Study On The Protective Effects And Mechanisms Of Dendrobium Nobile Lindle.Alkaloids On PC12 Cells Induced By A?_25-35

Objective: This study was designed to investigate the protective effect of Dendrobium nobile Lindle.Alkaloids?DNLA?on the oxidative stress and apoptosis of PC12 cells induced by A?_25-35,and further explore its potential mechanisms.Methods: PC12 cells were divided into 5 groups: control group,model group(10 ?M A?_25-35),DNLA-L group(0.035 mg/L DNLA+10 ?M A?_25-35),DNLA-M group(0.35 mg/L DNLA+10 ?M A?_25-35),DNLA-H group(3.5 mg/L DNLA+10 ?M A?_25-35).The cells were cultured in vitro and treated with A?_25-35 for 24 h to induce PC12 after 6 h DNLA?0.035³.5 mg/L?exposure.MTT assay was used to evaluate the effect of DNLA on cell viability induced by A?_25-35,meanwhile Annexin V/PI staining by flow cytometry was used to detect apoptotic cells.Based on the identification on that DNLA have anti-apoptosis function towards that model,do research on effects of DNLA on oxidative stress induced by A?_25-35 in PC12 cells by measuring intracellular ROS level,GSH level and SOD activity.Inverted immunofluorescence microscopy was used to examine the effect of DNLA on mitochondrial membrane potential?MMP?stained with JC-1.Bax,Bcl-2,Cleaved-caspase9,pro-caspase3 and Cleaved-caspase3 expression level,key proteins of mitochondrial apoptotic pathway,were detected by Western blot,respectively.Results: Compared with control group,the cell viability was markedly decreased and the apoptosis showed notable increase in model group;exposure of cells to A?_25-35 for 24 h elicited ROS production,while SOD activity,GSH expression level and MMP were markadly decreased,respectively;the protein expression of Bax,Cleaved-caspase9 and Cleaved-caspase3 were significantly increased and the protein expression of Bcl-2 and pro-caspase3 were decreased.However,compared with model,the cell viability ofDNLA-M and DNLA-H was significantly increased,and the apoptotic death was markedly decreased;ROS production was significantly reduced,as well as SOD activation;on the other hand,pretreatment with DNLA at the concentration of 3.5 mg/L dramatically enhanced both GSH content and MMP;the level of apoptosis relate protein Bax,Cleaved-caspase9 and Cleaved-caspase3 were significantly reduced;the protein expression of Bcl-2 and pro-caspase3 was notably increased.Conlusion: Under the experimental conditions,DNLA have significant inhabitation function on the apoptosis of PC12 cells induced by A?_25-35,and its protential mechanism can be related to reducing the level of oxidative stress to inhibit mitochondrial apoptosis-related protein expression.