异鼠李素对H_2O_2引起的H9C2细胞氧化应激损伤的保护作用研究

Protective effect of isorhamnetin on H9C2 cell line against oxidative stress

目的探讨异鼠李素对氧化应激诱导的心肌细胞损伤的保护作用及其作用机制。方法用MTT实验确定异鼠李素的心肌细胞毒作用浓度,以及抗H2O2氧化应激损伤的最佳作用浓度。将实验组分为正常组、模型组、治疗组、单给药组。治疗组、单给药组均用最佳浓度的异鼠李素预孵育12h,模型组、治疗组在预孵育结束后用300μmol·L-1H2O2作用4 h,模拟心肌细胞的氧化应激损伤。利用流式细胞术检测心肌细胞凋亡细胞比例、ROS产生等指标,利用荧光显微镜检测细胞的线粒体膜电位去极化,利用荧光酶标仪检测抗氧化酶与氧化产物MDA产量,利用Western blot检测线粒体凋亡信号通路与Nrf2/ARE信号通路的相关蛋白表达。结果与正常组相比,单给药组心肌细胞的凋亡细胞比例,ROS产生,线粒体膜电位去极化,细胞质细胞色素C、caspase-9、caspase-3、Bcl-2、Bax表达,抗氧化酶与氧化产物MDA产量差异均无显著性(P>0.05);与模型组相比,治疗组心肌细胞凋亡比例明显降低(P<0.01),ROS产生明显降低(P<0.05),线粒体膜电位去极化明显改善(P<0.01),细胞质细胞色素C、caspase-9、caspase-3、Bax表达明显降低(P<0.01),Bcl-2表达增加(P<0.01),抗氧化酶表达增加(P<0.01),氧化产物MDA表达降低(P<0.01);心肌细胞中Nrf2核转位与HO-1表达均随着异鼠李素预孵育时间的延长而增加。结论异鼠李素能够保护H2O2引起的心肌细胞损伤。其机制为影响线粒体凋亡通路以及激活Nrf2/ARE信号通路,实现抗氧化、抗凋亡功能。

Aim To investigate the protective effect of isorhamnetin on H9 C2 myocardial cell line and its mechanisms. Methods The toxicity and optimal pro-tective concentration of isorhamnetin were determined by MTT assay. The experimental subjects were divided into four groups：group N （ normal ） , group M （ mod-el） , group M ＋ ISO （ model ＋ isorhamnetin ） , and group ISO （ isorhamnetin only ） . Group M ＋ISO and ISO were pre-incubated with isorhamnetin for 12 hours while other groups with plain DMEM. Group M and M＋ ISO were treated with 300μmol · L^-1 H2 O2 for 4 hours after pre-incubation. Mitochondrial membrane potential depolarization of H9 C2 was measured by fluo-rescence microscope. Apoptotic rate and ROS produc-tion of injured myocardial cell line were detected using flow cytometry. The oxidative indictors were measured by spectrophotometry. The expressions of cytoplasmic cytochrome C, caspase-9, caspase-3, Bcl-2, Bax, Nrf2 and HO-1 were examined by Western blot. Result There was no difference in mitochondrial membrane potential depolarization, apoptotic rate, ROS produc-tion, oxidative indictors production and expressions of cytoplasmic cytochrome C, caspase-9,caspase-3, Bcl-2 , Bax between groups ISO and N （ P〉0. 05 ） . Apop-totic rate, ROS production, expressions of cytoplasmic cytochrome C, caspase-9, caspase-3, Bax, MDA pro-duction of group M＋ISO were significantly lower than those of group M （ P 〈 0. 01 ） . And mitochondrial membrane potential, Bcl-2, CAT, SOD and GSH-Px of group M ＋ ISO were increased compared to group M . Nuclear translocation of Nrf2 and expression of HO-1 in myocardial cell line were increased with the prolonged isorhamnetin incubation time. Conclusion Isorham-netin could protect myocardial cell line against H2 O2-induced oxidative injury and apoptosis through the interruption of mitochondrial dependent apoptotic path-way and activation of Nrf2/ARE pathway.