Tenuifolin Protects Against Aβ 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 Aβ secretion of AD model cell through the autophagy pathway.Our previous studies have found that Ten can reduce oxidative stress injury induced by Aβ 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 Aβ 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 Aβ 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 Aβ-induced activity and apoptosis of SH-SY5 Y cells.MTT assay was used to detect cell viability,25,50 μM of Ten can significantly increase cell viability,the minimal dose of Aβ significantly reduced cell viability was 20 μM,and the cell viability of Ten(25-200 μM)pretreatment group was significantly higher than that of Aβ(20 μM)model group,among them 50,100 μM dose of Ten has the best protective effect.The results showed that Ten 50,100 μM treatment group significantly reduced the apoptosis rate of Aβ-induced SH-SY5 Y cells.The 50 μM dose of Ten was the most significant,and 50 μM Ten and 20 μM Aβ were used for subsequent experiments.2.Effects of Ten on mitochondrial injury induced by Aβ in SH-SY5 Y cells.Fluorescence microscopy was used to observe the change of Δψm,the red fluorescence of Aβ-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 Aβ-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 Aβ.The results showed that pretreatment with Ten and CsA or siRNA could significantly inhibit the decrease of Δψm,the release of Cyt c,the enzyme activity and the mRNA expression of caspase-3 and caspase-9 induced by Aβ.3.Effects of Ten on Aβ-induced mitophagy of SH-SY5 Y cells.MDC staining showed that the Ten group significantly attenuated the enhancement of Aβ-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 Aβ-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 Aβ 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 Aβ.The results showed that pretreatment of Ten and CsA or siRNA could inhibit the increase of autophagy,LC3-II/I and mitophagy induced by Aβ.In summary,Ten can enhance the cell activity,decrease the apoptosis rate,reduce mitochondrial damage by increase Δψm 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 Aβ.In conclusion,Ten inhibits the expression of LC3 by downregulating the activation of PINK1/Parkin pathway induced byAβ,and reduces the release of Cyt c,thereby inhibits the activity of caspase and reduces Aβ-induced apoptosis of SH-SY5 Y cells.In this paper,we studied the protective mechanism of Ten in anti-Aβ-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 Aβ or AD drugs.