Explore The Total Saponins Of Trillium Tschonoskii Maxim In Rheumatoid Arthritis Based On PI3K/AKT Signaling Pathway

Research background Rheumatoid arthritis(RA)is an autoimmune disease characterized by chronic,destructive,polyarthritis.The adult incidence rate of RA is around1%,the disability rate is high,and the damage to the human body is irreversible.Objective By observing the effect of Total saponins of Trillium tschonoskii Maxim(TTM)on the inflammatory response and synovial cells of adjuvant arthritis(AA)rats,we will preliminarily explore the mechanism of TTM preventing rheumatoid arthritis;by observing TTM On the migration,invasion,cell cycle and apoptosis of human rheumatoid arthritis fibroblast-like synovial cells MH7 A,further explore its possible mechanism of action.To study the effect of Total saponins of Trillium tschonoskii Maxim(TTM)on the inflammatory response and synovium of adjuvant arthritis(AA)rats,and use in vivo and in vitro experiments to explore the possible mechanism of action.Methods(1)In vivo experiment: 50 SD rats were randomly divided into normal group(control group),model group(AA),trillium saponins low(TTM-L),high dose group(TTM-H),Tripterygium Glycosides(TG),gavage for 2 weeks,use vernier calipers to record the changes in the thickness of the left and right foot of the rat;enzyme-linked immunosorbent assay(ELISA)to detect serum interleukin-6(interleukin-6;IL-6)and tumor necrosis factor(Tumor necrosisi factor-α;TNF-α)values;hematoxylin-eosin(HE)staining to observe the pathological changes of synovial tissue in each group of rats;TUNEL staining The apoptosis index of synovial tissue in each group was detected;Western Blot was used to detect B-lymphocyte tumor-2(Bcl-2),Bcl-2 related X protein(Bax)and caspase-3(Caspase-3)protein expression level.(2)In vitro experiment: MH7 A cells were cultured,MH7 A cells were treated with different concentrations(0-16 μg/m L)TTM for 24 h,48h,72 h,and cell proliferation was detected by CCK-8 method;Cell scratch test and Transwell method to analyze the effect of TTM on the migration and invasion of MH7 A cells;PI single staining combined with flowcytometry to analyze the effect of TTM on the cell cycle of MH7A;PI/FITC-Annexin V double staining combined with flow cytometry was used to analyze the effects of MH7 A cell apoptosis;Western Blot was used to detect changes in MH7 A cell apoptosis-related proteins and PI3K/AKT signaling pathway-related proteins that were treated with different concentrations of TTM.Results(1)In vivo experiment: Compared with the AA group,the TTM-L,TTM-H and TG groups can all reduce the thickness of the left and right feet of AA rats;compared with the control group,the TNF-a and IL-6 in the AA group increased significantly,and the TTM-L,TTM-H,and TG groups can significantly reduce the values of TNF-a and IL-6 in AA rats;after HE staining,compared with the control group,the synovial hyperplasia on the left and right sides of the AA group is obvious,and the TTM-H group is compared with the control group.TG group can improve left synovial hyperplasia in AA rats;TUNEL staining shows that TTM-L,TTM-H groups and TG groups can induce apoptosis of synovial tissue cells in AA rats;compared with AA group,TTM-L,TTM-H group and TG group can significantly increase the expression level of Bax protein and Cleavd-Caspase-3 protein,and significantly reduce the expression level of Bcl-2 protein.(2)In vitro experiments: The CCK8 experiment showed that TTM significantly inhibited the proliferation of MH7 A cells,and the inhibitory effect was dose-and time-dependent.After 24 hours of action,the IC50 was 4.01±0.12μg/m L.After TTM acts on MH7 A cells for 24 h,it can inhibit cell migration and invasion in a concentration-dependent manner.TTM can induce MH7 A cells to undergo cycle changes,so that the cell cycle is blocked in S phase.After TTM treated MH7 A cells for 24 hours,the apoptosis ratio did not change significantly;when TTM treated MH7 A cells for 48 hours,the number of PI/Annexin-V double-positive cells in MH7 A cells increased significantly.Western Blot results showed that the expression of pro-apoptotic proteins Cleavd-Caspase 3 and Bax protein increased,Bcl-2 protein expression significantly decreased,Bax/Bcl-2 value increased,and p-PI3 K,p-AKTprotein expression levels decreased.Conclusion Total saponins of Trillium tschonoskii Maximcan can improve the synovial hyperplasia of RA and reduce the inflammatory response.The mechanism may be inhibiting the proliferation and migration of fibroblast-like synovial cells and inducing the apoptosis of fibroblast-like synovial cells by inhibiting the PI3K/AKT signaling pathway.