Effect Of Yishen Pills On Bone Metabolism And Immune Regulation In Rats With Collagen-induced Arthriti

1 BackgroundRheumatoid arthritis(RA)is chronic autoimmune disease mediated by inflammatory.Joint synovial inflammation,pannus formation and cartilage and bone destruction are the main pathological manifestations.Its clinical symptoms often manifeste as joint pain and tenderness,stiffness,and often accompanied by bone Loose,gradually appear irreversible joint injury,and even joint functional disability,joint deformity.Its pathogenesis has not yet been elucidated.Most scholor think that immune regulation disorder is the main mechanism of RA and an important part of RA.Immunosuppressive agents play a role in the treatment of RA.Regulatory T cells(Tregs)play an important role in improving RA progression and bone destruction.Previous experiments confirmed that Treg can release IL-10,TGF-β to enhance its inhibition effect on osteoclast(OC)differentiation.Yishen Juanbi pills is an important prescription for the treatment of RA.It has the effect of enhancing the kidney and getting rid of the pain.Its clinical effect is obvious.Clinical and experimental studies show that Yishen Juanbi pills can relieve bone pain and delay the progress of RA.Previous studies have shown that Yishen Juanbi pills can inhibit the development of bone destruction in rats with Collagen-induced arthritis(CIA)and regulate immune function,but the mechanism has not been studied.The aim of this study was to investigate the immune mechanism of Yishen Juanbi Pills in treating RA bone destruction.2 ObjectiveThe effect of YSJB on bone destruction in CIA rats was investigated from the perspective of differentiation and maturation of OC regulated by Tregs.3 Methods3.1 Effect of Yishen Juanbi Pill on Treg/Th17 Balance to Improve Bone Damage in CIA Rats The effect of YSJB on the arthritis index of CIA rats was observed by classical type Ⅱcollagen-induced rat arthritis model(CIA).HE staining was used to evaluate the pathological changes of ankle joint.Popliteal lymph node lymphocytes were isolated,FCM Were used to detect the ratio of Treg,Th17 and Thl immunocytes.The activity of bone metabolism in serum was detected by enzyme-linked immunosorbent assay(ELISA).The microstructure of ankle joint was analyzed by microCT.3.2 Effects of Serum Containing Yishen Juanbi Pills on Osteoclast Differentiation and Bone Resorption in OC-Tregs Co-culture System Bone marrow cells(BMMs)were obtained from the bilateral tibias and femus of C57BL/6 mice.The spleen of C57BL/6 mice was isolated aseptically.BMMs were induced by M-CSF into precursor osteoclasts(Pre-OC),then addition of RANKL induce it different into OC.The spleen is made into a single cell suspension,CD4+CD25+ Tregs were obtained by immunomagnetic beads sorting.OC+Tregs co-culture group was inoculated in Transwell at a ratio of OC:Tregs = 25:1.The group settings are as follows:OC+normal serum group,OC+CIA model serum group,OC +YSJB serum group,OC+LEF serum group,OC+Tregs+normal serum group,OC+ Tregs+CIA model serum group,OC +Tregs+YSJB serum group,OC+Tregs+LEF serum group.The coverslips or bone slices are preliminarily prepared in each culture system.Add different groups of rats containing serum.Cells were stained by TRAP to compare the number of osteoclasts on the 7th day.Tartrate-resistant acid phosphatase(TRAP)activity was measured to compare the activity of osteoclasts.The bone slices were stained by toluidine blue on the 10th day and quantified the resorption area by Leica Qwin system.4 Results4.1 Effect of Yishen Juanbi Pill on Bone Damage and Regulation of Immune Function in CIA RatsThe arthritis index increased significantly(P<0.01)at 15 days after immunization.Compared with the blank control group,the pathological score of the rats in the ankle joint was significantly increased(P<0.01),and the local bone destruction of the ankle joint was significantly increased(P<0.01),The area of BS and BS/BV increased significantly(P<0.01).Thl cells and Th17 cells were significantly increased(P<0.01,P<0.05),BALP,PICP and PINP were significantly decreased(P<0.01);TRACP,NTX and CTX were significantly increased(P<0.01).Th1 cells,Thl7 cells were significantly increased(P<0.01,P<0.05),Treg cells decreased significantly(P<0.05).Compared with the model group,the arthritis index of the rats treated with leflunomide and Yishen Juanbi pills decreased,and the arthroplasty was decreased(P<0.05),and the bone mass of the rats was significantly increased(P<0.01).The area of BS and BS/BV of ankle joint decreased significantly(P<0.01,P<0.05).After treatment with Yishen Juanbi Pills,PINP increased significantly(P<0.05),and BGP and PICP increased,but there was no statistically significant difference.Thl and Th17 cells were significantly decreased(P<0.01,P<0.05).Treg cells were significantly increased(P<0.05),and the levels of TRLP,CTX and NTX were significantly decreased(P<0.01,P<0.05).4.2 Effects of Serum Containing Yishen Juanbi Pills on Osteoclast Differentiation and Bone Resorption in OC-Tregs Co-culture SystemThe osteoclast differentiation rate of the OC+CIA model serum group was significantly larger than that of the OC+normal serum group,and the activity of TRAP was significantly increased and the area of bone resorption was larger(P<0.01).Compared with the OC+CIA model serum group,the number of osteoclasts differentiation and the activity of TRAP were decreased and the area of bone resorption pits was significantly decreased in OC+YSJB serum group(P<0.01).Compared with OC+YSJB serum group,the number of OC and the area of bone resorption were significantly decreased in OC+Tregs+YSJB serum group(P<0.05),suggesting that Tregs have a role in inhibiting OC differentiation.Compared with OC+Tregs+CIA model serum group,the OC differentiation of OC+Tregs+YSJB serum group was significantly reduced,and the area of bone resorption pits aea was small(P<0.05),suggesting that in Tregs-mediated co-culture environment,Yishen Juanbi pills strengthen the inhibition of OC differentiation.5 ConclusionCIA rat arthritis index increased,joint pathological damage,bone microstructure damage,bone metabolism disorders,Treg in PLNLs decreased,Thl and Th17 cells increased,Treg can inhibit OC differentiation and bone resorption function in co-culture environment.Yishen Juanbi Pills can reduce the index of arthritis in CIA rats,reduce the pathological damage of joints and improve the microstructure of bone.The possible mechanism may be through the increase of Treg cell ratio and the promotion of Treg inhibition of OC differentiation and bone resorption.