瓜蒌皮提取物对缺氧/复氧损伤H9c2心肌细胞的保护作用及其机制研究

Study on Protective Effects of Pericarpium Trichosanthis Extract on H9c2 Myocardial Cells Injured by Hypoxia/Reoxygenation and Its Mechanism

目的:研究瓜蒌皮提取物对缺氧/复氧损伤H9c2心肌细胞的保护作用及其机制。方法:以采用连二亚硫酸钠(Na_2S_2O_4)为化学缺氧剂,建立大鼠H9c2心肌细胞缺氧/复氧损伤模型,筛选不同浓度Na_2S_2O_4(0.625～10 mmol/L)和不同缺氧时间(10～60min)、复氧时间(2～8 h)的造模条件,以及瓜蒌皮提取物给药浓度(12.5～400μg/mL)。将细胞分为正常组、模型组、瓜蒌皮提取物不同剂量组(即TPE低、中、高剂量组,25、50、100μg/mL)和阳性对照组(槲皮素,25μmol/L),加入相应药物进行预处理24 h后,再建立缺氧/复氧损伤模型。采用MTT法检测各组细胞存活率;采用酶联免疫吸附法检测细胞中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;采用流式细胞术观察细胞凋亡情况;采用Western blotting法检测细胞中Bax、Bcl-2的蛋白表达水平。结果:心肌细胞缺氧/复氧损伤造模条件为Na_2S_2O_4造模浓度2.5 mmol/L,缺氧时间30 min,复氧时间4 h;12.5～400μg/mL的瓜蒌皮提取物对细胞均无毒性。分组处理后结果显示,与空白组比较,模型组细胞存活率显著降低、凋亡率显著升高,细胞中CK-MB、LDH、MDA水平均显著升高,SOD水平显著降低,Bax的蛋白表达水平及Bax/Bcl-2比值均显著升高,Bcl-2的蛋白表达水平均显著降低(P<0.05或P<0.01);与模型组比较,瓜蒌皮提取物各剂量组细胞上述变化均被逆转(P<0.05或P<0.01)。结论:瓜蒌皮提取物对缺氧/复氧损伤心肌细胞具有一定的保护作用;其机制可能与抑制细胞中脂质过氧化物的增加、提高细胞清除活性氧自由基的能力、上调Bcl-2/下调Bax蛋白表达等,从而抑制细胞凋亡有关。

OBJECTIVE:To study the protective effects of Pericarpium Trichosanthis extract(TPE)on H9c2 myocardial cells injured by hypoxia/reoxygenation(H/R). METHODS:H/R injury model of H9c2 myocardial cells was established by using sodium dithionite(Na2S2O4)as chemical hypoxia agent. The modeling conditions of different concentrations of Na2S2O4(0.625-10 mmol/L) and different time of hypoxia(10-60 min)and reoxygenation(2-8 h),as well as the concentration of TPE(12.5-400 μg/mL)were screened. The cultured H9c2 myocardial cells were randomly divided into normal group,model group,TPE different dose groups (TPE low-dose,medium-dose and high-dose groups,25,50,100 μ g/mL)and positive control group(quercetin,25 μmol/L). They were pre-treated with relevant medicine for 24 h,and then H/R injury model was established. Cell viability was measured by MTT assay. The levels of LDH,CK-MB,SOD and MDA in cells were tested by ELISA. Apoptosis of H9c2 myocardial cells were observed by flow cytometry. Western blotting was used to detect the protein expression of Bax and Bcl-2 in cells. RESULTS:The condition of H/R injury modeling included modeling concentration of Na2S2O4 2.5 mmol/L,hypoxia time 30 min,reoxygenation time 4 h. 12.5-400 μg/mL TPE showed no toxicity to H9c2 myocardial cells. After treatment,compared with blank group,survival rate and apoptotic rate of H9c2 myocardial cells in model group were increased significantly;the levels of CK-MB,LDH and MDA were increased significantly,while SOD level was decreased significantly;protein expression of Bax and Bax/Bcl-2 ratio were increased significantly,while that of Bcl-2 was decreased significantly(P<0.05 or P<0.01). Compared with model group,above changes of H9c2 myocardial cells were reversed in all dose groups of TPE(P<0.05 or P<0.01). CONCLUSIONS: TPE can protect H9c2 myocardial cells against H/R injury. Its mechanism may be associated with inhibiting the increase of lipid peroxide,improving the ability of scavenging reactive oxygen free radicals,up-regulating the protein expression of Bcl-2 or down-regulating protein expression of Bax,so as to inhibit the cell apoptosis.