乌头赤石脂丸汤方对心肌细胞氧化应激损伤的保护作用及其可能的分子机制

Effect of Wutou Chishizhiwan Decoction Durg-containing Serum on Oxidative Stress Injury of H9C2 Cells

为了观察乌头赤石脂丸汤方含药血清对H9C2细胞氧化应激损伤的保护作用与可能的分子机制,采用过氧化氢处理H9C2细胞构建氧化应激损伤模型:以乌头赤石脂丸汤、参附汤连续灌胃SD大鼠1周,制备含药血清.将H9C2细胞随机分成空白对照组、模型组、阳性对照组、实验组、空白抑制剂组、模型抑制剂组、阳性抑制剂组、实验抑制剂组。过氧化氢造模浓度为400μmol/L,GDC-0994浓度为1μmol/L,分组给药后培养4h。用CCK-8法检测各组细胞活力,用Western blot法检测细胞中Erk1/2、p-Erk1/2、Nrf2、HO-1蛋白表达水平。实验结果显示:(1)与空白对照组比,模型组的细胞活力显著降低(P<0.01),Erk1/2磷酸化的水平降低,Nrf2、HO-1蛋白的表达降低(P<0.01);(2)与模型组相比,实验组、阳性对照组细胞活力显著升高(P<0.01),Erk1/2磷酸化水平降低,Nrf2、HO-1蛋白的表达降低(P<0.01);(3)与阳性对照组相比,实验组细胞活力显著升高(P<0.01),Erk1/2磷酸化水平,Nrf2、HO-1蛋白的表达升高(P<0.01);各组之间Erk1/2总蛋白表达无差异。(4)GDC-0994能够抑制乌头赤石脂丸汤方对细胞活力的保护作用(P<0.01),抑制乌头赤石脂丸汤方对p-Erk1/2,Nrf2,HO-1表达的上调作用(P<0.01)。研究表明乌头赤石脂丸汤方可以通过抑制氧化应激途径发挥对H9C2心肌细胞的保护作用,对抗再灌注引起的心肌细胞损伤。该作用是通过促进Erk1/2磷酸化,增强Nrf2、HO-1蛋白的表达,活化Erk1/2/Nrf2/HO-1信号通路进行的。本研究为阐明乌头赤石脂丸的心血管保护作用及治疗急性心肌梗死的作用提供了实验依据。

To evaluate the protective effect and possible molecular mechanism of Wutou Chishizhiwan decoction drug-containing serum on oxidative stress injury of H9C2 cells,the oxidative stress injury model was constructed by treating H9C2 cells with hydrogen peroxide.SD rats were fed with Wutou Chishizhiwan decoction and Shenfu decoction for one week to prepare drug-containing serum.H9C2 cells were randomly divided into different groups,including control group,model group,positive control group,experimental group,inhibitor group,model inhibitor group,positive inhibitor group,and experimental inhibitor group.The concentration of hydrogen peroxide was 400μmol/L,and the concentration of GDC-0994 was 1μmol/L.Both were cultured for 4 h.The cell viability of each group was measured by CCK-8 method,and the expression levels of Erk1/2,p-Erk1/2,Nrf2,and HO-1 proteins were examined by Western blot.The results were as follows.Firstly,compared to the control group,the cell viability of the model group was significantly reduced(P<0.01).The level of Erk1/2 phosphorylation in model group was also reduced,as well as the expression level of Nrf2 and HO-1 protein(P<0.01).Secondly,compared to the model group,the cell viability of the experimental group and the positive control group was significantly increased(P<0.01),the phosphorylation level of Erk1/2 was reduced,and the expression of Nrf2 and HO-1 protein was also reduced(P<0.01).Thirdly,compared to the positive control group,the cell viability of the experimental group was significantly increased(P<0.01),the phosphorylation level of Erk1/2,and the expression of Nrf2 and HO-1 proteins were increased(P<0.01).No significant differences were found in total protein expression of Erk1/2 among different groups.Finally,GDC-0994 inhibited the protective effect of Wutou Chishizhiwan decoction on cell vitality(P<0.01),and inhibited the up-regulation of Wutou Chishizhiwan decoction for p-Erk1/2,Nrf2,HO-1 expression(P<0.01).The results showed that Wutou Chishizhiwan decoction could play aprotective role on H9C2 cardiomyocytes by inhibiting oxidative stress pathways and counteract myocardial cell damage caused by reperfusion.This effect was carried out by promoting the phosphorylation of erk1/2,enhancing the expression of Nrf2 and ho-1 proteins,and activating the erk1/2/Nrf2/ho-1 signaling pathway.This study provides an experimental basis for elucidating the cardiovascular protective effect of Wutou Chishizhiwan decoction and its role in treating acute myocardial infarction.