白藜芦醇通过MEK/ERK-SIRT1通路的激活调节酒精诱导的HepG2细胞凋亡损伤

Resveratrol inhibits alcoholinduced apoptosis via MEK/ERK-SIRT1 signaling in human HepG2 cells

目的:探讨白藜芦醇在酒精诱导的HepG2细胞凋亡中的调节能力,揭示白藜芦醇抗酒精性肝损伤的作用机制.方法:白藜芦醇预处理HepG2细胞24 h后,用酒精诱导凋亡的产生.MTT方法检测白藜芦醇处理组与非处理组HepG2细胞的细胞活力;用ELISA试剂盒检测不同实验组的细胞内总超氧化物浓度(total superoxide)和细胞总抗氧化能力(oxygen radical antioxidant capacity,O R A C);同时检测了含半胱氨酸的天冬氨酸蛋白水解酶3(cysteine-requiring aspartate protease,Caspase3)蛋白表达及应用荧光倒置显微镜观察了吖啶橙(acridine orange,AO)/碘化丙啶(propidium iodide,PI)染色的细胞形态学改变;采用RT-PCR方法检测氧化应激调节凋亡通路中关键基因Caspase3,细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK),ERK激酶(mitogen-activated protein kinase kinase,MEK)和沉默信息调节因子1(silent mating type information regulation 1,SIRT-1)mRNA的表达.结果:MTT结果显示,与对照组比较,25-100μmol/L白藜芦醇可有效对抗300 mmol/L酒精对HepG2引起的细胞不良反应使细胞保持较高的活力;AO/PI活细胞凋亡检测显示了酒精处理组有大量的橙色凋亡细胞,而白藜芦醇预处理组橙色细胞明显减少;Caspase3结果显示不同浓度的白藜芦醇均可以降低被酒精激活的Caspase3的活性,其Caspase3的活性降为2.12、1.46、0.90和0.75倍;细胞内总超氧化物浓度结果显示预处理100、50、25μmol/L白藜芦醇组含量明显低于酒精诱导组;而未经白藜芦醇预处理的酒精诱导组ORAC(45.26±2.75)明显低于100、50、25μmol/L白藜芦醇预处理组(65.74±1.64、68.14±6.06、70.81±6.35).RT-PCR结果显示,与300 mmol/L酒精比较,不同浓度白藜芦醇均可上调SIRT1与ERK mRNA表达量;50μmol/L和100μmol/L白藜芦醇均可明显上调MEK mRNA表达量;50μmol/L和100μmol/L白藜芦醇均可显著下调Caspase3基因mRNA的表达量.结论:本研究提示酒精可诱导氧化应激相关的凋亡产生,而白藜芦醇通过调节MEK/ERKSIRT1通路中基因的表达发挥抗凋亡作用从而削弱酒精诱导的氧化应激及凋亡的损伤作用.

AIM： To investigate the effects of resveratrol on alcohol induced apoptosis of HepG2 cells and to explore the possible mechanisms involved. METHODS： HepG2 ceils were pretreated withresveratrol for 24 h before treatment with alcohol to induce apoptosis. MTT assay was then performed to detect cell cytotoxicity and viability. Inverted fluorescence microscopy was used to detect cell morphologic changes after AO/PI staining. ELISA was performed to detect the presence of intracellular superoxide anion and oxygen radical antioxidant capacity levels. RT-PCR was performed to detect the mRNA expression of Caspase3, mitogen-activated pro- tein kinase kinase （MEK）, extracellular signal- regulated kinase （ERK） and silent mating type information regulation 2 homolog （SIRT1）. RESULTS： In comparison with control cells （non-treated with resveratrol）, resveratrol at concentrations between 25-100 μmol/L exerted an antagonistic effect against cytotoxicity of 300 mmol/L alcohol to HepG2 cells. AO/PI apoptosis assay showed alcohol-treated cells contained many orange apoptotic cells, but resveratrol treated cells had less orange cells. Different concentrations of resveratrol could decrease the activity of activated Caspase3 in alcohol-treated cells by 2.12, 1.46, 0.90 and 0.75 times. Intracel- lular superoxide anion concentrations in cells treated with 100, 50 or 25 μmol/L resveratrol were obviously lower than those in alcohol- treated cells. However, the total oxygen radical antioxidant capacity was significantly higher in cells pre-treated with 100, 50, or 25 μmol/L resveratrol compared with alcohol-treated cells （65.74 ±1.64, 68.14 ± 6.06, 70.81 ± 6.35 vs 45.26 ± 2.75）. In addition, resveratrol increased the expression of SIRT1 and ERK mRNAs and decreased the expression of Caspase3 mRNA. CONCLUSION： Alcohol induces oxidative stress related apoptosis of HepG2 cells, and resveratrol exerts anti-apoptosis effects by regulating the expression of genes involved in the MEK/ERK- SIRT1 pathway.