目的 研究葛根素( puerarin, Pue)抗心肌缺血/再灌注损伤作用,及其可能涉及的线粒体机制.方法 建立H9c2心肌细胞缺氧/复氧( anoxia/reoxygenation, A/R)损伤模型.构建VDAC1 重组质粒pFLAG-VDAC1.细胞随机分为4组,正常对照组(Control)、缺氧/复氧组(A/R)、葛根素组(Pue+A/R)、VDAC1高表达组(pFLAG-VDAC1-葛根素),采用Real-time PCR法检测VDAC1 mRNA的表达情况,West-ern blot法检测其蛋白水平.流式细胞术观察线粒体膜电位(Δψm)和细胞凋亡,自动生化分析仪检测LDH、CK活性,线粒体肿胀实验检测各组细胞线粒体通透性转换孔道( mito-chondria permeability transition pore, mPTP)的开放情况.结果 与Control组相比,A/R组VDAC1 mRNA及蛋白表达水平均上调,LDH、CK水平升高,Δψm崩溃,mPTP开放,细胞凋亡明显增多.葛根素预处理则可下调VDAC1 表达,维持Δψm,防止mPTP开放,减少细胞凋亡.转染VDAC1高表达载体pFLAG-VDAC1,可取消葛根素的保护作用.结论 葛根素抗A/R损伤作用与 VDAC1 密切相关,其可通过下调VDAC1,防止 mPTP 开放,从而减少细胞凋亡,保护心肌细胞.%Aim To study the effect of puerarin( Pue) against myocardial ischemia/reperfusion injury and in-volved mitochondrial mechanism. Methods Anoxia/reoxygenation( A/R) injury model was established in H9c2 cell. Recombinant plasmid pFLAG-VDAC1 was constructed. Cells were randomly divided into 4 groups, normal control group ( Control) , A/R group, puerarin group ( Pue + A/R ) , and pFLAG-VDAC1-Pue group. Real-time PCR was used to investigate the expression of VDAC1 at mRNA level, and the expres-sion of protein level was detected by Western blot. LDH and CK activities were measured by automatic bi-ochemical analyzer. Mitochondrial membrane potential ( Δψm) and cell apoptosis were observed by flow cy-tometry method. Mitochondrial swelling test was used to detect the opening of mitochondria permeability tran- sition pore ( mPTP) . Results Compared with control group, the expression of VDAC1 and mRNA was up-regulated in A/R group, LDH and CK activity were el-evated, and then mPTP opened, Δψm collapsed, cell apoptosis was significantly increased. Puerarin pre-treatment can lower the expression of VDAC1, main-tain Δψm, prevent the opening of mPTP, and reduce apoptosis. However, the protective effect of Puerarin could be cancelled by transfection of pFLAG-VDAC1. Conclusions The cardioprotection of Puerarin against A/R injury is closely related to down-regulation of VDAC1 and prevention of mPTP opening.
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