Alkaloids Of Dendrobium Nobile Lindl Attenuate Cell Damage Induced By Aβ Via Activating Autophagy

ObjectiveThe present study focused on autophagy to explore the mechanisms of protective effects of alkaloids of Dendrobium nobile Lindl（ADNL）on axonal degeneration induced by Aβ_25-35 in primary hippocampal neurons of rat,at the same time,to explore whether dendrobine is the main component in ADNL induced autophagy by investigating the effects of dendrobine on mTOR/ULK1 signaling pathway.Methods（1）The protective effects of ADNL on axon degeneration induced by Aβ_25-35 in primary hippocampus neurons of rats in vitro was evaluated by measuring cell viability,degeneration index,postsynaptic density 95（PSD95）and synaptophysin（SYN）protein levels.The effects of ADNL on autophagy in hippocampus neurons were assessed at key step of autophagy process including:the formation of autophagosome,autophagosome-lysosome fusion,lysosomal acidification,activity of cathepsins and degradation of autophagosome.Lastly,two autophagy inhibitors bafilomycin A1（BAFA）and hydroxychloroquine（HCQ）were employed to interfere effects of ADNL on axonal degeneration to confirm the role of autophagy in neuroprotection of ADNL.（2）Effects of dendrobine on autophagic flux were evaluated by testing formation,ultrastructure and degradation of autophagosome in PC12 cells.Subsequently,the relationship between effects of dendrobine on autophagy and mTOR/ULK1 signaling pathway was confirmed in PC12 which ULK1 was knockdown by lentivirus mediated ULK1 siRNA technology.The protective effects of dendrobine on cell damage induced by Aβ_25-35 were assessed by detecting cell viability,lactate dehydrogenase（LDH）leakage rate and necrosis and apoptosis in PC12 cells,and the relationship was analyzed between the protection of dendrobine from Aβ_25-355-35 cytotoxicity and autophagy in PC12 cells.Results（1）ADNL treatment reduced the degeneration index,increased cell viability and protein levels of PSD95 and SYN in axonal degeneration of primary hippocampus neurons of rats induced by Aβ_25-35,suggesting that ADNL have protective effects on axonal degeneration.ADNL could increase formation of autophagosome,promote autophagosome-lysosome fusion,reduce the lysosomal pH to activate CTSB and CTSD,urge autophagolysosome degradation,suggesting that ADNL induce autophagy flux in hippocampal neurons of rats.The protective effect of ADNL on axonal degeneration could be antagonized by two fully different autophagy inhibitors HCQ and BAFA respectively.Taken together,these results indicated that ADNL prevent Aβ_25-35-induced axonal degeneration via activation of autophagy.（2）Dendrobine treatment increased formation and degradation of autophagosome in PC12 cells,which were significantly inhibited in ULK1 siRNA PC12 cells.Meanwhile,dendrobine treatment induced phosphorylation cascade of mTOR,ULK1 and Belin1protein,which were significantly inhibited in ULK1 siRNA PC12 cells,suggesting that dendrobine induced autophagy by activating mTOR/ULK1 signaling pathway in PC12cells.Dendrobine treatment improved cell viability,reduce LDH leakage and proportion of cell necrosis and apoptosis induced by Aβ_25-355-35 in PC12 cell,which could be inhibited in ULK1 siRNA PC12 cells,suggesting that the protective effect of dendrobine on PC12damage induced by Aβ_25-35 be related with activation of autophagy.Conclusion（1）ADNL prevents Aβ_25-35-induced axonal degeneration via activation of autophagyc in primary hippocampus neurons of rats.（2）Dendrobine activates autophagy through mTOR/ULK1 signaling pathway to protect PC12 cell damage from Aβ_25-355-35 cytotoxicity.