活血荣络方含药血清对氧糖剥夺/复氧糖损伤PC12细胞线粒体自噬的影响及机制研究  

Study on the Effects and Mechanism of Drug-containing Serum Huoxue Rongluo Decoction in Mitochondrial Autophagy in Oxygen-glucose Deprivation/Reoxygenation-damaged PC12 Cel

作　　者：颜思阳 杨仁义[2] 陈瑶[1] 刘利娟[1] 高晓峰[1] 周德生[1] YAN Siyang;YANG Renyi;CHEN Yao;LIU Lijuan;GAO Xiaofeng;ZHOU Desheng(The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]湖南中医药大学,湖南长沙410208

出　　处：《中国中医药信息杂志》2023年第7期101-107,共7页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金(81874463、82104766);湖南省自然科学基金(2021JJ30521、2021JJ40424);湖南省教育厅一般项目(20C1405);湖南省卫健委科研项目(202103071190);中国卒中学会基金项目(CSA-SF-2021-03);湖南中医药大学科研基金项目(2021XJJJ052)。

摘　　要：目的基于氧糖剥夺/复氧糖(OGD/R)损伤的PC12细胞模型观察活血荣络方含药血清对线粒体自噬的影响及作用机制。方法采用OGD/R损伤PC12细胞模拟体外脑缺血再灌注损伤模型,将细胞分为正常对照组、模型组、空白血清组、活血荣络方含药血清组和雷帕霉素组,采用相应血清进行干预,JC-1荧光探针检测线粒体膜电位(MMP),免疫荧光染色检测线粒体-LC3B共定位、线粒体-PINK1-Parkin共定位,Western blot检测微管蛋白1轻链3B(LC3B)、p62、线粒体外膜转位酶20(TOMM20)、PTEN诱导假定激酶1(PINK1)、Parkin蛋白表达。结果与正常对照组比较,模型组细胞MMP明显降低(P<0.01),线粒体-LC3B共定位无明显变化,线粒体-PINK1-Parkin共定位增强,LC3B、PINK1、Parkin蛋白表达明显升高,p62、TOMM20蛋白表达明显降低,差异均有统计学意义(P<0.05,P<0.01);与模型组比较,活血荣络方含药血清组细胞MMP明显升高(P<0.01),线粒体-LC3B共定位和线粒体-PINK1-Parkin共定位增强,LC3B、PINK1、Parkin蛋白表达明显升高(P<0.05,P<0.01),p62、TOMM20蛋白表达明显降低(P<0.05,P<0.01)。结论活血荣络方含药血清可减轻OGD/R诱导的PC12细胞损伤,其机制可能与调控PINK1/Parkin通路,增加Parkin蛋白的线粒体转位,上调LC3B,下调p62、TOMM20蛋白表达,激活线粒体自噬相关。Objective To observe the effects and mechanism of drug-containing serum Huoxue Rongluo Decoction on mitochondrial autophagy based on PC12 model of oxygen-glucose deprivation/reoxygenation(OGD/R)damage.Methods The model of cerebral ischemia-reperfusion injury in vitro was established by simulating PC12 cells injured by OGD/R.The cells were divided into normal control group,model group,blank serum group,drugcontaining serum Huoxue Rongluo Decoction group and rapamycin group,and the corresponding serum was used for intervention.JC-1 was used to detect mitochondrial membrane potential(MMP),immunofluorescence was used to analyze the mitochondrial-LC3B colocalization and mitochondrial-PINK1-Parkin colocalization,Western blot was used to detect the expressions of LC3B,p62,TOMM20,PINK1 and Parkin protein.Results Compared with the normal control group,the MMP of the cells in model group significantly decreased(P<0.01),the mitochondrial-LC3B colocalization had no significant change,mitochondrial-PINK1-Parkin colocalization was enhanced,the expression of LC3B,PINK1 and Parkin protein increased,the expression of p62 and TOMM20 protein decreased,all with statistical significance(P<0.05,P<0.01).Compared with the model group,the MMP of the drug-containing serum Huoxue Rongluo Decoction group significantly increased(P<0.01),the mitochondrial-LC3B colocalization and mitochondrial-PINK1-Parkin colocalization were enhanced,the protein expression of LC3B,PINK1 and Parkin significantly increased(P<0.05,P<0.01),the protein expressions of p62 and TOMM20 significantly decreased(P<0.05,P<0.01).Conclusion Drug-containing serum Huoxue Rongluo Decoction can alleviate the injury of PC12 cells induced by OGD/R.The mechanism may be related to the regulating of PINK1/Parkin pathway,increasing mitochondrial translocation of Parkin protein,up-regulating LC3B,down-regulating p62 and TOMM20 protein expressions,and activating mitochondrial autophagy.

关 键 词：活血荣络方 含药血清 脑缺血再灌注损伤 线粒体自噬 神经保护 PC12细胞 

分 类 号：R285.5[医药卫生—中药学]