The Protect Effects And The Underlying Mechanisms Of Panax Notoginseng Saponins On Nerve Cells

ObjectiveParkinson’s disease(PD)is a degenerative disease of the nervous system.The pathology of PD is complex and the etiology remains poorly understood.α-synuclein has been demonstrated to be implicated in formation of Lewy body and the mainly pathogenesis of PD.Study showed that α-synuclein point mutations is being considered as the main cause to the formation of aggregated a-synuclein.The aggregated α-synuclein interfere with the normal cell function and lead to the apoptosis in dopaminergic neurons.How to stop the formation of aggregated α-synuclein or improve its degradation were highly noticed by researchers.Panax Notoginseng Saponins(PNS)is as a main component of Panax.In recent years,domestic and foreign scholars conduct research on PNS,and they find it has pharmacological effects on the central nervous system,cardiovascular system,anti-inflammatory,anti-neoplastic,anti-fatigue,etc.The treatment of PNS on Parkinson’s nervous systemic disease and Alzheimer’s disease play a multifaceted role,which has a wide development prospect.In the current study,we constructed SH-SY5Y cells lines expressing wild type and A53T mutant a-synuclein,to investigate whether PNS can enhance mitophagy of transfected WT or A53T SH-SY5Y cells and the potential mechanisms.MethodsEstablishment of a model of PD induced by 6-OHDA in SH-SY5Y cells.The protective effects of PNS were examined by MTT assay,relative kits,TUNEL assay,JC-1 and ROS staining,and Western blotting.The viability of WT or A53T SH-SY5Y cells was estimated by MTT.MTT method also was employed to determine the viability in PNS(6.25、12.5、25、50、100μg·mL-1)treated cells for 24h and 48h.Mitochondrial membrane potential was measured by JC-1,the change of the number of autophagosome,mitochondria and autolysosome were detected by immunofluorescence.The expression of PINK1,Parkin and P62 were also analyzed by Western blotting to evaluate whether PNS could enhance the mitophagy of transfected WT or A53T SH-SY5Y cells.Western blotting method was used to detect the protein expression of phosphorylation AMPK,AMPK in the AMPK signaling pathway and phosphorylation PI3K,PI3K,phosphorylation Akt,Akt,phosphorylation mTOR,mTOR in the PI3K/Akt/mTOR signaling pathway.Results1.PNS has significant protective effects in SH-SY5Y cells(1)PNS has significant protective effects against 6-OHDA-induced in SH-SY5Y cells:the viability of 6-OHDA treated SH-SY5Y cell was increased by PNS(6.25、12.5、25μg · mL-1)for 24h.The level of LDH was reduced(P<0.01).(2)PNS has significant anti-apoptosis effects against 6-OHDA-induced in SH-SY5Y cells:PNS inhibits DNA fragmentation,the decrease of mitochondrial and the activation of caspase-3(P<0.01).(3)PNS has significant anti-oxidant effects against 6-OHDA-induced in SH-SY5Y cells:PNS could reduce the levels of ROS,MDA and increase the activities of SOD,GAT,GSH-Px(P<0.01).2.Without significant differences were detected among the growth curves of three kinds of SH-SY5Y cells.Both 50μg · mL-1 and 100 u g · mL-1 PNS decreased the cell viability of SH-SY5Y cells and in a time-and dose-dependent manner(P<0.05).But had no obvious effects by PNS(6.25、12.5、25 μg · mL-1)for 24h and 48h.3.The mitochondrial membrane potential of A53T SH-SY5Y cell was increased by PNS(P<0.01).PNS enhanced the number of autophagosome and autolysosome,up-regulated the expression of LC3(P<0.01).When the cells were pretreated 3-MA for 2h,the autophagy induced by PNS was inhibited.4.PNS up-regulated the p-AMPK protein expression and down-regulated the p-PI3K,p-Akt,p-mTOR protein expression.ConclusionPNS had significant anti-apoptosis and anti-oxidant protective effects against 6-OHDA-induced in SH-SY5Y cells.PNS could enhance the mitophagy of A53T SH-SY5Y cell by the AMPK signaling pathway and PI3K/Akt/mTOR signaling pathway.