Regulation Of Autophagy Induced By Polygala Tenuifolia Saponins On Aβ Clearance And Cell Cycle In APP/PS1 Mice Hippocampus Based On AMPK/mTOR/ULK1 Pathway

Objective:To investigate the effect of tenuifolin(TEN)on Aβ protein clearance and cell cycle regulation in APP/PS1 double transgenic mice hippocampus by inducing autophagy,and to explore the protective effect of TEN on nerve cells by regulating autophagy and its related mechanism.Methods:1.Grouping and administration: 60 APP/PS1 T transgenic mice of AD model animals were selected and divided into six experimental groups,namely Model group(model group),TEN-L group(low-dose TEN group),TEN-M group(middle-dose TEN group),TEN-H group(high-dose TEN group),RP group(autophagy activator group),and TEN-M+MA group(autophagy inhibitor group),with 10 mice in each group;in addition,10APP/PS1 W mice of wild type were used as the control group(normal control group).The mice in each administration group were intragastrically administrated with the corresponding solution,and the mice in the model group and the normal control group were intragastrically administrated with 0.3% sodium carboxymethyl cellulose solution.The drug was fixed once a day,and the administration volume was 0.01 m L/g for three consecutive months.After the last administration,the mice were sacrificed by cervical dislocation,and the fresh brain tissues of each group were collected.2.Detection of related indicators: The changes of Aβ deposition in hippocampus of mice in each group were detected by sulfur S staining and immunohistochemical staining;flow cytometry was used to detect the cell cycle distribution in hippocampus of mice in each group;the expressions of Aβ transport related proteins(LRP-1 and RAGE),apoptosis related proteins(Bcl-2/Bax and Caspase-3)and cell cycle related proteins(CDK4,Cyclin D1,CDK2,Cyclin B1,E2F1,P21 and PCNA)were detected by Western blot.3.Molecular mechanism research: Pharm Mapper and Gene Cards were used to predict the possible targets of TEN against AD,and Bioinformatics Database was used to predict the biological process of TEN against Alzheimer’s disease and related molecular mechanisms;the expression levels of autophagy-related proteins(LC3)and mTOR signaling pathway-related proteins(AMPK,p-AMPK,mTOR,p-mTOR and ULK1,p-ULK1)were detected by Western blot.Results:1.The results of sulfur S staining and immunohistological staining showed that the green spots and Aβ protein deposition in CA1 region of mice in each dose group of TEN and RP groups decreased,and the green spots and Aβ protein deposition in CA1 region of mice in TEN-M + MA group were more than those in TEN-M dose group;Western blotting results showed that TEN could increase the expression level of transport-related protein LRP-1 and reduce the expression level of RAGE protein.The results showed that TEN and RP could reduce the deposition of Aβ protein in the hippocampus of APP / PS1 double transgenic mice and improve the clearance and transportation level of Aβ,which was related to autophagy.2.Western blotting results showed that TEN could increase the ratio of Bcl-2 / Bax and decrease the expression level of Caspase-3 in APP /PS1 transgenic mice.The results showed that TEN could improve the anti-apoptotic level of brain neurons in APP / PS1 double transgenic mice and reduce neuronal apoptosis.3.The results of flow cytometry showed that the proportion of G0 /G1 cells in each TEN dose group and RP group was high,and that in the TEN-M + MA group was lower than that in the TEN-M dose group.Western blotting results showed that TEN and RP could reduce the expression levels of CDK4,Cyclin D1,CDK2,Cyclin B1,E2F1 and PCNA,and increase the expression level of P21,while MA antagonized the effect of TEN.The results showed that TEN and RP could block the cell cycle of APP / PS1 double transgenic mouse hippocampal cells in G0 / G1 phase and inhibit the cell cycle restart process,which was related to autophagy.4.The results of network pharmacology analysis showed that 130 possible targets of TEN and 2579 possible targets of AD were obtained by Pharm Mapper and Gene Cards database platforms,and 78 possible targets of TEN and AD were obtained by intersection of the two.Bioinformatics Database analysis showed that the effect of TEN on AD may be regulated by mTOR-based autophagy signaling pathway.5.Western blotting results showed that TEN and RP could promote the expression of LC3 protein,inhibit mTOR hyperphosphorylation,increase the phosphorylation level of AMPK and ULK1,reduce the ratio of p-mTOR / mTOR,and increase the ratio of p-AMPK / AMPK and p-ULK1 / ULK1,while MA antagonized the effect of TEN.The results showed that TEN and RP could improve the autophagy level of APP / PS1 double transgenic mice by increasing the expression of LC3 protein and regulating the AMPK / mTOR / ULK1 signaling pathway.Conclusion:TNE can reduce the deposition of Aβ protein in the hippocampus of APP/PS1 double transgenic mice by regulating autophagy,improve the transport and clearance ability of Aβ,inhibit cell cycle reactivation and reduce neuronal apoptosis,so as to protect the brain neurons of APP/PS1 double transgenic mice.The mechanism may be by regulating AMPK/mTOR /ULK1 signaling pathway to increase the level of autophagy.