| Background:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by invasive synovial hyperplasia leading to progressive joint destruction.Synovitis and articular cartilage/bone destruction are the most important factors in the pathogenesis of RA.Two links.The study of RANKL/OPG/TRAF6 axis in bone destruction is becoming more popular.It can affect the balance of bone metabolism of RA by affecting the proliferation and differentiation of osteoclasts.In recent years,studies have found that mesenchymal stem cell-derived microvesicles(MSC-MVs)have a clear tissue repair function and the ability of regulating cell growth and differentiation.MSC-MVs may serve as a novel non-cellular therapy in terms of depressing the progress of bone destruction in RA.Objective:To investigate the effects of human umbilical cord mesenchymal stem cell derived microvesicles(hUCMSC-MVs)on the RANKL/OPG/TRAF6 bone destruction pathway in CIA rats,and to elucidate the possible mechanism of inhibition of RA bone destruction by hUCMSC-MVs from the perspective of bone metabolism.Methods:1.Acquisition and identification of hUCMSC-MVs: The hUCMSCs obtained in the previous experiments were subjected to “hunger” pretreatment.Microvesicle are separated from mesenchymal stem cells conditioned supernatant using a differential centrifugation methods and identified by transmission electron microscopy.The protein content was determined using a Nano Drop 2000 ultra-micro spectrophotometer.2.Experimental grouping: Type II collagen + Freund's complete adjuvant was used to immunize female Wistar rats twice for establishing a collagen induced arthritis rats model.The immunological rats were randomly divided into 6 groups,inclouding CIA group(n=6),MTX group(n=6),hUCMSCs group(n=6),low concentration of hUCMSC-MVs group(n=6),high concentrations of hUCMSC-MVs group(n=6),and set up a normal control group.3.Processing method: MTX group was injected with 0.9 mg/kg intraperitoneally once a week for 3 weeks;MSC group was injected with 2×106 MSCs/100 ?l in the tail vein once a time;low and high concentration of hUCMSC-MVs groups were injected with 30 ug hUCMSC-MVs/100?l saline and 90 ug hUCMSC-MVs/100?l saline once a time,respectively.4.Detection Indicator: Rats in each group were sacrificed and the ankle synovial tissues were retained.Using RT-qPCR to detect the relative expression of RANKL mRNA,OPG mRNA,and TRAF6 mRNA in synovial tissues of each group for statistical analysis.Res?lts:1.The effect of hUCMSC-MVs on RANKL/OPG/TRAF6 system in ankle synovial membrane of CIA rats in protein expression level:1)Changes in RANKL/OPG ratio: Compared with HC(healthy controls)group,the expression of RANKL was up-regulated,the expression of OPG was down-regulated,and the RANKL/OPG ratio was significantly increased in the CIA group(P<0.05);Compared with CIA group,the level of RANKL was decreased and the level of OPG level were increased,RANKL/OPG ratio was decreased(P<0.05)in hUCMSC-MVs high,low concentration group,hUCMSCs group and MTX group.RANKL/OPG ratio was similar between hUCMSC-MVs high concentration group,hUCMSCs group,and MTX group(P>0.05),suggesting that the effect of high concentration of hUCMSC-MVs is similar to that of MTX and hUCMSCs.The ratio of RANKL/OPG in hUCMSC-MVs high concentration group is lower than that of hUCMSC-MVs low concentration group(P<0.05).2)Changes in TRAF6: The expression of TRAF6 in CIA group was significantly higher than that in HC group(P<0.05).The level of TRAF6 was decreased in hUCMSC-MVs high,low concentration group,hUCMSCs group,and MTX group compared with CIA group(P<0.05),and the level of TRAF6 was similar in hUCMSC-MVs high concentration group,h UCMSCs group and MTX group(P>0.05).The level of TRAF6 in hUCMSC-MVs high concentration group was lower than hUCMSC-MVs low concentration group(P<0.05).2.Effect of hUCMSC-MVs on RANKL/OPG/TRAF6 system in ankle synovial tissue of CIA rats at gene transcriptional level:1)Changes in RANKL/OPG mRNA ratio: Compared with the HC group,the RANKL mRNA level was up-regulated,the OPG mRNA level was down-regulated,and the ratio of RANKL/OPG mRNA was significantly higher in the CIA group(P<0.05).Compared with the CIA group,the level of RANKL mRNA in hUCMSC-MVs high and low concentration groups,the hUCMSCs group and the MTX group was up-regulated,the level of OPG mRNA was down-regulated,and the RANKL/OPG mRNA ratio was decreased(P<0.05).The ratio of RANKL/OPG mRNA was close in the hUCMSC-MVs high-concentration group,the hUCMSCs group,and the MTX group(P>0.05),suggesting that the effect of high concentration of hUCMSC-MVs was similar to that of MTX and hUCMSCs.The ratio of RANKL/OPG mRNA in hUCMSC-MVs high concentration group was lower than that hUCMSC-MVs low concentration group(P<0.05).2)Changes in TRAF6mRNA: The level of TRAF6 mRNA in CIA group was significantly higher than that in HC group(P<0.05).TRAF6 mRNA expression was down-regulated in hUCMSC-MVs high,low concentration group,hUCMSCs group,and MTX group compared with CIA group(P<0.05).But the effect of hUCMSC-MVs high concentration group was weaker than hUCMSCs and MTX(P<0.05).The hUCMSC-MVs high concentration group had lower TRAF6 mRNA level than hUCMSC-MVs low concentration group(P<0.05).Conclusions:1.There is an imbalance between RANKL and OPG mRNA levels in CIA rats and affects the expression of intracellular receptor TRAF6 mRNA.2.The hUCMSC-MVs intervention mechanism is similar to that of hUCMSCs.It can correct RANKL/OPG mRNA imbalance by down-regulating RANKL mRNA and up-regulating the expression of OPG mRNA.3.hUCMSC-MVs can correct the imbalance of bone metabolism in CIA rats by acting on the RANKL/OPG/TRAF6 regulatory axis,and the effect of intervention is related to its concentration. |
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