| Objective:Rheumatoid arthritis(RA)is one of the most common rheumatoid immune diseases in clinic.At present,the prevalence of RA in China is 0.32%-0.36%,and that in the world is 0.5%-1.0%,which is increasing year by year.The high incidence and disability rate of rheumatoid arthritis seriously affect the quality of life of patients.Current studies suggest that Th17/Treg cell imbalance plays a key role in rheumatoid arthritis,and regulating the imbalance of Th17 cells and Treg cell subsets is a core target for the treatment of RA.Interleukin-38(IL-38)is a new member of the IL-1 family,which inhibits the inflammatory immune process.Some studies have found that IL-38can play an important role in a variety of autoimmune diseases.In this study,we established a rat model of collagen-induced arthritis(CIA)and observed that IL-38inhibits inflammatory response of collagen-induced arthritis rats through SIRT1/HIF-1αsignaling pathway,which provides a new theoretical basis for the treatment of rheumatoid arthritis.Methods:1.Thirty SD rats were randomly divided into control group(CON group),CIA model group(CIA group),CIA+IL-38(5ng/g)group(IL-38 group)with 10 rats in each group.CIA rat model was established by subcutaneous injection of bovine collagen II emulsion into rat tail root.The hind foot condition of rats was continuously observed,and the arthritis symptom score was evaluated by arthritis index integral method,and the average arthritis score of each group was calculated.After administration,the pathological changes of synovial tissue were observed by HE staining,and the expression of related factors was observed by immunohistochemistry and immunofluorescence;the expression of inflammatory factors,cytokines and cell differentiation-related factors in serum was detected by ELISA;Treg/Th17 cells were detected by flow cytometry;The expression of Th17/Treg related genes was measured.2.30 SD rats were randomly divided into CIA model group(CIA group),CIA+IL-38(5ng/g)group(IL-38 group),CIA+IL-38(5ng/g)+SIRT1 inhibitor(EX527 1ug/kg)group(SIRT1i group),10 rats in each group;CIA rat model was established by subcutaneous injection of bovine collagen type II emulsion into the tail root of rats;arthritis score of rats in each group was observed by HE staining;synovial cells were observed by HE staining.Pathological changes;ELISA detection of inflammatory factors,cytokines and cell differentiation related factors in serum;flow cytometry detection of Treg/Th17;q RT-PCR detection of Th17/Treg related gene expression;immunohistochemistry and Western Blot detection of SIRT1/HIF-1αsignal pathway related proteins.Results:1.1)Arthritis index score:The arthritis index score of each model group was significantly higher than that of the blank control group at the same time point(P<0.05).Compared with the CIA group,the arthritis index score of the IL-38 group was significantly lower(P<0.05);2)HE:The structure of the ankle joint in the control group was intact and no obvious injury was observed.In CIA group,the structure of the ankle joint was disordered with obvious proliferation of synovial cells,a large number of inflammatory cells were infiltrated in the interstitium,and pannus was formed.Some of them even eroded to the surface of cartilage and damaged the joint.Compared with CIA model group,joint injury and inflammatory cells of synovial tissue cells were significantly reduced in IL-38 group.3)Serum cytokine levels:compared with the control group,the expressions of IL-17,IL-21,IL-22,IL-6 and IL-23 in CIA group were increased,while the expressions of TGF-βand IL-10 were decreased(P<0.05);Compared with CIA group,the expressions of IL-17,IL-21,IL-22,IL-6 and IL-23 in IL-38 group were decreased,while the expressions of TGF-βand IL-10 were increased(P<0.05).4)Flow cytometry:compared with CIA group,the percentage of spleen MNCs CD4~+IL-17A~+cells in IL-38 group decreased,while the percentage of CD4~+CD25~+Foxp3~+cells increased(P<0.05);The ratio of Treg/Th17 cells in IL-38 group was increased compared with CIA group(P<0.05).5)Th17/Treg related genes:Compared with CIA group,the m RNA expression of transcription factor RORγt in Th17 cells in IL-38 group was decreased,while the m RNA expression of transcription factor Foxp3 in Treg cells was increased(P<0.05).The m RNA expression of Th17 cytokines IL-17,IL-21 and IL-22 decreased,the m RNA expression of IL-6 and IL-23,which promoted Th17 cell differentiation,decreased,the m RNA expression of Treg cytokines TGF-βand IL-10 increased(P<0.05).6)Immunofluorescence and immunohistochemical results showed that the expressions of NF-κB and AP-1 in CIA group were significantly increased compared with the control group(P<0.05),and the expressions of NF-κB and AP-1 in IL-38 group were significantly decreased compared with CIA group(P<0.05);7)Immunofluorescence results showed that the positive expression of SIRT1 was decreased and the positive expression of HIF-1αwas increased in CIA group compared with the control group(P<0.05);Compared with CIA group,the positive expression of SIRT1and HIF-1αin IL-38 group was decreased(P<0.05).2.Part II:1)Arthritis index score:the arthritis index score of rats in the IL-38 group was significantly lower than that in the CIA group,while the arthritis score of rats in SIRT1i group was higher than that in the IL-38 group(P<0.05);2)HE staining results:Compared with CIA group,the ankle joint of the IL-38 group was significantly improved,while the structure of the ankle joint of the SIRT1i group was significantly hyperplasia and disorganized,with a large number of inflammatory cells infiltrating in the interstitium,and the focal degeneration of the cartilage surface was severe,even destroying the joint.3)Serum cytokines:Compared with CIA group,the expression levels of cytokines IL-17,IL-21 and IL-22 secreted by Th17 in IL-38 group were all decreased,while the expression levels of cytokines TGF-βand IL-10 secreted by Treg cells were increased(P<0.05).Compared with the IL-38group,the expressions of cytokines IL-17,IL-21 and IL-22 secreted by Th17 were increased in SIRT1i group,while the expressions of cytokines TGF-βand IL-10 secreted by Treg cells were decreased(P<0.05).Compared with CIA group,the expression levels of cytokines IL-6 and IL-23 that promote Th17 differentiation were decreased in IL-38group(P<0.05),and the expression levels of IL-6 and IL-23 were increased in SIRT1i group compared with IL-38 group(P<0.05).4)Flow cytometry:The percentage of spleen MNCs CD4~+IL-17A~+cells was down-regulated,and the percentage of spleen MNCs CD4~+CD25~+Foxp3~+cells was up-regulated,meanwhile the ratio of Treg/Th17cells was higher in IL-38 group(vs.IL-38 group,P<0.05).The percentage of cells in the spleen MNCs CD4~+IL-17A~+cells was higher in SIRT1i group,and the percentage of CD4~+CD25~+Foxp3~+cells was decreased,meanwhile the ratio of Treg/Th17 cells was in lower(vs.IL-38 group,P>0.05).There was no significant difference in the CIA group and SIRT1i group.5)Th17/Treg-related genes:Treated with IL-38,the expression of the transcription factor RORγt,Th17 cytokines IL-17,IL-21 and IL-22,together with Th17differentiation promoting cytokines IL-6,IL-23 in the synovial tissue of rats were down-regulated,while the transcription factor Foxp3,Treg cytokines TGF-βand IL-10were up-regulated(P<0.05 vs CIA).In the SIRT1i group,the level of the transcription factor RORγt,Th17 cytokines IL-17,IL-21 and IL-22,Th17 differentiation promoting cytokines IL-6,IL-23 in the synovial tissue of rats in CIA group was increased,while transcription factor Foxp3,Treg cytokines TGF-βand IL-10 were decreased(vs.IL-38groups,P<0.05).There was no significant difference in the CIA group and SIRT1i group;6)SIRT1/HIF-1αrelated proteins:Immunofluorescence and Western Blot results showed that the expression of SIRT1 was higher and HIF-1α,Myd88,NF-κb and AP-1 was lower in IL-38 group(P<0.05 vs CIA),while the expression of HIF-1α,Myd88,NF-κb and AP-1 in SIRT1i group was significantly increased,and the expression of SIRT1 was decreased(P<0.05 vs IL-38 group).Conclusions:IL-38 can alleviate CIA joint injury,inhibit inflammation and improve Th17/Treg imbalance.Its mechanism may be related to the regulation of SIRT1/HIF-1αsignaling pathway. |
