The Effect Of PSTPIP2 On Adjuvant-Induced Arthritis And The Probable Mechanisms

Rheumatoid arthritis(RA)is a chronic,auto-inflammatory and autoimmune disease characterized by synovial hyperplasia,chronic inflammation,and secretion of various pro-inflammatory cytokines.Fibroblast-like synoviocytes(FLSs)play an important role in joint damage and synovitis in RA.PSTPIP2,proline-serine-threonine-phosphatase interacting protein 2,previously shown to be predominantly expressed in macrophages,which is associated with inflammation.This study focused on the effects of PSTPIP2 on FLSs in vitro and on adjuvant-induced arthritis(AIA)in rats.We first established AIA model and then explored the role of PSTPIP2 in FLSs and AIA and its possible mechanisms.The main research contents are summarized as follows:(1)The expression of PSTPIP2 in FLSsOn day 0,0.1 ml of complete Freund’s adjuvant(CFA)was injected into the left hind paw of the rats.The rats were sacrificed on 24 th day,and the relevant specimens were collected for subsequent experiments.The expression of PSTPIP2 in synovial tissue or FLSs was detected by WB or RT-qPCR respectively.Secondly,immunohistochemistry was used to further detect the expression of PSTPIP in synovialtissue.The results showed that PSTPIP2 was highly expressed in normal synovial tissue and FLSs,and significantly decreased in synovial tissue and FLSs of AIA rat.(2)PSTPIP2 inhibits the inflammatory response of FLSsRT-qPCR,WB and ELISA techniques were used to detect the expression of TNF-α,IL-6 and IL-1β in FLSs with PSTPIP2 overexpression or silencing.The results indicated that the expression of TNF-α,IL-6 and IL-1β is decreased with PSTPIP2 overexpression in FLSs,but increased with PSTPIP2 silencing.These indicate that PSTPIP2 inhibits the inflammatory response of FLSs.(3)PSTPIP2 inhibits the proliferation of FLSsCFDA SE assay and WB were used to examine the effects on the proliferation of FLSs with PSTPIP2 overexpression or silencing.The results showed that overexpression of PSTPIP2 inhibited the proliferation of FLSs,and silencing of PSTPIP2 promoted the proliferation of FLSs.These results indicate that PSTPIP2 inhibits the proliferation of FLSs.(4)Study on the relationship between PSTPIP2 and NF-κB pathwayAs above,after overexpression or silencing of PSTPIP2 in FLSs,we examined the expression of p-P65 and p-IkB in FLSs using WB.The results showed that the expression of p-P65 and p-IkB was decreased with PSTPIP2 overexpression,but increased with PSTPIP2 silencing.(5)Overexpression of PSTPIP2 in vivo improves the severity of AIA in ratsThe AIA model(on Day 0)was first established,and then the adeno-associatedvirus(AAV)was injected into the knee joint cavity of the rat(on Day 8)to overexpress PSTPIP2 in vivo.The experiments were grouped into Normal,Model,AAV-empty,and AAV-PSTPIP2.Rats were sacrificed on day 18 and relevant specimens were collected for subsequent experiments.Rat body weight,paw swelling and arthritis index were measured at different time points.The results of WB showed that the expression of PSTPIP2 in the synovial tissue of rats was significantly increased after AAV was injected into the joint cavity,which suggested that overexpression of PSTPIP2 was successful in vivo.Immunohistochemistry showed that PSTPIP2 was highly expressed in normal FLSs,and decreased in AIA FLSs,and was higher in FLSs of AAV-PSTPIP2 group than in AAV-empty group.The body weight,paw swelling,and arthritis index of the rats were improved in AAV-PSTPIP2 group compared with AAV-empty group.These results indicate that overexpression of PSTPIP2 in FLSs in vivo improves the severity of rat AIA.(6)Overexpression of PSTPIP2 in vivo improves morphological characteristics of AIAAfter the rats were sacrificed on the day 18,the right hind paws(paws on the non-injection side)of the rats were used for HE staining,safranin green staining and toluidine blue staining.The experimental results showed that overexpression of PSTPIP2 in vivo can improve the joint morphology of AIA.(7)Overexpression of PSTPIP2 in vivo reduces bone destruction in ratsAs above,the right hind paws of the rat were used for X-ray and micro-CT analysis.The experimental results showed that overexpression of PSTPIP2 in vivo reduces the bone destruction of knee.(8)Overexpression of PSTPIP2 in vivo reduces the release of inflammatory cytokines in ratsThe expressions of TNF-α,IL-6,IL-1β and IL-17 in serum or synovial tissues of rats were detected by ELISA and RT-qPCR respectively.The results indicated that overexpression of PSTPIP2 in vivo reduces the release of inflammatory cytokines.(9)Overexpression of PSTPIP2 in vivo reduces the number of osteoclasts and the ratio of RANKL:OPGTo further investigate the relationship between PSTPIP2 overexpression and AIA,we examined the number of osteoclasts and the expression of RANKL and OPG in rat.The results showed that overexpression of PSTPIP2 in vivo reduces the number of osteoclasts and decreases the ratio of RANKL:OPG.