| Background:Rheumatoid arthritis(RA)is an autoimmune disease that characterized by chronic inflammation of the synovial tissue,progressive destruction of cartilage and bone,leading to joint malformation.Fibroblast-like synoviocytes(FLSs)tumor-like growth is the central link in the pathogenesis of RA.Sonic Hedgehog(Shh)signaling pathway is abnormally activated in both RA patients and their animal models,and participates in the pathogenesis of RA by promoting excessive proliferation of FLSs,and hypermethylation of Ptch1 gene can overactivate this pathway,targeting Shh therapy is expected to serve as a new strategy for RA therapy.Mesenchymal stem cell microvesicles(MSC-MVs)are soluble biological media isolated from mesenchymal stem cells(MSCs)in vitro.They not only promote the repair of damaged tissues,inhibit inflammation,and regulate immunity,but also can avoid the adverse biological effects of direct injection of living cells in vivo.Previous studies have confirmed that human umbilical cord mesenchymal stem cell microvesicles(hUCMSC-MVs)transplantation can improve the arthritis symptoms,imaging and pathological manifestations of RA animal model-collagen-induced arthritis(CIA)rats.And can regulate immune disorders in RA patients.However,the effect of hUCMSC-MVs on the function of RA-FLSs and its mechanism of action are still unclear.Objective:To investigate the effect of hUCMSC-MVs on the proliferation,apoptosis and migration of RA-FLSs,further investigate whether the effect of hUCMSC-MVs on RA-FLSs is through targeting Shh signaling pathway,or whether the effect of hUCMSC-MVs on Shh signaling pathway is achieved by targeting the methylation level ofthe Ptch1 gene.In this study,we would use in vivo experiments to study the mechanism of hUCMSC-MVs on synovial fibroblasts of rheumatoid arthritis from the cell,protein,gene level.In order to provide a theoretical and experimental basis for the treatment of RA with hUCMSC-MVs.Methods:1.Acquisition and identification of hUCMSC-MVs: The first generation of hUCMSCs cell line was purchased,and the culture was passaged to the 4-5 generation for experimental use.hUCMSC-MVs were obtained by differential centrifugation conditioned medium by "starvation" treatment of hUCMSCs,and identified by morphology,protein concentration and surface molecule expression.2.Acquisition and identification of FLSs: The fourth generation of RA synovial fibroblast cell line(MH7A)used for experiments.The synovial tissue of osteoarthritis(OA)patients was collected under aseptic conditions,and the tissues were cut into chyle-like tissues,the cells were inoculated into DMEM culture medium after treated by trypsin digestion.The fluid was changed in 3-4 days,the cell started to passage when 90%of the cell adhered,and the 4-5 generation was selected for the experiment.Flow cytometry was used to detect the surface marker molecules CD14,CD45 and CD90 of OA-FLSs.3.Experimental group:(1)Detection of RA-FLSs functional grouping: divided into blank control group,Cyclopamine intervention group(Shh signaling pathway inhibitor),5-Azadc intervention group(DNA methylation inhibitor),hUCMSC-MVs intervention groups with different concentrations(10μg/μL,50μg/μL,100μg/μL,150μg/μL,200μg/μL);(2)Detection of Shh signaling pathway molecular expression group: divided into OA group,RA blank control group,Cyclopamine intervention group,5-Azadc intervention Group,hUCMSC-MVs intervention group(150μg/μL).4.Detection methods:(1)CCK8 method was used to detect the proliferation of each group,flow cytometry was used to detect the apoptosis,and the scratch test was used to compare the cell migration.(2)Western blot was used to detect the expression of Shh,Gli1 and Ptch1 in Shh signaling pathway.Real-time PCR was used to detect the expression ofSHH,GLI1 and PTCH1 mRNA in each group.Results:1.Acquisition and identification of hUCMSC-MVs: hUCMSC-MVs were obtained by gradient centrifugation and successfully identified.The hUCMSC-MVs were mostly between 40 nm and 300 nm in diameter.They were tea-like structure with lipid bilayer.The protein concentration after resuspension was about 2.00μg/μL.The surface molecules expressed CD63 and Hsp70 by Western blot.These results meet its identification criteria,provideing a basis for further research.2.Acquisition and identification of OA-FLSs: OA-FLSs were successfully isolated and cultured in this experiment.The expression of CD90 on surface molecules was more than 90%,and the expression rate of CD14 and CD45 was less than 1%,which was consistent with FLSs identification criteria.3.Effects of hUCMSC-MVs on RA-FLSs functions:1)Effects of hUCMSC-MVs on the proliferation of RA-FLSs:(1)Compared with the blank control group,the proliferation rate of RA-FLSs in the intervention group of hUCMSC-MVs 10μg/mL,50μg/mL and 100μg/mL was significantly increased(P<0.001),and the proliferation rate of the intervention group 150μg/mL and200μg/mL was decreased compared with the blank control group(P<0.001).There was no significant difference in the proliferation rate of RA-FLSs between the 150μg/mL and200μg/mL intervention groups(P>0.05).(2)Compared with the blank control group,the proliferation rate of RA-FLSs in the Cyclopamine intervention group and the 5-Azadc intervention group was significantly decreased(P<0.001);the proliferation rate of FLSs was lower in the hUCMSC-MVs150μg/mL intervention group compared with the Cyclopamine intervention group(P<0.001),at the same time,the proliferation rate of FLSs in the intervention group of hUCMSC-MVs 150μg/mL and 5-Azadc intervention group was not significantly different(P>0.05).The hUCMSC-MVs 150 μg/mL intervention group was selected for subsequent experiments.2)The effect of hUCMSC-MVs on the apoptosis of RA-FLSs:Compared with the blank control group,the apoptosis rate of RA-FLSs in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group was significantly increased(P<0.01,P<0.01,P<0.01).There was no significant difference in apoptosis rate between hUCMSC-MVs intervention group and other intervention groups(P>0.05).3)Effects of hUCMSC-MVs on RA-FLSs migration:(1)After 12 hours of intervention,the mobility of RA-FLSs in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group were decreased compared with the blank control group(P<0.001,P<0.01,P<0.05);The mobility of the Cyclopamine intervention group was significantly lower than that of the5-Azadc intervention group and the hUCMSC-MVs intervention group(P<0.01,P<0.001).There was no significant difference in mobility between the 5-Azadc intervention group and the hUCMSC-MVs intervention group(P>0.05).);(2)After 24 hours of intervention,the mobility of RA-FLSs in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group decreased compared with the blank control group(P<0.001,P<0.001,P<0.01);The mobility of the Cyclopamine intervention group was significantly lower than that of the5-Azadc intervention group and the hUCMSC-MVs intervention group(P<0.001,P<0.001).The migration rate of the 5-Azadc intervention group was lower than that of the hUCMSC-MVs intervention group(P<0.001).(3)After 48 hours of intervention,the migration rate of RA-FLSs in the Cyclopamine intervention group and the 5-Azadc intervention group was lower than that in the blank control group(P<0.001,P<0.05).There was no significant difference in the rate between the hUCMSC-MVs intervention group and the blank control group(P>0.05).4.Effect of hUCMSC-MVs on RA-FLSs Shh signaling pathway:1)Protein expression level:(1)Compared with the OA group,the expression of Shh and Gli1 protein in the blank control group was significantly increased(P<0.001,P<0.001).There was no significant difference in the expression level of Ptch1 protein(P>0.05).(2)Compared with the blank control group,the expression of Shh protein in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group was decreased(P<0.05,P<0.05,P<0.01);There was no significant difference between the intervention groups of Shh protein expression(P>0.05).(3)Compared with the blank control group,the expression of Gli1 protein in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group was decreased(P<0.05,P<0.05,P<0.001);compared with Cyclopamine intervention group,the expression of Gli1 protein in hUCMSC-MVs intervention group was significantly decreased(P<0.05).There was no significant difference in Gli1 protein expression between 5-Azadc intervention group and hUCMSC-MVs intervention group(P>0.05).(4)There was no significant difference in Ptch1 protein expression between the intervention groups and the blank control group(P>0.05).4)mRNA expression changes:(1)Compared with the OA group,the expression of GLI1 and SHH mRNA in the blank control group increased(P<0.05,P<0.05),and the expression of PTCH1 mRNA decreased(P<0.05).(2)Compared with the blank control group,the expression of SHH mRNA in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group was decreased(P<0.001,P<0.01,P<0.01),and there was no significant difference between the intervention groups of the SHH mRNA expression(P>0.05).(3)Compared with the blank control group,the expression of GLI1 mRNA in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group decreased(P<0.05,P<0.05,P<0.05),and there was no significant difference between the intervention groups of the GLI1 mRNA expression(P>0.05);(4)Compared with the blank control group,the expression of PTCH1 mRNA in the Cyclopamine intervention group,5-Azadc intervention group and hUCMSC-MVs intervention group increased(P<0.001,P<0.05,P<0.05),The expression of PTCH1 mRNA in Cyclopamine intervention group was higher than that in 5-Azadc intervention group andhUCMSC-MVs intervention group(P<0.001,P<0.001).There was no difference in PTCH1 mRNA expression between 5-Azadc intervention group and hUCMSC-MVs intervention group(P>0.05);Conclusion:1.The hUCMSC-MVs and OA-FLSs obtained in this experiment meet the identification criteria;2.Abnormal activation of Shh signaling pathway promotes proliferation and migration of RA-FLSs,and inhibits its apoptosis;3.The elevated level of Ptch1 methylation mediates the abnormal activation of the Shh signaling pathway;4.hUCMSC-MVs may affect the function of RA-FLSs by inhibiting the activation of Shh signaling pathway and methylation of Ptch1 gene. |
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