Based On Autophagy Of PPI3K/AKT/mTOR Signaling Pathway To Explore The Mechanism Of Toddalia Asiatica Inhibiting The Proliferation Of MH7A Cells

Objective To investigate the effects of Toddalia asiatica alcohol extract（TAAE）on the proliferation,autophagy and apoptosis of rheumatoid arthritis（RA）fibroblast-like synoviocytes（FLS）cell line MH7 A death and its mechanism,and to provide a theoretical basis for the further development and application of TA in clinic.Methods MH7 A cells were cultured in vitro,Cell Countin Kit-8（CCK-8）was used to detect the effect of TAAE on the survival rate of MH7 A cells,and the concentration and time of drug action were screened out.Flow cytometry was used to detect the effect of TAAE on cells.The effect of autophagy on apoptosis was further verified by using the early autophagy inhibitor 3-methyladenine（3-MA）and the late autophagy inhibitor chloroquine（CQ）to further verify the effect of autophagy on apoptosis;Western blot（WB）was used to detect microtubule-associated protein1 light chain3（LC3）,P62 and apoptosis-related protein Bcl-2 associated X protein（Bax）,B cell lymphoma/lewkmia-2（Bcl-2）,cleaved cysteinyl aspartate-specific protease-3（cleaved Caspase-3）,phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin（PI3K/AKT/m TOR）signaling pathway and phosphorylation（p）-PI3 K,p-AKT,p-m TOR protein expression;3-MA and CQ were used to verify the effect of TAAE on autophagy and apoptosis and used PI3 K agonists 740YPDGFR（740Y-P）to verify the inhibitory effect of TAAE on signaling pathways.Results CCK-8 showed that TAAE dose-dependently inhibited the proliferation of MH7 A cells in the concentration range of 0-2 mg/m L（P<0.05）,and the cell viability rate decreased with time;Flow cytometry showed that both TAAE and CQ induced apoptosis,and CQ+TAAE was more obvious than that of single use of the drug（P<0.05）.Compared with 740Y-P combined with TAAE,the apoptosis rate decreased（P<0.01）,and 3-MA had no significant effect on the apoptosis rate of TAAE cells.WB also showed that TAAE up-regulated the expression of pro-apoptotic protein Bax,autophagy-related protein LC3-II and P62 in a dose-time-dependent manner,and down-regulated the protein expressions of Bcl-2,p-PI3 K,p-AKT and p-m TOR（P<0.05）;CQ up-regulated the protein expression of P62,LC3-II,Bax,cleaved Caspase-3,and down-regulated the protein expression of Bcl-2（P<0.05）;3-MA and740Y-P weakened the accumulation of P62 and LC3-II（P<0.05）;740Y-P up-regulated the protein expressions of p-PI3 K,p-AKT,p-m TOR and Bcl-2,and down-regulated the expression of cleaved Caspase-3（P<0.05）.Conclusion TAAE can block autophagic flux through PI3K/AKT/m TOR signaling pathway,thereby inhibiting MH7 A cell proliferation and inducing apoptosis.