| BackgroundRheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease,which is primarily manifested by pathological synovitis and bone erosion.The main cause of articular bone erosion is synovitis,accompanied by massive secretion of proinflammatory cytokines and receptor activator of nuclear factor-κB ligand(RANKL),which subsequently stimulates the migration of osteoclast precursor cells and the differentiation of osteoclasts in joint.Effectively anti-rheumatic therapy for synovitis is the most valid strategy for the prevention of bone erosion.But it should be noted here that OC is an essential cell type in the bone erosive process,and it is difficult to reverse the progression of bone erosion by only targetting the synovitis.In the new perspective,bone destruction has been converted from an irreversible terminal lesion into a dynamic pathological change and could develop even at the early stage of RA.Is there a mechanism that responsible for the synovitis and bone loss simultaneously? Rho A belongs to the Rho small GTPase family whose downstream effector molecule is ROCK.Rho A/ROCK performs as an indispensable role in the non-canonical Wnt/PCP pathway,and has the intimate relation with multiple signal pathways such as MAPK and NF-κB.It is believed that Wnt/PCP pathway not only participates in the process of OC differentiation,but also plays a critical role in the proliferation,inflammation and invasion of synovial in RA.Previous studies on Rho A/ROCK were mainly concentrated on the area of cancer,while only few studies in RA or inflammatory arthritis animal models were reported.Here,this study firstly explored the effect and mechanism of Rho A/ROCK on the biological phenotype of RA-FLS and mouse OC differentiation in vitro,and its role on the CIA model in vivo was further demonstrated.Our study may provide a potential basis for novel therapeutic targets of ameliorating synovial inflammation and repairing bone erosion in RA.ObjectivesPart I: To clarify the expressions of Rho A and ROCK2 in RA synovial tissue and synovial cells.Part II: To clarify the effect of Rho A on RA-FLS proliferation,apoptosis,cell cycle,migration and invasion,MMPs and inflammatory factor secretion in vitro.Part III: To study the effect of Rho A on BMMC fusion and OC differentiation in vitro.Part IV: To study the effect of Rho A on joint symptoms,synovial pathology,cartilage erosion,OC formation and inflammatory factor secretion in CIA mice.Part V: To investigate the molecular mechanism of Rho A in RA-FLS and OC.MethodsPart I: 1.Knee synovial tissue of RA and trauma patients were collected,and FLSs were extracted and identified;2.The pathological changes of synovium were compared between the two groups by HE staining;3.The Rho A expression of synovial tissue was detected by IF and IHC 4.The expressions of Rho A and ROCK2 in synovium of the two groups were compared by WB;5.The expressions of Rho A and ROCK2 in the FLS were detected by IF and WB.Part II: 1.Humanized Rho A was overexpressed and knocked down by lentivirus and RA-FLSs were transfected and selected by puromycin;2.We verified the transfection effect of lentivirus using q PCR and WB;3.The cell viability of Rho A overexpressing and interfering RA-FLS was detected by CCK8 assay,and the change of cell cycle was measured by flow cytometry;4.The ratio of apoptotic cells was detected by Tunel,and apoptosis-related proteins were studied by WB;5.The migration and invasion abilities of RA-FLS were compared by wound healing test and Transwell experiments.The expression of MMPs were investigated by WB;6.The expressions of TNF-α,IL-1β,IL-17 and IL-21 m RNA were tested by q PCR.Part III: 1.The ratio of OPG/RANKL in RA-FLS after transfection was compared by q PCR;2.Mouse BMMCs were extracted and cultured;3.Rho A m RNA expression was detected by q PCR during BMMC differentiation induced by RANKL and M-CSF;4.Murine Rho A was upregulated and downregulated by lentivirus and mouse BMMCs were infected;5.The transfection effect was determined by q PCR;6.The effect of Rho A on OC differentiation was studied by TRAP staining;7.The influence of Rho A on BMMC membrane fusion course was compared by Dil cell membrane staining.Part IV: 1.A mouse CIA model was established,and the expressions of Rho A and ROCK2 in synovium of CIA mice were detected by IHC;2.The mouse knees were injected with murine Rho A lentivirus,and the expression of EGFP was detected by IHC to verify the transfection effect.The expression of Rho A in mouse joints was detected by WB;3.The effect of Rho A on the inflammatory index of CIA mouse joints was observed between different groups;4.The knees of mice were taken,and the effect of Rho A on the pathological manifestations was observed by HE staining;5.The effect of Rho A on knee cartilage erosion was observed by Saffron O-Fast green staining,and the expressions of MMP3,MMP9,MMP13 in mouse joints were compared with WB and IHC.6.The effect of Rho A on the apoptosis of knee synovium was detected by Tunel fluorescence staining;7.Micro-CT showed the effect of Rho A on subchondral bone microstructure in CIA mice;8.TRAP staining was used to observe the numbers of infiltrating OC precursor cells and OC of the interaction of synovium and bone;9.Blood was collected from the eyeball,and the effect of Rho A on serum IL-17 and IL-21 was detected by ELISA.Part V: 1.Related molecules m RNA of JAK/STAT,MAPK and NF-B pathway in RA-FLS after infected were detected by q PCR;2.The protein expressions of related molecules in RA-FLS were further verified by WB;3.The changes of pathway molecules during the OC differentiation mediated by RANKL were studied using q PCR;4.The interaction among Rho A,ROCK2,and p STAT3 was detected by CO-IP.ResultsPart I: 1.FLS was successfully extracted and identified by IF,with antigen Vimentin positive and CD68 negative;2.The HE scores of RA synovium was higher than the trauma group obviously,which was mainly evaluated according to the degree of synovial hyperplasia and inflammatory cells infiltration;3.RA synovium expressed more Rho A than the control verified by IF,IHC and WB,while the difference of ROCK2 expression was not obvious compared with the trauma patients;4.IF co-staining showed that Rho A and ROCK2 were highly expressed in RA-FLS than trauma FLS.However,WB showed that RA-FLS expressed more Rho A significantly than trauma FLS,but there was no difference in ROCK2 between two groups.Part II: 1.The suitable MOI values of Lv-Rho A,Lv-ctr,Sh-Rho A,Sh-ctr lentivirus were fined out,and the transfection efficiency of RA-FLS was about 80%;2.q PCR and WB verified that the expression of Rho A was upregulated or downregulated after infection.3.The cell activity of the Lv-Rho A group was higher than that of the Lv-ctr group,and the proportion of cells in S phase was increased significantly.As a result,the proliferation of RA-FLS was promoted;The cell activity of the Sh-Rho A group was significantly lower than that of the Sh-ctr group,and the cells were blocked in phase G0/1,thereby inhibiting the proliferation of RA-FLS;4.The apoptosis ratio in the Lv-Rho A group was significantly decreased than that of the Lv-ctr group,and the expressions of caspase-9 and-3 were significantly reduced correspondingly.The percentage of apoptosis of RA-FLS was significantly upregulated in the Sh-Rho A group,and the expressions of caspase-9 and-3 were increased significantly;5.Lv-Rho A lentivirus promoted the migration and invasion of RA-FLS,as well as the expression of MMP3 and 13;Sh-Rho A significantly inhibited the migration and invasion abilities of RA-FLS,and the expressions of MMP3 and 13 were reduced accordingly;6.Lv-Rho A promoted the secretions of IL-1β,IL-17,and IL-21 m RNA in RA-FLS.But only the expression of IL-17 m RNA was inhibited by Sh-Rho A lentivirus in RA-FLS.Part III: 1.Lv-Rho A down-regulated the ratio of OPG/RANKL in RA-FLS,while Sh-Rho A significantly up-regulated this ratio;2.BMMCs were successfully isolated,and the expression of Rho A on Day3 was significantly higher than that of Day0 during the OC differentiation,but no difference between Day3 and Day5;3.The MOI value suitable for BMMC was explored,and q PCR result showed that the expression of Rho A was adjusted accordingly;4.The result of TRAP staining showed that LV-Rho A boosted the differentiation of BMMC into OC,while Sh-Rho A inhibited the OC differentiation compared with the control group;5.Dil staining indicated that Lv-Rho A facilitated the membrane fusion of BMMC,while Sh-Rho A significantly prevented the membrane fusion induced by RANKL.Part IV: 1.The expressions of Rho A and ROCK2 in the synovium of CIA mice were significantly increased than those of normal mice;2.IHC indicated that the lentivirus carrying EGFP had been stably expressed in the knees of mice.The result of WB showed that Lv-Rho A up-regulated the expression of Rho A in mice,while Sh-Rho A down-regulated the expression of Rho A;3.The joint score of mice in the Lv-Rho A group was significantly higher than that in the Lv-ctr group on day43 after the primary immunization.And the score of the Sh-Rho A group was significantly decreased than that of the Sh-ctr group on day47.4.HE staining showed that Lv-Rho A significantly aggravated the pathological changes of knees in CIA mice,while HE score of the Sh-Rho A group was lower than that of the Sh-ctr group;5.The result of Saffron O-Fast green showed that the cartilage erosion in the Lv-Rho A group was aggravated compared with the Lv-ctr group,while the degree of cartilage erosion in the Sh-Rho A group was lighter than that of the Sh-ctr group.Besides,Lv-Rho A can significantly elevate the expression of MMP3,MMP9,MMP13 detected by WB and IHC.And Sh-Rho A repressed the MMPs expressions obviously.6.Tunel staining showed that Lv-Rho A inhibited the apoptosis of synovial cells in CIA,while Sh-Rho A significantly facilitated the apoptosis of synovial cells;7.Micro-CT indicated that the subchondral bone structure in the Lv-Rho A group was damaged obviously,and trabecular bone was sparse and bone volume fraction(BV/TV)were decreased significantly.In the Sh-Rho A group,the subchondral bone structure was intact,and the trabecular arrangement was dense and the BV/TV was significantly increased.8.TRAP staining showed that the degree of OC precursor cells and OC infiltration at synovium in the Lv-Rho A group was more severe than that of the Lv-ctr group,while the Sh-Rho A group showed a significant lightning effect of OC infiltration and differentiation.9.The levels of serum IL-17 and IL-21 in CIA were boosted by Lv-Rho A,while Sh-Rho A inhibited the secretions of IL-17 and IL-21.Part V: 1.Lv-Rho A significantly up-regulated the expressions of ROCK2,JAK2 and STAT3 m RNA in RA-FLS,while Sh-Rho A inhibited the expressions of ROCK2,JAK2 and STAT3 m RNA;2.The expressions of GTP-Rho A,ROCK2 and p STAT3 were significantly enhanced in the Lv-Rho A group detected by WB,while the expressions of GTP-Rho A,ROCK2 and p STAT3 were down-regulated by Sh-Rho A compared with Sh-ctr lentivirus;3.The result of q PCR showed that among the process of OC differentiation induced by RANKL,the expressions of ROCK2 and c-Fos m RNA on day3 were increased by murine Lv-Rho A,and NFATc1 m RNA on day3 and day5 were improved at the same time by Lv-Rho A.Compared with Sh-ctr,Sh-Rho A significantly down-regulated the expressions of ROCK2 and c-Fos on day3,and the expression of NFATc1 on day3 and day5;4.Rho A mediated signal transduction by binding to ROCK2,which could directly activate p STAT3.ConclusionThe above results showed that the expression of Rho A was significantly increased in the synovium and FLS of RA patients and CIA mice.In vitro,Rho A could promote the proliferation,invasion and secretion of inflammatory factors in RA-FLS.Rho A directly facilitated OC differentiation by promoting BMMC membrane fusion and indirectly stimulated OC formation by downregulating OPG/RANKL ratio in RA-FLS.The similar effects of Rho A were observed in vivo.Rho A can significantly aggravate synovial hyperplasia,cartilage destruction and bone erosion in CIA mice.Further mechanism studies suggested that Rho A mediated signal transduction through ROCK2,and Rho A/ROCK2 pathway can be coupled with JAK2/STAT3 pathway in RA-FLS.During the differentiation of mouse OC,Rho A increased the expression of c-Fos and NFATc1 through ROCK2 to promote the fusion of BMMC and the formation of OC. |
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