Study On The Mechanism Of BRD4 In Rheumatoid Arthritis Bone Destruction

Objective: To investigate the effect of bromodomain-containing protein 4(BRD4)on bone damage in Rheumatoid arthritis(RA).In this study,the expression of BRD4 in articular synovial tissues of human RA and Osteoarthritis(OA)was firstly compared.Then,the role and mechanism of BRD4 in bone destruction of RA joint were discussed through RA mouse model,so as to further elucidate the pathological mechanism of bone destruction of RA and provide a theoretical basis for its treatment.Methods: The histological morphology and BRD4 expression distribution of RA and OA synovium were first detected by HE staining and immunohistochemistry.Then Western Blot was used to detect whether there were differences in the expression of BRD4 in RA and OA synovium.Collagen-induced arthritis(CIA)mice model was established,and the mice were randomly divided into 3 groups: control group(CONT),arthritis model group(CIA),and arthritis intervention group(CIA +JQ1).The expression of BRD4 in synovial tissues of mice was detected by qrt-pcr and western-blot.HE staining was used to observe the differences of bone destruction in the joints of mice,and VG staining and acid phosphatase staining(TRAP)were used to detect the differences of bone mass and osteoclast expression in the three groups of mice.Qrt-pcr was used to compare the expression differences of osteoclast-related factors in the three groups of mice.The results of the experimental data were analyzed by one-way anova or t-test,and P<0.05 was considered to be statistically significant.Results: HE staining showed that the inflammatory response of RA synovial tissue was significantly higher than that of OA synovial tissue.Immunohistochemical detection found that BRD4 was expressed in both RA and OA synovium,but the expression in RA group was significantly higher than that in OA group(P<0.05).Western-blot results showed that the expression of BRD4 in RA group was significantly higher than that in OA group(P<0.05).The results of the CIA mouse model showed that the mice in the model group and the intervention group began to develop arthritis symptoms after the second injection and the symptoms gradually worsened over time.The symptoms were relieved after the JQ1 intervention.Two mice died of intolerance.The qRT-PCR to detect the expression of BRD4 mRNA in synovial tissue in mice,the results showed that compared with the control group,model group significantly higher,expressing differences statistically significant(P<0.05).By Western Blot on the above two groups of synovial tissues BRD4 protein for testing,the results showed that compared with the control group,BRD4 protein expressed in model group increased obviously,statistically significant difference(P<0.01).The results of arthritis index score showed that the arthritis index score of mice in the intervention group was significantly lower than that in the model group(P<0.01).After JQ1 intervention,bone destruction in the intervention group was significantly decreased compared with that in the experimental group.The comparison of bone histopathological scores between the model group and the intervention group showed that JQ1 significantly reduced the bone histopathological scores of CIA mice(P<0.05).VG staining results showed that compared with the control group,bone mass production in the model group was significantly reduced,and bone mass was significantly increased after JQ1 intervention.Again through the bone volume fraction(BV/TV),bone trabecular number(Tb.N),trabecular spacing(Tb.Sp)and bone trabecular thickness(such as Tb.Th)index analysis,compare the difference of bone mass,the result shows: compared with control group,model group BV/TV decrease obviously,model group Tb.Sp increased obviously,with significant statistical difference(P<0.01): when given JQ1 after the intervention,compared with model group,BV/TV in the intervention group was obviously increased,Tb.Sp was decreased,with statistical difference(P<0.05).However,there was no significant difference between Tb.N and Tb.Th among the three groups(P>0.05).TRAP staining results showed that compared with the control group and the intervention group,there were a large number of multinucleated osteoclasts on the bone surface in the erosion area of the model group.Through morphometric analysis of ankle bone,the results showed that,compared with the control group,the osteoclast surface area(oc.s /BS),osteoclast number(n.occ /BS)and erosion surface area(ES/BS)in the model group increased significantly(P<0.05),but JQ1 inhibited such changes P<0.05).Qrt-pcr was used to detect the expression of osteoclast related genes in the three groups.Compared with the control group,the mRNA expression levels of RANKL,TRACP,Cathepsin K and the ratio of RANKL/OPG in the synovial tissues of the model group were all increased.After JQ1 intervention,their expression levels were significantly decreased compared with the model group(P<0.05).Conclusion: The expression of BRD4 in synovium of RA patients was significantly higher than that of OA patients.It was found in the CIA mouse model that BRD4 was involved in the pathogenesis of RA bone destruction by activating osteoclasts,and the inhibitor of BRD4 protein JQ1 had a protective effect on bone destruction in CIA mice.