The Function Of Interleukin-35 In Osteoclasts Differentiation And Bone Destruction In Collagen-induced Arthritis

Objective: rheumatoid arthritis(RA)is a chronic systemic inflammatory disease with unknown etiology.Abnormal synovial hyperplasia at the affected site,chronic synovila inflammation,irreversible erosion and destruction of cartilage and bone tissue are the key features of RA.70% of patients with RA will have varying degrees of osteoporosis and bone destruction within 2 years of onset.In severe cases,pathological fractures or physical disabilities may occur,which seriously affects the quality of life.A large number of studies have shown that the abnormal activation of osteoclast(OC)function under the participation of various factors is the initiating factor of RA bone destruction.OC is derived from the bone marrow mononuclear macrophage system,and the unique ability of OC is dissolving bone tissues.It migrated to the surface of bone,invaded and absorbed bone,and became the “culprit” of osteoporosis and bone destruction in RA patients.Therefore,the relevant research on the differentiation and function of OC is the key to prevent bone destruction in RA.Interleukin(IL)– 35 plays an important role in several autoimmune diseases.Recent studies have shown that IL-35 plays a negative regulation role in the pathological process of RA.Our research group found that IL-35 participated in the regulation of bone metabolism by down-regulating receptor activator of nuclear factor--κB ligand(RANKL)and up-regulating osteoprotegerin(OPG).At the same time,IL-35 can also inhibit the proliferation and promote its apoptosis of mature OC.It had also been reported in the literature that IL-35 inhibited the formation of bone lacuna caused by mature OC through the NF-κB pathway.However,the current researches on the role of IL-35 in the formation and differentiation of OC were inadequate.Therefore,in this study,we induced RAW264.7 cell line to generate OC in vitro and establish the collagen-induced arthritis(CIA)model to explore the effect of IL-35 on OC differentiation and bone destruction in CIA model,so as to provide a basis for the prevention and treatment of clinical RA complicated with bone destruction.Methods:1.We selected mouse monocyte macrophage leukemia cell line RAW264 7 as OC precursor cells,added different concentrations of IL-35(0,25,50,100,150 and 200ng/ml)and RANKL and macrophage colony stimulating factor(M-CSF)to induce mature OC.(1)Cell counting kit-8(CCK-8)assay detected the influence of different concentrations of IL-35 on cell activity of RAW264.7.(2)Trap staining evaluated the effect on differentiation from RAW264.7 into OC with different concentrations of IL-35 induced by RANKL and M-CSF.(3)Transwell chamber method tested the affects on migration and invasion of OC with different concentrations of IL-35.(4)Real time-PCR and Western blot tested the expression of marked factors of OC treated with different concentrations of IL-35.2.After added fludarabine,a STAT1 specific inhibitor,we detected the effect on signal pathways of OC differentiation managed by IL-35,and explored the possible mechanism of IL-35 affecting OC differentiation and function.3.CIA models were induced by DBA/1J mice.The experimental group and the model group were injected intraperitoneally with IL-35 and phosphate buffer saline(PBS)respectively.The articular bone tissues were stained with TRAP and hematoxylin eosin(HE),micro computed tomography(Micro-CT)was also used for observation the role of IL-35 in CIA bone destruction.Results: 1.(1)The results of CCK-8 showed that IL-35 was effective on RAW264.7cells did not significantly promote or inhibit proliferation,indicating that IL-35 had no effect on cell activity of RAW264.7.(2)TRAP staining results suggested that RANKL and M-CSF induced RAW264.7 cells into mature OC.Compared with the control group(i.e.IL-35 of 0ng/ml),IL-35 in different concentrations inhibited production of OC in a manner concentration dependence(P < 0.05),and there was almost no TRAP positive cell in the 200ng/ml group.(3)The results of Transwell chamber showed that IL-35 could reduce the number of transmembrane cells migrated and invaded by OC,indicating that IL-35 could inhibit the migration and invasion of OC.(4)Results of RT-PCR and Western blot suggested that IL-35 could significantly reduce RANK、TRACP、Cathepsin K,C-fos and NFATc1(P < 0.05).2.(1)TRAP staining showed that the number of trap positive cells in the fludarabine group was higher than that in the IL-35 group(P < 0.05),but less than control group.(2)RT-PCR and Western blot suggested that IL-35 could promote the phosphorylation of STAT1,inhibit PI3K/Akt signaling pathway and downstream transcription factors C-fos and NFATc1,which would be weakened by fludarabine(P < 0.05).3.(1)The weight of mice in CIA +IL-35 and CIA groups obviously decreased when compared with the control group,and the weight of CIA group was lower than that of CIA + IL-35 group as a whole;The degree of joint swelling and arthritis index in CIA +IL-35 group were significantly lower than those in CIA group.(2)TRAP staining of joint bone tissues showed that IL-35 significantly reduced the number of TRAP positive cells round joint synovium and bone tissues.(3)HE staining demonstrated IL-35 significantly reduced synovial hyperplasia,infiltration and aggregation of inflammatory cells and destruction of bone tissue.(4)Micro-CT showed that IL-35 up-regulated bone volume fraction(BV/TV),bone mineral density(BMD)and reduced joint bone damage in CIA mice.Conclusion: 1.(1)IL-35 had no obvious cytotoxicity on RAW264.7 cell lines;(2)IL-35 reduced the differentiation of mature OC;(3)IL-35 inhibited the migration and invasion of OC;(4)IL-35 reduced the expression of RANK,TRACP,Cathepsin K,C-fos and NFATc1 in OC.2.IL-35 inhibited differentiation of OC induced by RANKL and M-CSF by activating STAT1 pathway,inhibiting the expression of PI3K/AKT signaling pathway and downstreaming transcription factors.3.IL-35 played a protective role in the destruction of CIA joint bone.