采用SM-actin免疫熒光法對(duì)VSMCs進(jìn)行細(xì)胞鑒定，陽(yáng)性表達(dá)為胞質(zhì)呈細(xì)顆粒狀綠色沉淀反應(yīng)。

1.3 細(xì)胞分組 選擇第3～5代VSMCs進(jìn)行實(shí)驗(yàn)。胰蛋白酶消化制備細(xì)胞懸液，調(diào)整細(xì)胞密度為5.0×107/L，接種于培養(yǎng)板備用。在37℃、5%CO2條件下經(jīng)10%胎牛血清預(yù)培養(yǎng)24h和0.5%胎牛血清預(yù)處理12h后進(jìn)行分組。在檢測(cè)白藜蘆醇對(duì)AngⅡ誘導(dǎo)的VSMCs增殖和細(xì)胞活力的影響作用實(shí)驗(yàn)中，將細(xì)胞隨機(jī)分為對(duì)照組(不加特殊處理因素)、AngⅡ組(1μmol/L AngⅡ處理)、不同濃度白藜蘆醇組(10、30、100μmol/L白藜蘆醇處理)、不同濃度白藜蘆醇+AngⅡ組(1μmol/L AngⅡ+10、30、100μmol/L白藜蘆醇處理)，各組細(xì)胞反應(yīng)0、6、12、24h后進(jìn)行檢測(cè)。在檢測(cè)復(fù)合物C對(duì)VSMCs生物活性影響的實(shí)驗(yàn)中，將細(xì)胞隨機(jī)分為對(duì)照組、AngⅡ組(1μmol/ L AngⅡ處理)、白藜蘆醇+AngⅡ組(1μmol/L AngⅡ和100μmol/L白藜蘆醇處理)和復(fù)合物C+白藜蘆醇+AngⅡ組(以1μmol/L復(fù)合物C預(yù)處理細(xì)胞24h后，再加入100μmol/L白藜蘆醇+1μmol/L AngⅡ)，各組細(xì)胞反應(yīng)24h后進(jìn)行檢測(cè)。

1.4 細(xì)胞計(jì)數(shù)[9]VSMCs培養(yǎng)24h后換用含0.5%胎牛血清的DMEM培養(yǎng)液同步化24h。按實(shí)驗(yàn)分組換用含不同干擾因素及10%胎牛血清的DMEM培養(yǎng)液繼續(xù)培養(yǎng)。分別在給予干擾因素后0、6、12、24h計(jì)數(shù)各組細(xì)胞。實(shí)驗(yàn)重復(fù)3次，每次計(jì)數(shù)3個(gè)復(fù)孔。

1.5 MTT法檢測(cè)細(xì)胞活力[10]按每孔1×103個(gè)細(xì)胞接種于96孔板，用含10%胎牛血清的DMEM培養(yǎng)液培養(yǎng)24h后換用含0.5%胎牛血清的DMEM培養(yǎng)液同步化24h。按實(shí)驗(yàn)分組用含不同干擾因素及10%胎牛血清的DMEM培養(yǎng)液繼續(xù)培養(yǎng)。分別在給予干擾因素后6、12、24h后用MTT法檢測(cè)，于490nm波長(zhǎng)處測(cè)定各孔A值。實(shí)驗(yàn)重復(fù)3次，每次6個(gè)復(fù)孔。

1.6 Western blotting檢測(cè)PCNA蛋白的表達(dá)[11]提取細(xì)胞總蛋白，以SDS-PAGE凝膠行蛋白電泳，上樣量為50μg。電泳完畢將蛋白轉(zhuǎn)到聚偏氟乙烯膜(PVDF)上。轉(zhuǎn)膜后在含5%脫脂奶的TBST中封閉1h，TBST洗滌3次，與1:250稀釋的PCNA及β-actin抗體反應(yīng)，4℃過(guò)夜。洗膜3次，加1:2000稀釋的二抗，室溫孵育1h，ECL化學(xué)發(fā)光法顯色，X線膠片曝光、顯影、定影，采用Quantity One軟件行灰度分析，以兔抗鼠β-actin作為內(nèi)參照。

1.7 統(tǒng)計(jì)學(xué)處理 采SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行分析。計(jì)量資料結(jié)果以±s表示，組間比較采用單因素方差分析，進(jìn)一步兩兩比較采用Tukey多重比較法，P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 VSMCs的體外培養(yǎng)及鑒定 倒置相差顯微鏡下觀察，細(xì)胞為梭形或長(zhǎng)梭形，排列成放射狀、旋渦狀，呈典型“谷”與“峰”生長(zhǎng)現(xiàn)象(圖1A)。SM-actin單克隆抗體免疫組化染色可見胞質(zhì)肌絲呈典型棕黃色細(xì)顆粒狀沉淀，陽(yáng)性率達(dá)95%以上，表明VSMCs純度高(圖1B)。

圖1 體外培養(yǎng)的VSMCs形態(tài)及鑒定Fig. 1 Morphology and identification of VSMCs cultured in vitro

2.2 白藜蘆醇對(duì)AngⅡ誘導(dǎo)VSMCs增殖的影響在6、12、24h時(shí)間點(diǎn)，AngⅡ組細(xì)胞數(shù)均明顯高于對(duì)照組(P＜0.05)，而10、30、100μmol/L白藜蘆醇組細(xì)胞數(shù)在各時(shí)間點(diǎn)與對(duì)照組比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。AngⅡ+30μmol/L白藜蘆醇組和AngⅡ+100μmol/L白藜蘆醇組各時(shí)間點(diǎn)細(xì)胞數(shù)均低于AngⅡ組(P＜0.05)，而AngⅡ+10μmol/L白藜蘆醇組細(xì)胞數(shù)在各時(shí)間點(diǎn)與AngⅡ組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05，表1)，表明30μmol/L和100μmol/L白藜蘆醇均可阻斷AngⅡ誘導(dǎo)的VSMCs增殖，而10μmol/L白藜蘆醇無(wú)此作用。

2.3 白藜蘆醇對(duì)AngⅡ調(diào)節(jié)VSMCs細(xì)胞活力的影響 MTT法檢測(cè)顯示，AngⅡ組A值在6、12、24h均明顯高于對(duì)照組(P＜0.05)，而10、30、100μmol/L白藜蘆醇組A值在各時(shí)間點(diǎn)與對(duì)照組比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。與AngⅡ組比較，AngⅡ+30μmol/L白藜蘆醇組和AngⅡ+100μmol/L白藜蘆醇組各時(shí)間點(diǎn)A值均明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)，而AngⅡ+10μmol/L白藜蘆醇組A值在各時(shí)間點(diǎn)與AngⅡ組比較無(wú)明顯差異(P＞0.05，表2)，表明30、100μmol/L白藜蘆醇均可阻斷AngⅡ增強(qiáng)VSMCs細(xì)胞活力的作用，而10μmol/L白藜蘆醇則無(wú)此作用。

表1 各組血管平滑肌細(xì)胞計(jì)數(shù)結(jié)果(×105/ml，±s，n=3)Tab. 1 VSMCs count in different groups (×105/ml,±s, n=3)

表1 各組血管平滑肌細(xì)胞計(jì)數(shù)結(jié)果(×105/ml，±s，n=3)Tab. 1 VSMCs count in different groups (×105/ml,±s, n=3)

(1)P＜0.05 compared with control group; (2)P＜0.05 compared with AngⅡgroup

2.4 復(fù)合物C對(duì)白藜蘆醇抑制AngⅡ誘導(dǎo)VSMCs細(xì)胞增殖及PCNA蛋白表達(dá)的影響 采用1μmol/L復(fù)合物C預(yù)處理后，與AngⅡ+白藜蘆醇組比較，復(fù)合物C+AngⅡ+白藜蘆醇組VSMCs細(xì)胞數(shù)增加38.0%，細(xì)胞活力增加22.1%，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。Western blotting檢測(cè)顯示：與對(duì)照組比較，AngⅡ組VSMCs內(nèi)PCNA蛋白表達(dá)顯著升高，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。與AngⅡ組比較，白藜蘆醇+AngⅡ組PCNA蛋白表達(dá)顯著降低(P＜0.05)。采用復(fù)合物C預(yù)處理后，復(fù)合物C+AngⅡ+白藜蘆醇組VSMCs細(xì)胞內(nèi)PCNA蛋白表達(dá)顯著高于白藜蘆醇+AngⅡ組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖2)。

表2 MTT法檢測(cè)細(xì)胞活力(A值，n=3)Tab. 2 Activity of VSMCs measured by MTT assay (A value, n=3)

圖2 不同處理對(duì)VSMCs內(nèi)PCNA蛋白表達(dá)的影響Fig. 2 Effects of resveratrol and AngⅡ on the protein expression of PCNA in VSMCs

3 討 論

動(dòng)脈粥樣硬化是由多種病因造成的始發(fā)于動(dòng)脈壁內(nèi)膜的一系列復(fù)雜的分子和細(xì)胞改變的總結(jié)果，也是誘發(fā)冠心病、心肌梗死、腦梗死等心腦血管疾病的主要原因[12]。流行病學(xué)調(diào)查發(fā)現(xiàn)，有飲用適量葡萄酒習(xí)慣的人其心血管疾病的發(fā)病率和病死率相對(duì)較低，進(jìn)一步研究發(fā)現(xiàn)這一現(xiàn)象可能與葡萄酒中含有較高含量的白藜蘆醇有關(guān)[13]。現(xiàn)代藥理學(xué)研究證實(shí)，白藜蘆醇具有多種藥理學(xué)作用，如抗炎、抑制血小板聚集、調(diào)節(jié)脂質(zhì)代謝、保護(hù)心血管缺血性損傷和抗腫瘤等[14]。

VSMCs異常增殖是高血壓、冠狀動(dòng)脈粥樣硬化和冠狀動(dòng)脈介入治療術(shù)后再狹窄等疾病的主要病理生理機(jī)制[15]。AngⅡ是一種重要的血管活性物質(zhì)，在體內(nèi)和體外均可誘導(dǎo)VSMCs過(guò)度增殖，而抑制此作用可阻止多種心血管疾病的發(fā)生及發(fā)展[16]。本研究發(fā)現(xiàn)AngⅡ能夠顯著增加VSMCs細(xì)胞的增殖能力、細(xì)胞活性及增殖標(biāo)志物PCNA蛋白的表達(dá)，且30、100μmol/L白藜蘆醇能夠抑制AngⅡ誘導(dǎo)的VSMCs增殖，而10μmol/L白藜蘆醇則無(wú)此作用，提示白藜蘆醇對(duì)VSMCs增殖的影響呈濃度依耐性。Schreiner等[17]研究發(fā)現(xiàn)白藜蘆醇可抑制AngⅡ誘導(dǎo)的VSMCs信號(hào)轉(zhuǎn)導(dǎo)分子激活，進(jìn)而發(fā)揮相應(yīng)的生物學(xué)作用，且這一作用與白藜蘆醇抗氧化作用有關(guān)，但其具體機(jī)制尚未闡明。

本課題組前期研究發(fā)現(xiàn)，100μmol/L白藜蘆醇可顯著減輕高糖對(duì)血管舒張活動(dòng)的抑制作用，進(jìn)一步研究發(fā)現(xiàn)白藜蘆醇能夠激活A(yù)MPK，進(jìn)而誘導(dǎo)內(nèi)皮細(xì)胞內(nèi)一氧化氮等生物活性物質(zhì)釋放，起到抗氧化和抗炎癥反應(yīng)的作用[5]。AMPK是廣泛存在于真核生物細(xì)胞中的一種信號(hào)通路分子，介導(dǎo)多種細(xì)胞生物學(xué)功能，本研究結(jié)果顯示，給予復(fù)合物C抑制細(xì)胞內(nèi)AMPK活性后，白藜蘆醇抑制細(xì)胞增殖、活力和PCNA蛋白表達(dá)的作用被逆轉(zhuǎn)。該結(jié)果提示AMPK是白藜蘆醇發(fā)揮細(xì)胞保護(hù)作用的重要信號(hào)分子，激活A(yù)MPK后抑制氧化應(yīng)激可能是白藜蘆醇抑制平滑肌細(xì)胞增殖的重要作用途徑。

綜上所述，白藜蘆醇能夠抑制血管平滑肌細(xì)胞的異常增殖，進(jìn)而起到預(yù)防高血壓、冠心病等心腦血管疾病的作用，而AMPK信號(hào)激活在白藜蘆醇細(xì)胞保護(hù)過(guò)程中起到重要作用。

[1] Little PJ, Getachew R, Rezaei HB, et al. Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFbeta receptor phosphorylation[J]. Arch Biochem Biophys, 2012, 525(1): 25-31.

[2] Badimon L, Vilahur G. LDL-cholesterol versus HDL-cholesterol in the atherosclerotic plaque: inflammatory resolution versus thrombotic chaos[J]. Ann N Y Acad Sci, 2012, 1254(2): 18-32.

[3] Chu LM, Lassaletta AD, Robich MP, et al. Resveratrol in the prevention and treatment of coronary artery disease[J]. Curr Atheroscler Rep, 2011, 13(6): 439-446.

[4] Paffett ML, Lucas SN, Campen MJ. Resveratrol reverses monocrotaline-induced pulmonary vascular and cardiac dysfunction: a potential role for atrogin-1 in smooth muscle[J]. Vascul Pharmacol, 2012, 56(1-2): 64-73.

[5] Xu Q, Hao X, Yang Q, et al. Resveratrol prevents hyperglycemiainduced endothelial dysfunction via activation of adenosine monophosphate-activated protein kinase[J]. Biochem Biophys Res Commun, 2009, 388(2): 389-394.

[6] Li L, Xu YF. Effect of Angiotensin Ⅱ on the expression of Kv4.2 potassium channel and its underlying mechanism in cardiomyocytes of neonatal rat[J]. Acta Acad Med CPAPF, 2010, 19(1): 26-28. [李亮, 許彥芳. Ang Ⅱ?qū)π募〖?xì)胞Kv4.2鉀通道蛋白表達(dá)的影響及機(jī)制[J]. 武警醫(yī)學(xué)院學(xué)報(bào), 2010, 19(1): 26-28.]

[7] Putnam K, Shoemaker R, Yiannikouris F, et al. The reninangiotensin system: a target of and contributor to dyslipidemias, altered glucose homeostasis, and hypertension of the metabolic syndrome[J]. Am J Physiol Heart Circ Physiol, 2012, 302(6): H1219-H1230.

[8] Guo RW, Wang H, Gao P, et al. An essential role for stromal interaction molecule 1 in neointima formation following arterial injury[J]. Cardiovasc Res, 2009, 81(4): 660-668.

[9] Li CY, Jiang S, Yue YJ, et al. Effect and pathway of Id1 on the cell growth of nasopharyngeal carcinoma[J]. Med J Chin PLA, 2012, 37(6): 569-572. [李春燕, 江山, 岳渝娟, 等. 分化抑制因子1對(duì)鼻咽癌細(xì)胞增殖的影響及其途徑[J]. 解放軍醫(yī)學(xué)雜志, 2012, 37(6): 569-572.]

[10] Feng F, Gao XD, Lu YY, et al. Regulatory effect of LINE-1 ORF-1p on hepatocellular carcinoma cells and proliferation of immortalized hepatocellular cells[J]. Med J Chin PLA, 2012,37(3): 213-216. [馮帆, 高旭東, 陸蔭英, 等. LINE-1 ORF-1p對(duì)肝臟腫瘤細(xì)胞系和肝源永生化細(xì)胞系增殖的調(diào)控作用[J].解放軍醫(yī)學(xué)雜志, 2012, 37(3): 213-216.]

[11] Jia QH, Yang XP, Hui L, et al. Effect of lead acetate on apoptosis of human renal mesangial cells and caspase-3 expression[J]. Med J Chin PLA, 2012, 37(6): 628-631. [賈慶華, 楊霄鵬, 惠玲, 等.醋酸鉛對(duì)人腎小球系膜細(xì)胞凋亡及Caspase-3表達(dá)的影響[J]. 解放軍醫(yī)學(xué)雜志, 2012, 37(6): 628-631.]

[12] Campbell KA, Lipinski MJ, Doran AC, et al. Lymphocytes and the adventitial immune response in atherosclerosis[J]. Circ Res, 2012, 110(6): 889-900.

[13] Kroon PA, Iyer A, Chunduri P, et al. The cardiovascular nutrapharmacology of resveratrol: pharmacokinetics, molecular mechanisms and therapeutic potential[J]. Curr Med Chem, 2010, 17(23): 2442-2455.

[14] Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging[J]. Cell, 2011, 146(5): 682-695.

[15] Jing QM, Huang GQ, Han YL, et al. Comparative study on percutaneous coronary intervention of unprotected left main coronary artery disease: transradial versus transfemoral approach[J]. Med J Chin PLA, 2011, 36(11): 1149-1153. [荊全民, 黃貴奇, 韓雅玲, 等. 經(jīng)橈、股動(dòng)脈途徑行無(wú)保護(hù)左主干病變介入治療的對(duì)比研究[J]. 解放軍醫(yī)學(xué)雜志, 2011, 36(11): 1149-1153.]

[16] Wang ZM, Wu X. Influence of atorvastatin on left ventricular hypertrophy in spontaneous hypertension rats[J]. J Zhengzhou Univ (Med Sci), 2012, 47(4): 554-556. [王智敏, 吳瑕. 阿托伐他汀對(duì)自發(fā)性高血壓大鼠左心室肥厚的影響[J]. 鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版), 2012, 47(4): 554-556.]

[17] Schreiner CE, Kumerz M, Gesslbauer J, et al. Resveratrol blocks Akt activation in angiotensin Ⅱ- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner[J]. Cardiovasc Res, 2011, 90(1): 140-147.

Inhibitory effect of resveratrol on proliferation of vascular smooth muscle cells induced by angiotensin Ⅱ and its underlying mechanism

GAO Pan, SI Liang-yi*, XU Qiang, WANG Xiao-mei
Department of Geriatrics, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
*

, E-mail: doctorsly@126.com
This work was supported by the National Natural Science Foundation of China (81000132), and the Special Fund of Chongqing Key Laboratory (CSTC) from Institute of Cardiovascular Science in Xinqiao Hospital (CQZDSYS201202)

ObjectiveTo explore the effect of resveratrol on proliferation of vascular smooth muscle cells (VSMCs) as induced by angiotensin Ⅱ (Ang Ⅱ) and its underlying mechanism.MethodsVSMCs of rat′s thoracic aorta were primarily cultured in vitro. For detecting the effects of resveratrol on Ang Ⅱ-induced VSMC proliferation, the cultured cells were divided into a control group, Ang Ⅱ group (1μmol/L), gradient concentrations of resveratrol groups, and Ang Ⅱ + gradient concentration of resveratrol groups. The gradient concentrations of resveratrol were set as 10, 30 and 100μmol/L. Cells in each group were treated for 0, 6, 12 and 24h, respectively. VSMC proliferation was detected by cell count assay, and the viability of the VMSC was measured by MTT assay. For detecting the effects of compound C [adenine monophosphate-activated protein kinase (AMPK) inhibitor] on biological effects of VSMCs, the cultured cells were divided into a control group, Ang Ⅱ group, resveratrol + Ang Ⅱ group and compound C + resveratrol + Ang Ⅱ group. The protein expression of proliferated cell nuclear antigen (PCNA) in each group was detected by Western blotting after being treated for 24 hours.ResultsCompared with control group, the cell viability and cell number in Ang Ⅱ group were significantly increased (P＜0.05) after Ang Ⅱ stimulation for 6, 12 and 24 hours. Compared with AngⅡ group, the cell vitality and cell number were significantly decreased in different concentrations of resveratrol + Ang Ⅱ groups (P＜0.05). On the other hand, compared with the control group, the expression of PCNA protein in Ang Ⅱ group was significantly increased (P＜0.05); as compared with Ang Ⅱ group, the expression of PCNA protein in resveratrol + Ang Ⅱ group was significantlydecreased (P＜0.05); as compared with resveratrol + Ang Ⅱ, the cell number, cell viability, and PCNA protein expression in compound C + resveratrol + Ang Ⅱ group was significantly increased (P＜0.05).ConclusionResveratrol can inhibit Ang Ⅱstimulated cell proliferation through activation of AMPK.

resveratrol; myocytes, smooth muscle; angiotensin Ⅱ; adenosine monophosphate-activated protein kinase

R541.4

A

0577-7402(2013)04-0269-05

2012-10-03；

2013-01-09)

(責(zé)任編輯：張小利)

國(guó)家自然科學(xué)基金(81000132)；重慶市新橋醫(yī)院全軍心血管研究所重點(diǎn)實(shí)驗(yàn)室專項(xiàng)經(jīng)費(fèi)資助項(xiàng)目(CQZDSYS201202)

郜攀，醫(yī)學(xué)博士，主治醫(yī)師。主要從事冠心病及其發(fā)病機(jī)制研究

400038 重慶 第三軍醫(yī)大學(xué)西南醫(yī)院老年病科(郜攀、司良毅、徐強(qiáng)、王笑梅)

司良毅，E-mail：doctorsly@126.com