基于VDAC1/mPTP调节机制探讨葛根素抗心肌细胞缺氧/复氧损伤的保护作用  

Protective effect of puerarin against anoxia reoxygenation injury in cardiomyocytes based on VDAC1/mPTP regulation mechanism

作　　者：马文 徐勇民 孟艳 贺敏 杨小梅 何明[1] 廖章萍[1] MA Wen;XU Yong-min;MENG Yan;HE Min;YANG Xiao-mei;HE Ming;LIAO Zhang-ping(Department of Pharmacology,Nanehang University School of Pharmaceutical Science,Nanchang 330006,China;Department of Anesthesiology,The Third Hospital of Nanchang,Nanehang 330006,China;Department of Nursing,The Third Hospital of Nanehang,Nanehang 330006,China)

机构地区：[1]南昌大学药学院药理学教研室,江西南昌330006 [2]南昌市第三医院麻醉科,江西南昌330006 [3]南昌市第三医院护理部,江西南昌330006

出　　处：《中国药理学通报》2018年第7期964-969,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81460495;81660601);南昌大学研究生创新基金资助项目(No cx2016381)

摘　　要：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,Western 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(mitochondria 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 involved 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 expression of protein level was detected by Western blot.LDH and CK activities were measured by automatic biochemical analyzer.Mitochondrial membrane potential（ Δψm） and cell apoptosis were observed by flow cytometry method.Mitochondrial swelling test was used to detect the opening of mitochondria permeability transition pore（ mPTP）.Results Compared with control group,the expression of VDAC1 and mRNA was upregulated in A/R group,LDH and CK activity were elevated,and then mPTP opened,Δψm collapsed,cell apoptosis was significantly increased.Puerarin pretreatment can lower the expression of VDAC1,maintain 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.

关 键 词：葛根素 缺氧/复氧损伤 细胞凋亡 电压依赖性阴离子通道1 线粒体通透性转换孔道 心肌细胞 

分 类 号：R-332[医药卫生] R284.1