The Protective Effects Of Tenuifolin On A?-induced Cell Injury Through Regulate Autophagy And Potential Related Mechanism

Tenuifolin is a major component of Polygala tenuifolia.Polygala tenuifolia has a brain protective function and traditionally used to improve memory and treat dementia.Our previous studies showed that Polygala tenuifolia has neuroprotective effects of reduce abnormal deposition of A?,inhibit the phosphorylation of tau protein,regulate the function of cholinesterase system,inhibit the production of inflammatory mediators,inhibit the neurotoxicity caused by excessive release of glutamate,antagonize the oxidation and inhibit cell apoptosis,etc.,thereby exerting anti-AD and neuroprotective effects.However,the specific role and molecular mechanism of Ten's anti-AD activity need to be further clarified,especially the influence and molecular mechanism of Ten on A?production and A? toxicity.This paper explored the role and molecular mechanism of Ten through regulating autophagy on the cell damage induced by A?,and elucidated the signal pathway of Ten-regulated autophagy,providing a new target for the application of Polygala tenuifolia and scientific basis in AD therapy.The contents are as follows:1.Effect of Ten regulated autophagy on A?-induced morphology and activity of SH-SY5 Y cells: MTT assay was used to detect cell viability,the results showed that pretreatment of 50?M Ten can significantly increase the cell viability and improve the cell morphology of 20?M A?25-35 group,but the autophagy inhibitor 3-MA can reverse the neuroprotective effects of Ten.The results suggest that Ten can improve A?-induced cellmorphology and increase A?-induced cell viability through activation of autophagy.2.Effect of Ten regulated autophagy on A?-induced oxidative stress of SH-SY5 Y cells:DCFH-DA fluorescence probe was used to measure ROS level,thiobarbituric acid?TBA?was used to measure the level of MDA,WST-8 was used to determine the activity of SOD,NADPH method was used to determine the activity of GSH-Px,and the activity of CAT was measured by the method of ultraviolet absorption.The results showed that Ten can reduce the concentration of ROS and MDA,and increase the activity of SOD,GSH-Px and CAT induced by A?25-35 in SH-SY5 Y cells,but adding 3-MA will reverse the anti-oxidation effect of Ten.The results suggest that Ten can improve the antioxidant capacity induced by A?25-35 through activation of autophagy.3.Effect of Ten regulated autophagy on A?-induced inflammation of SH-SY5 Y cells:The expression levels of IL-1?,IL-6 and TNF-? m RNA were detected by RT-PCR.The results showed that Ten can significantly reduce the m RNA expression levels of IL-1?,IL-6 and TNF-? induced by A?25-35 in SH-SY5 Y cells,but 3-MA will reverse the effect of Ten on antagonizing neuroinflammation.The results suggest that Ten can significantly reduce A?25-35-induced inflammatory damage by inducing autophagy.4.Effect of Ten regulated autophagy on A? production in SH-SY5 Y cells:the expression levels of APP and BACE1 m RNA were detected by RT-PCR,the concentration of BACE1,A?_1-40 and A?_1-42 were detected by Elisa.The results showed that Ten can significantly reduce the expression level of BACE1 m RNA,the concentration of BACE1,A?_1-40 and A?_1-42 induced by A?25-35 in SH-SY5 Y cells,but Ten and A?25-35 have no effect on the expression level of APP m RNA,whereas adding 3-MA will reverse the effects of Ten.The results suggest that Ten can inhibit the activity of BACE1 in SH-SY5 Y cells by inducing autophagy,thereby reducing A?production.5.Mechanisms of Ten on A?-induced autophagy in SH-SY5 Y cells:The m RNA and protein levels of Beclin-1,LC3,m TOR,AMPK and ULK1 were detected by RT-PCR and western.The results shows that A?can increase the m RNA expression of Beclin-1 and LC3 and the protein expression of Beclin-1 in SH-SY5 Y cells but has no effect on the expression level of LC3-II/I.Ten can further increase A?-induced the expression of Beclin-1 and LC3,while 3-MA reversed Ten-induced autophagy.A? also can reduce the expression levels of m RNA and protein of AMPK and ULK1,and increase the m RNA expression and protein levels of m TOR in SH-SY5 Y cells,whereas Ten can increase the expression of AMPK and ULK1,and reduce the expression of m TOR.The results suggest that Ten can enhance autophagy by regulating the AMPK/m TOR/ULK1 pathway and increasing the expression of Beclin-1and LC3-II/I.In summary,Ten can inhibit A?-induced neurotoxicity.The possible molecular mechanism are that Ten activates autophagy through regulates AMPK/m TOR/ULK1 pathway and increases the protein levels of Beclin-1and LC3-II/I,thereby reducing the activity of BACE1 and the secretion of A?,thereby improving A?25-35-induced cell morphology damage and decline of cell viability,improving cellular antioxidant capacity and reducing inflammatory injury,and exerting neuroprotective roles.