Study on the Protective Effects and Mechanisms of Dendrobium Nobile Lindle.Alkaloids on PC12 Cells induced by Aβ25-35

摘要

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 Aβ25-35,and further explore its potential mechanisms. Methods:PC12 cells were divided into 5 groups:control group,model group(10 μmol·L-1Aβ25-35),DNLA-L group(0.035 mg·L-1 DNLA+10 μmol·L-1 Aβ25-35),DNLA-M group(0.35 mg·L-1 DNLA+10 μmol·L-1 Aβ25-35), DNLA-H group(3.5 mg·L-1 DNLA+10μmol·L-1 Aβ25-35). The cells were cultured in vitro and treated with Aβ25-35 for 24 h to induce PC12 after 6 hDNLA(0.035～3.5 mg·L-1)exposure. MTT assay was used to evaluate the effect of DNLA on cell viability induced by Aβ25-35,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 byAβ25-35in 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-caspase-9 and cleaved-caspase-3 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 Aβ25-35 for 24 h elicited ROS production, while SOD activity,GSH expression level and MMP were markadly decreased,respectively;the protein expression of Bax,cleaved-caspase-9 and cleaved-caspase-3 were significantly increased and the protein expression of Bcl-2 were decreased. However,compared with model,the cell viability of DNLA-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-1 dramatically enhanced both GSH content and MMP;the level of apoptosis relate protein Bax,cleaved-caspase-9 and cleaved-caspase-3 were significantly reduced;the protein expression of Bcl-2 was notably increased. Conclusions:Under the experimental conditions,DNLA have significant inhabitation function on the apoptosis of PC12 cells induced by Aβ25-35,and its protential mechanism can be related to reducing the level of oxidative stress to inhibit mitochondrial apoptosis-related protein expression.