Effect Of Human Umbilical Cord Mesenchymal Stem Cell Microbvesicles On Autophagy In Synovial Fibroblasts Of Rheumatoid Arthritis

Objective:To investigate whether hUCMSC-MVs have a regulatory effect on the autophagy level of RA-FLSs,and explore the potential mechanism of hUCMSC-MVs acting on RA.Methods:The hUCMSCs cell line(SM-MSC)and the RA-FLSs cell line(MH7A)were purchased and used for experiments after passage.hUCMSC-MVs were obtained by gradient centrifugation,and the form of the MVs was observed by transmission electron microscopy the surface antigen phenotype was identified by Western Blot.The experiment contained control group,3-MA intervention group(autophagy inhibitor group)and hUCMSC-MVs intervention group.Cell proliferation was detected by CCK8 kit,cell apoptosis was evaluated by flow cytometry,cell migration was assessed by scratch assay,mRNA expression of autophagy-related genes BECN1 and MAP1LC3B was used by qPCR,and autophagy-related protein Beclin1、LC3II expression and the ratio of LC3Ⅱ/Ⅰ was analyzed by Western Blot.Result:1.The influence of hUCMSC-MVs in the cell proliferation,apoptosis,migration ablility of RA-FLSs.(1)Cell proliferation: Compared with the control group,the cell viability of the10μg/mL intervention MVs group,the 50μg/mL MVs intervention group,and the100μg/mL MVs intervention group increased significantly(P<0.001);Compared with the control group,the cell viability of the 3-MA intervention group,150μg/mL MVs intervention group and 200μg/mL MVs intervention group decreased significantly(P<0.001).There was no significant difference between the 150μg/mL MVs intervention group and 200μg/mL MVs intervention group(P>0.05).There was no significant difference between the survival rate of the 150μg/mL MVs intervention group and the 3-MA intervention group(P>0.05).(2)Cell apoptosis: Compared with control group,the apoptosis rate of 3-MA intervention group and hUCMSC-MVs intervention group increased significantly(P<0.01);there was no significant difference between the 3-MA intervention group and hUCMSC-MVs intervention group(P>0.05).(3)Cell migration ability: After 12 h and 24 h intervention,compared with the control group,the migration rate of cells in the 3-MA intervention group and the hUCMSC-MVs intervention group decreased,which was significantly different(P<0.01,P<0.01).There was no significant difference between the hUCMSC-MVs intervention group and the 3-MA intervention group(P>0.05).After 48 hours of intervention,there was no statistical difference among the control group,3-MA intervention group and the hUCMSc-MVs intervention group.(P>0.05).2.Effects of hUCMSC-MVs on the expression of BECN1 and MAP1LC3 mRNA in RA FLSs.(1)BECN1 mRNA expression: Compared with the control group,the expression of BECN1 mRNA in the 3-MA intervention group and the hUCMSC-MVs intervention group was significantly lower(P<0.01,P<0.05);and compared with the hUCMSC-MVs intervention group,the expression of BECN1 mRNA in the 3-MA intervention group was increased significantly(P<0.05).(2)MAP1LC3B mRNA expression: Compared with the control group,the expression of MAP1LC3 B mRNA was decreased in the 3-MA intervention group and the hUCMSC-MVs intervention group(P<0.01,P<0.01);and compared with the3-MA intervention group,the expression of MAP1LC3 B mRNA in thehUCMSC-MVs intervention group was increased significantly(P<0.05).3.Effects of hUCMSC-MVs intervention on the level of Beclin1,LC3 II protein and the ratio of LC3II/I in RA-FLSs(1)Beclin1 protein expression: Compared with the control group,the expression of Beclin1 protein in the 3-MA intervention group and the hUCMSC-MVs intervention group was decreased(P<0.05);the difference between the hUCMSC-MVs group and the 3-MA group was not statistically significant.(P>0.05).(2)LC3II protein expression: Compared with the control group,the expression of LC3 II protein in the 3-MA intervention group and the hUCMSC-MVs intervention group was significantly lower(P<0.01,P<0.05);and there was no significant difference in LC3 protein expression between the 3-MA intervention group and hUCMSC-MVs intervention group(P>0.05).(3)LC3II/I ratio: Compared with the control group,the ratio of LC3II/I in the3-MA intervention group and the hUCMSC-MVs group was significantly lower(P<0.001,P<0.001);There was no significant difference between the 3-MA interwention group and hUCMSC-MVs intervention group(P>0.05).Conclusion:1.hUCMSC-MVs intervention can inhibit the proliferation and migration of RA-FLSs and promote the apoptosis of RA-FlSs;2.There are abnomal autophagy in RA-FLSs,hUCMSC-MVs play a role in RA by down-regulating autophagy of RA-FLSs,Provide theoretical basis and basis for hUCMSC-MVs for clinical treatment of RA.