Tenuifolin Protects Against A? Induced Apoptosis Of Cells Through Regulating Mitophagy

Polygala tenuifolia has the effects of anti-dementia,brain protection,sedation,antidepressant,protect cardiovascular and cerebrovascular and so on.Its main active component of tenuifolin(Ten)can exert its neuroprotective effects through a variety of ways,has anti-oxidative,anti-aging,inhibiting cell apoptosis,improve cognitive and reduce the A? secretion of AD model cell through the autophagy pathway.Our previous studies have found that Ten can reduce oxidative stress injury induced by A? by reducing the activity of ROS,MDA and increasing the concentration of SOD,GSH-Px,reduce cell apoptosis and inhibition of caspase apoptosis protein expression by upregulating Bcl-2/Bax ratio,improve the transport and clearance of A? by regulating the expression of RAGE,LRP1 protein,and prevent nerve cell cycle disorders by regulating the expression level of cyclin,and thus play a protective role in nerve cells.At present,with the further research on the neuroprotective effect of Ten,the molecular mechanism and the related signal pathway still need to be further elucidated.This study explored the neuroprotective mechanism of Ten against A? induced apoptosis of cells through regulate mitophagy and elucidated its signal pathway.To provide experimental basis for further study on the role and mechanism of autophagy in Ten resistance to Alzheimer's Disease.The contents are as follows:1.Effects of Ten on A?-induced activity and apoptosis of SH-SY5 Y cells.MTT assay was used to detect cell viability,25,50 ?M of Ten can significantly increase cell viability,the minimal dose of A? significantly reduced cell viability was 20 ?M,and the cell viability of Ten(25-200 ?M)pretreatment group was significantly higher than that of A?(20 ?M)model group,among them 50,100 ?M dose of Ten has the best protective effect.The results showed that Ten 50,100 ?M treatment group significantly reduced the apoptosis rate of A?-induced SH-SY5 Y cells.The 50 ?M dose of Ten was the most significant,and 50 ?M Ten and 20 ?M A? were used for subsequent experiments.2.Effects of Ten on mitochondrial injury induced by A? in SH-SY5 Y cells.Fluorescence microscopy was used to observe the change of ??m,the red fluorescence of A?-induced SH-SY5 Y cells was significantly decreased,the green fluorescence significantly increased,and the red fluorescence changed to green fluorescence.After pretreatment with Ten and CsA,the trend of red fluorescence to green fluorescence was suppressed.Western blot was used to detect the changes of Cyt c,CsA and Ten pretreatment groups significantly inhibited A?-induced protein expression in Cyt c.The activity and the mRNA expression of caspase-3 and caspase-9 were detected by kit and qPCR.Ten and siRNA pretreatment significantly inhibited the activity and the mRNA expression of caspase-3 and caspase-9 induced by A?.The results showed that pretreatment with Ten and CsA or siRNA could significantly inhibit the decrease of ??m,the release of Cyt c,the enzyme activity and the mRNA expression of caspase-3 and caspase-9 induced by A?.3.Effects of Ten on A?-induced mitophagy of SH-SY5 Y cells.MDC staining showed that the Ten group significantly attenuated the enhancement of A?-induced fluorescence intensity of SH-SY5 Y cells.Western blot was used to detect the changes of autophagy marker protein LC3,CsA and Ten pretreatment groups significantly inhibited A?-induced increase in LC3-II/I.MTG and LTR staining were used to detect co-localization of mitochondrial and lysosomes,Ten,Cs A and Ten+CsA pretreatment groups significantly inhibited the increase of A? induced red and green fluorescence co-localization.Through qPCR method to detect the mRNA expression of PINK1 and Parkin,Ten and siRNA pretreatment significantly inhibited the mRNA expression of PINK1 and Parkin induced by A?.The results showed that pretreatment of Ten and CsA or siRNA could inhibit the increase of autophagy,LC3-II/I and mitophagy induced by A?.In summary,Ten can enhance the cell activity,decrease the apoptosis rate,reduce mitochondrial damage by increase ??m and reduce the release of Cyt c,inhibit the enzyme activity and the m RNA expression of caspase-3 and caspase-9,reduce autophagic bodies,the expression of LC3-II/I protein,mitochondrial and lysosomes co-localization and the mRNA expression of PINK1 and Parkin induced by A?.In conclusion,Ten inhibits the expression of LC3 by downregulating the activation of PINK1/Parkin pathway induced byA?,and reduces the release of Cyt c,thereby inhibits the activity of caspase and reduces A?-induced apoptosis of SH-SY5 Y cells.In this paper,we studied the protective mechanism of Ten in anti-A?-induced apoptosis by regulating the mitophagy and mitochondrial apoptosis pathway and discussed the application of Ten in anti-neuronal apoptosis strategy to provide theoretical basis for the study of A? or AD drugs.