Biological Inhibition Of SPRY2’s On Rheumatoid Atrhritis

Background: Rheumatoid arthritis (RA) is a chronic systemic autoimmunedisease mainly affecting the joints, characterized by abnormal proliferation offibroblast-like synovial cells (FLS) and infiltration of inflammatory cells leading tothe destruction of cartilage and bone, even causing joint damage and even loss offunction. Acting as the final target of RA pathological course, FLS plays an importantrole in RA development process. FLS have characteristics of abnormal proliferationlike tumor cells. Likewise, growth behavior of synovial tissue based on FLS is similarto tumor growth locally. Therefore, it is suggested that inhibition of tumor cellproliferation can be used in the treatment of RA.SPRY2can antagonize tyrosine kinase receptor (receptor tyrosine kinase, RTK)signal. The research found that the expression of SPRY2was down-regulated in sometumor cells. Overexpressing SPRY2molecules in tumor cells, proliferation of tumorcell can be effectively suppressed. SPRY2suppressed proliferation of rumor cell bydown-regulating the activities of RAS/ERK and PI3K/AKT signal pathway. This twopaths are just the main pathways which promote inflammatory response andproliferation of FLS. Therefore, it suggests that SPRY2may impact development ofRA by inhibiting proliferation of FLS.Purpose：To study the influence of SPRY2on abnormal proliferation of RA FLS.And inhibition SPRY2rheumatoid arthritis rat model validation in RA. Exploring theSPRY2’s inhibit function to RA in RA rat model.Methods：1.SPRY2gene cloning and activity identification: Using PCR to amplify humanSPRY2gene, and cloned it into pDC315vector, constructing the recombinant plasmidpDC315/hSPRY2. Then pDC315/hSPRY2was transfected to293T, and CCK-8wasused to detect the proliferation and survival of293T. 2.Construction and identification of recombinant adenovirus AdSPRY2:pDC315/hSPRY2and pBHGlox(delta)E1,3Cre was co-transfected to HEK293, topackage the recombinant adenovirus AdSPRY2. The changes of SPRY2expressionlevel under stimulation with different concentrations of TNF-α were tested byRT-PCR, and the regulatory effect of SPRY2on TNF-α cytotoxicity was observedthrough SPRY2gene overexpression mediated by recombinant adenovirus..3.In vitro, SPRY2inhibition of proliferation and inflammatory response in RAFLS: We use TNF-α to stimulate FLS, observing the gene expression changes ofSPRY2. FLS cells were infected with AdSPRY2, then the regulatory effect of SPRY2on TNF-α-induced cell proliferation was test by Brdu.4. Influence of AdSPRY2on adjuvant-induced arthritis in rat: AdSPRY2wasinjected to articular of rat. Then ankle thickness, articular index score and bodyweight of rats were obersved every3days. The rats were executed at the end time ofobservation, then tissue section obtained from ankle joints and stained with H&E. Finally,we use ELISA to detect secretion level of pro-inflammatory cytokines TNF-α and IL-1β.Results:1.Recombinant plasmid pDC315/hSPRY2was successfully constructed: Doubledigestion and sequencing verified that pDC315/hSPRY2was constructed successfully.RT-PCR and Western blot analysis confirmed that SPRY2can be expressed inHEK293T transfected with pDC315/hSPRY2.; CCK-8cell proliferation assayconfirmed that SPRY2can promote proliferation and survival of HEK293T cell.2.AdSPRY2was packaged successfully and having immunological activity: PCRand sequencing confirmed that AdSPRY2was constructed successfully; RT-PCR andWestern blot showed that SPRY2was expressed naturally in L929cells, and heexpression level of SPRY2correlated with concentrations of TNF-α positively.Enhanced sensitivity of L929to TNF-α by SPRY2was obersved via CCK-8. 3.Overexpression of SPRY2inhibits the abnormal proliferation of FLS: RT-PCRand Western blot find natural expression of SPRY2molecules in FLS cells. In FLSgene expression levels of SPRY2decreased with increasing concentration of TNF-α.After infecting FLS cells with AdSPRY2, we found that abnormal proliferation of FLSwas inhibited in the experimental group compared with control significantly (p<0.05).4.AdSPRY2can inhibit rat adjuvant-induced arthritis effectively: clinicalindicators showed that, compared with control ankle thickness and articular indexscore of rat in treatment group was significantly lower (p <0.05); tissue sectionsstained with H&E showed that intra-articular infiltration of treatment group wassignificantly less than the control group; ELISA assay confirmed that IL-1βexpression in treatment group was significantly lower than the control group(p <0.05).Conclusion: Overexpression of SPRY2molecules in vitro can inhibit theabnormal proliferation of human FLS. SPRY2molecules can effectively inhibit thedevelopment of rheumatoid arthritis in rats.