The Protective Effect Of Escitalopram On The MPTP-treated PC12 Cells And Its Mechanism

Objective: Parkinson’s disease(PD)is a degenerative disease of the central nervous system(CNS)that often occurs in middle-aged and elderly people,the main clinical manifestations are tremor,ankylosis,motor relaxation,postural balance disorder and other motor symptoms.In addition,it is often accompanied by some non-motor symptoms such as depression,anxiety,constipation,sleep disorders,cognitive impairment.Autophagy is a process that depends lysosomal pathway for degradation of cytoplasmic proteins and organelles,it is an important mechanism of self protection.Autophagy occurs in the basic physiological state and participates in the basic physiological process of the cell,but it can also be induced by different types of stress.However,excessive autophagy can lead to dysfunction.Studys have shown that autophagy disorder is associated with the pathogenesis PD.Escitalopram(ESC)is a selective serotonin reuptake inhibitor(SSRI),it is used in the treatment of generalized anxiety and depression,it also shows neuroprotective effect.Studys have shown that ESC can activate ubiquitin mediated autophagy lysosomal degradation pathway in hippocampal primary neuron cells.This study aimed to investigate the ESC on the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-treated PC12 cells and its mechanism.Method: According to the purpose of the study,ESC(final concentration5μM,10μM,20μM,40μM,60μM,80μM)or / and MPTP(final concentration500μM,750μM,1000μM,1250μM,1500μM)were added to the PC12 cells.After 1m M 3-MA was used,MTS were used to detect cell viability,Hoechst33258 staining for the apoptosis,Western blot were used to detectthe expression of autophagy marker LC3B-Ⅰ,LC3B-Ⅱ and apoptotic protein Bcl-2,Bax.Result:1 MTS was used to detect the cell viability of PC12 cells.1.1 The effect of MPTP on the cell viability of PC12 cells.PC12 cell were treated with the MPTP 500μM,750μM,1000μM,1250μM,1500μM for 24 h,48h,72 h,the cell viability of PC12 cells showed a dose-dependent and time-dependent manner,with the increase of MPTP concentration and the prolongation of time,the cell viability of PC12 cells decreased significantly.1.2 The effect of ESC on the cell viability of PC12 cells.PC12 cells were treated with the ESC 5μM,10μM,20μM,40μM,60μM,80μM for 24 h,there was no statistical difference in cell viability compared with CON group(P>0.05).Although there is no statistical difference,we can see that the trend of cell survival is first rising and then decreasing,and the increase is most obvious at 10μM.For 48 h,80μM ESC had damaged the PC12 cells,and the cell viability decreased by about 14.3% compared to CON group,the difference was statistically significant(P<0.05).There was no significant difference in the cell viability of PC12 cells between the other concentration of ESC and CON group(P>0.05).For 72 h,5μM,10μM,20μM ESC increased cell viability,especially at 10μM,increased about 12%,while60μM,80μM ESC decreased cell viability,respectively by 18.7% 和 32.9%compared to CON group,the difference was statistically significant(P<0.05).40μM ESC was no statistical difference in cell viability compared with CON group(P>0.05).Therefore,large dose of ESC can also cause cell apoptosis.1.3 ESC pretreatment on the cell viability of PC12 cells.PC12 cells were pretreated with 10μM and 20μM ESC for 1h,followed by MPTP 1000μM for another 24 h,or 5μM and 10μM ESC for 1h,followed by MPTP 1000μM for another 72 h,the cell viability was significantly increased compared with MPTP group.1.4 Effects of autophagy inhibitor 3-MA on cell viability in each group.The autophagy inhibitor 3-MA could significantly inhibit the neuroprotective effect of ESC pretreatment on PC12 cells.The cell viability of ESC+MPTP+3-MA group(65.23 + 2.96%)was significantly different from ESC+MPTP group(73.18 + 2.27%)(P<0.05).2 Detection of cell apoptosis by Hoechst33258 staining.Hoechst33258 staining showed that the nucleus of PC12 in 1000μM MPTP group was obviously constricted,and the nucleus showed bright blue fluorescence.There was no obvious change in the nucleus of PC12 in 10μM ESC group compared with CON group.ESC pretreatment group could significantly reduce the rate of apoptosis compared with MPTP group,the difference was statistically significant(P<0.05).3 Western blot detection of the expression of LC3B-Ⅱ/Ⅰand Bcl-2/Bax protein.Western blot detection showed that the level of LC3B-Ⅱ/Ⅰ in MPTP group was significantly higher than that in CON group,after 10 μM ESC pretreatmentand,the level of LC3B-Ⅱ/Ⅰ higher than that in MPTP group,this phenomenon was reversed by the application of autophagy inhibitor3-MA,both the level of LC3B-Ⅱ/Ⅰ and Bcl-2/Bax were all decreased.Conclusion:1.The proper time of ESC treatment at a certain concentration can promote cell viability.2.ESC pretreatment can increase the cell viability of PC12 cells and decrease the apoptosis rate of PC12 cells treated with MPTP,which indicates that ESC has a certain neuroprotective effect.3.ESC plays a neuroprotective role on PC12 cells mediated by MPTP via PI3K/AKT/Bcl-2 pathway.Autophagy inhibitor 3-MA inhibits PI3K/AKT/Bcl-2 pathway and inhibits the protective effect.So we speculated that the protective mechanism may be partly related to autophagy.