IL-35 Inhibits The Expression Of VEGF And Its Receptor In Osteoclasts Through Th17/IL-17 Related Pathway

Objective:It is reported that interleukin(IL)-35 is an immunosuppressive cytokine secreted by regulatory T cells(Treg),which can inhibit the proliferation of T helper(Th)-17 cells,and Th17 cells are involved in the pathogenesis of a variety of autoimmune diseases.Angiogenesis and bone erosion occur in the early stage of rheumatoid arthritis(RA)and throughout the course of the disease,and uncontrolled expression of vascular endothelial growth factor(VEGF)in bone can lead to increased bone resorption of osteoclasts,thus accelerating bone destruction.Recent studies have shown that Th17 cells can directly stimulate angiogenesis by producing IL-17,or indirectly stimulate angiogenesis by inducing RA macrophages and fibroblasts to secrete angiogenic factors.The aim of this study was to investigate the effects of IL-35 on osteoclast(OC)activity,apoptosis and the expression of VEGF and its receptors fms-like tyrosine kinase(Flt-1)and fetal liver kinase(Flk-1).Methods:In this study,mouse monocyte macrophage leukemia cell line RAW264.7 was used as OC precursor,which was induced to OC by receptor activator of nuclear factor kappa-B ligand(RANKL)and macrophage colony stimulating factor(M-CSF).The OCs were first stimulated with tumor necrosis factor(TNF)-? 20 ng / ml for 2 hours,then cultured with different concentrations of IL-35(0,25,50,100 ng / ml)for 48 hours.The activity and apoptosis of OCs were measured by cell counting kit(CCK)-8 and flow cytometry method(FCM)respectively.The expression of VEGF protein in the supernatant of OCs was measured by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression of VEGF,Flt-1 and Flk-1 were detected by real-time quantitative polymerase chain reaction(RT-PCR)and western blot respectively.Plumbagin was used as an inhibitor of Th17/IL-17 related pathway.Mature OCs were induced and inoculated into the six pore plate of cell culture.After the cells adhered to the wall,plumbagin 1uM was added for 24 hours to block Th17/IL-17 related pathway.After removing the blocking,TNF-? 20 ng/ml was added to stimulate for 2 hours.After removing the stimulation,the cells were cultured with different concentrations of IL-35(0,25,50,100 ng/ml)for 48 hours.After obtaining the cell samples,RT-PCR,ELISA,Western blot were used to detect the expression of VEGF,Flt-1 and Flk-1 mRNA and protein.Results:1.The OC activity of 100 ng/ml IL-35 group was lower than that of 0 ng/ml IL-35 control group,the difference was statistically significant(P<0.01),indicating that IL-35 can inhibit OC activity.2.The apoptosis rate of 25 ng/ml,50 ng/ml,100 ng/ml IL-35 in experimental group was higher than that of 0ng/ml IL-35 group(P<0.01),indicating that IL-35 can promote OC apoptosis in a dose-dependent manner.3.After IL-35 treatment,the expression of VEGF and Flt-1 in OCs in experimental group was lower than that in control group(P<0.05).4.The expression of Flk-1 mRNA and protein was not detected in OCs.5.In order to explore the cellular mechanism,after blocking with plumbagin,the expression of VEGF and Flt-1 in the experimental group and the control group were the same(P >0.05).Conclusion : 1.IL-35 can inhibit OC activity and promote OC apoptosis in a dose-dependent manner in vitro.2.IL-35 can inhibit OC expression of VEGF and its receptor through Th17/IL-17 related pathway in vitro.