异甘草素抑制SETD7表达可保护缺氧/复氧诱导心肌细胞的氧化损伤

Isoliquiritigenin inhibits SETD7 expression against oxidative damage in cardiomyocytes induced by hypoxia/reoxygenation

背景:缺血性心脏病已成为威胁人体健康的重要疾病之一,但其发生发展的分子机制尚未阐明。目的:探讨异甘草素对心肌细胞缺氧/复氧损伤后氧化应激及细胞凋亡的影响及其可能的作用机制。方法:体外培养大鼠心肌细胞H9C2,随机分为空白对照组、缺氧/复氧组、缺氧/复氧+异甘草素10,20,40,80μmol/L组,缺氧/复氧+si-con组(si-con为阴性对照)、缺氧/复氧+si-SETD7组、缺氧/复氧+异甘草素+pcDNA组、缺氧/复氧+异甘草素+pcDNA-SETD7组。采用MTT法检测细胞存活率;采用流式细胞仪检测细胞凋亡情况;测定细胞中活性氧、谷胱甘肽过氧化物酶、丙二醛水平;蛋白免疫印迹法检测细胞中凋亡相关蛋白活化半胱氨酸蛋白酶3表达。结果与结论:①与空白对照组比较,缺氧/复氧组细胞存活率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),活化半胱氨酸蛋白酶3蛋白表达水平与活性氧、丙二醛水平显著升高(P<0.05),而谷胱甘肽过氧化物酶水平显著降低(P<0.05);②与缺氧/复氧组比较,缺氧/复氧+异甘草素10,20,40,80μmol/L组细胞存活率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),SETD7、活化半胱氨酸蛋白酶3蛋白表达水平与活性氧、丙二醛水平显著降低(P<0.05),而谷胱甘肽过氧化物酶水平显著升高(P<0.05);③与缺氧/复氧+si-con组相比,缺氧/复氧+si-SETD7组心肌细胞存活率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),SETD7与活化半胱氨酸蛋白酶3蛋白的表达水平显著降低(P<0.05),活性氧、丙二醛水平显著降低(P<0.05),谷胱甘肽过氧化物酶水平显著升高(P<0.05);④与缺氧/复氧+异甘草素+pcDNA组相比,缺氧/复氧+异甘草素+pcDNA-SETD7组心肌细胞存活率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),SETD7、活化半胱氨酸蛋白酶3蛋白表达水平显著升高(P<0.05),活性氧、丙二醛水平显著升高(P<0.05),谷胱甘肽过氧化物酶水平显著降低(P<0.05);⑤提示异甘草素可通过抑制SETD7表达而抵抗细胞凋亡及增强其抗氧化能力,从而对缺氧/复氧损伤心肌细胞发挥保护作用。

BACKGROUND:Ischemic heart disease has become one of the important diseases that threaten human health,but the molecular mechanism of its occurrence and development has not yet been elucidated.OBJECTIVE:To investigate the effects of isoliquiritigenin(ISL)on oxidative stress and apoptosis in cardiomyocytes after hypoxia/reoxygenation(H/R)injury and its possible mechanism.METHODS:Rat cardiomyocytes H9C2 were cultured in vitro and randomly divided into normal control group,H/R group,H/R+ISL 10,20,40,80μmol/L groups,H/R+si-con group,H/R+si-SETD7 group,H/R+ISL+pcDNA group,H/R+ISL+pcDNA-SETD7 group.Cell viability was measured using methylthiazolyl tetrazolium(MTT)assay.Apoptosis was detected by flow cytometry.The levels of reactive oxygen species(ROS),glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)in the cells were measured.The expression of apoptosis-related protein Cleaved Caspase-3 was detected by western blot assay.RESULTS AND CONCLUSION:Compared with the normal control group,the cell survival rate of H/R group was significantly decreased(P<0.05),the apoptosis rate was significantly increased(P<0.05),Cleaved caspase-3 expression and ROS and MDA levels were significantly increased(P<0.05),while the GSH-Px content was significantly reduced(P<0.05).Compared with the H/R group,the cell survival rate of H/R+ISL 10,20,40,80μmol/L groups was significantly increased(P<0.05),and the apoptosis rate was significantly decreased(P<0.05),and the expression of SETD7 and Cleaved caspase-3 was significantly decreased(P<0.05),and the levels of ROS and MDA were significantly decreased(P<0.05),while the content of GSH-Px was significantly increased(P<0.05).Compared with the H/R+si-con group,the cell survival rate of H/R+si-SETD7 group was significantly increased(P<0.05),the apoptosis rate was significantly decreased(P<0.05),the expression of SETD7 and Cleaved caspase-3 was significantly decreased(P<0.05),and the levels of ROS and MDA were significantly decreased(P<0.05),while the content of GSH-Px was significantly increased(P<0.05).Compared with the H/R+ISL+pcDNA group,the cell survival rate of H/R+ISL+pcDNA-SETD7 group was significantly decreased(P<0.05),the apoptosis rate was significantly increased(P<0.05),the expression of SETD7 and Cleaved caspase-3 was significantly increased(P<0.05),and the levels of ROS and MDA were significantly increased(P<0.05),while the content of GSH-Px was significantly decreased(P<0.05).These findings indicate that ISL can inhibit the expression of SETD7 to reduce the apoptosis of cardiomyocytes induced by H/R and enhance the cell antioxidant capacity,thereby protecting cardiomyocytes against H/R injury.