Study On The Effect Of Tofacitinib Combined With Iguratimod In Rheumatoid Arthritis Model

Background and PurposeRheumatoid arthritis(RA)is a chronic systemic autoimmune disease with a poor prognosis,mainly causing the degeneration of joints with a variety of complications such as osteoporosis,interstitial lung disease and cardiovascular disease.The main pathological features of RA are abnormal proliferation of the synovial cells,pannus formation,and mononuclear cell infiltration,which trigger each other and constantly aggravate bone erosion and joint destruction in a vicious cycle.The core of the lesion is uncontrolled proliferation of fibroblast-like synoviocytes(FLS).At present,specific immune-targeted therapeutics including biologics and kinase inhibitors have made significant progress in RA.However,the suboptimal response to targeted therapies in some refractory RA patients suggests that we should search for new pathogenic mechanisms and therapeutic targets.Pyroptosis is a form of programmed cell death,which can trigger the release of inflammatory cytokines via NLRP3/caspase-1 pathway,thereby interacting with the inflammatory response in RA and exacerbating the condition.Reversing the deregulation of pyroptosis may be a new treatment strategy for active RA.TNF-α is a proinflammatory cytokine that plays a fundamental role in the pathogenesis of RA,which could induce pyroptosis in mononuclear macrophages and cause the destruction of bone and cartilage.Iguratimod a small molecular drug,has been confirmed as a highly efficacious and safe conventional synthetic disease-modifying anti-rheumatic drug for RA in Asia due to its potent anti-inflammation effect.Therefore,we sought to explore the therapeutic effect of Iguratimod and Tofacitinib on arthritis and secondary osteoporosis in the collageninduced arthritis(CIA)+TNF model,as well as to explore the effects of Iguratimod and Tofacitinib on pyroptosis in synovial tissue and RA-FLS.Methods1.Effect of Tofacitinib combined with Iguratimod on synovitis and secondary osteoporosis in CIA+TNF rats(1)A total of 40 Sprague Dawley(SD)male rats were randomly allocated into five groups:the normal control group(Control),the CIA+TNF model group(CIA),the TOF treatment group(TOF),the IGU treatment group(IGU),and the TOF+IGU combination treatment group(TOF+IGU).(2)The arthritis score and the degree of ankle joint swelling of hind paws was recorded every 3 days.(3)ELISA analysis was performed to detect levels of interleukin(IL)-18,IL-1β,and IL-6 in the plasma.(4)HE staining was used to evaluate the pathological changes in the ankle and knee joints.(5)The trabecular bone changes in distal femoral metaphyses and the destruction of the knee joint were analyzed by Micro-computed tomography(Micro-CT).(6)Immunohistochemistry(IHC)and tartrate resistant acid phosphatase(TRAP)staining were used to evaluate the changes of osteoblast and osteoclast in bone tissue.2.Effect of Tofacitinib combined with Iguratimod on pyroptosis in synovial tissue and RA-FLS.(1)In vitro,RA-FLS was isolated,cultured and allocated into five groups:the normal control group(Control),the TNF-α(TNF-α),the Tofacitinib treatment group(TOF),the Iguratimod treatment group(IGU),and the Tofacitinib+Iguratimod combination treatment group(TOF+IGU).(2)The effects of Tofacitinib and Iguratimod on TNF-α-induced pyroptosis on RA-FLS were detected by quantitative reverse-transcription polymerase chain reaction(qRT-PCR)and Western blotting.(3)ELISA analysis was performed to detect levels of inflammatory cytokines and pyroptosis-related cytokines in the cell supernatants.(4)IHC and Immunofluorescence(IF)were used to evaluate the level of pyroptosis-related proteins in synovial tissues.Results1.Tofacitinib synergized with Iguratimod to alleviate synovitis and reduce bone erosion(1)The degree of joint swelling and the level of interleukin(IL)-18,IL-1β,and IL-6 in the plasm were significantly increased in the CIA+TNF model.After 6 weeks of single or combination treatment with TOF or IGU,arthritis score,and plasma inflammatory cytokines were significantly reduced,and the reduction in the combination group was more significant than in the monotherapy group.(2)HE staining showed that obvious synovial hyperplasia,pannus,and a large amount of inflammatory cell infiltration were observed in the synovial tissue of the CIA+TNF group.The number of trabecular bones was reduced and the cartilage growth plate was severely damaged in the knee joints.The combination group significantly reduced pannus and inflammatory cell infiltration,as well as increased trabecular density and resulted in good repair of the growth plate.(3)Micro-CT revealed that the CIA+TNF group displayed severe bone erosion,including decreased trabecular bone number and thickness,and increased trabecular spacing.The combination therapy efficiently increased bone density and reduced bone loss.(4)IHC and TRAP staining showed that both the osteoblast bone formation and osteoclast bone absorption were sharply ruined in the CIA+TNF model.Bone destruction was significantly alleviated and bone turnover rate was remarkably increased in the combination group.2.Tofacitinib synergized with Iguratimod to inhibit pyroptosis in synovial tissue and RAFLS(1)In vitro,the mRNA and protein expression of pyroptosis-related molecules(NLRP3、IL-1β and GSDMD)were higher in the TNF-α group,and both Tofacitinib and Iguratimod played an opposite role by downregulating the expression of these cytokines.(2)The levels of IL-18,IL-1β and IL-6 in cell culture supernatants were significantly higher in the TNF-α group.The combination of Tofacitinib and Iguratimod showed markedly downregulated levels of these cytokines.(3)IHC and IF staining revealed substantially higher NLRP3,GSDMD,IL-1β and Caspase-1 expression in CIA+TNF rats,and NLRP3 was abundantly expressed in FLS.Treatment with Tofacitinib and Iguratimod had a stronger inhibitory effect on these proteins than monotherapy group.Conclusion1.The combination of Tofacitinib and Iguratimod effectively relieved synovitis.2.The combination of Tofacitinib and Iguratimod had a good protective effect against osteoporosis.3.The combination of Tofacitinib and Iguratimod significantly suppressed the NLRP3/GSDMD-mediated pyroptosis on RA-FLS.