黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

Effects of Berberine Preconditioning on PC12 Cells Exposed to 6-OHDA

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。

Objective:To observe the effect of berberine(BBR)preconditioning on the PC12 cells exposed to 6-hydroxydopamine(6-OHDA),and investigate whether type 2 superoxide dismutase(SOD2)is involved in the protectiive effect of BBR.Methods:PC12 cells were divided into 5 groups,including the normal cultured control group,25μM 6-OHDA injury group(6-OHDA),1μM BBR preconditioning for 24 h group(BBR+6-OHDA),SOD2-siRNA interfering group(SOD2-siRNA+BBR+6-OHDA)and scrambled siRNA treatment group(SC-siRNA+BBR+6-OHDA),after 24-h incubation,methylthiazolyldiphenyl-tetrazolium bromide(MTT)was used to assess cell viability,reagent kits were taken to evaluate lactic dehydrogenase(LDH),intracellular reactive oxygen species(ROS),glutathione(GSH)and catalase(CAT)levels,flow cytometry was used to measure cell apoptosis rate,and western blot was taken to evaluate SOD2 and cleaved caspase-3 expression.Results:Compared with the control group,6-OHDA exposure for 24 h significantly reduced the cell viability,markedly decreased the SOD2 protein expression,intracellular GSH and CAT levels,meanwhile,increased LDH activity in the medium,up-regulated cell apoptosis rate and intracellular ROS level(P<0.05);however,the BBR preconditioning restored cell viability,SOD2 expression,intracellular GSH and CAT levels,and decreased LDH release,cell apoptosis,cleaved caspase-3 expression and ROS levels(P<0.05);however,SOD2-siRNA reversed the BBR preconditioning-induced cytoprotective effects(P<0.05),but the SC-siRNA did not induce significant effects on the BBR preconditioning-induced effects mentioned above(P>0.05).Conclusion:BBR preconditioning reduces 6-OHDA-induced injury in PC12 cells,and SOD2 mediates BBR preconditioning-induced protection in PC12 cells exposed to 6-OHDA.