Study On Negative Feedback Mechanism Of JAK-STAT Signaling Pathway Of Regulating RA Synovial Cell Function By Moxibustion

Aim:On the basis of explicit function of anti-inflammatory of RA,the study is under the guidance of traditional Chinese acupuncture and moxibustion theory to treat experimental RA by moxibustion on "Shenshu" and "Zusanli". Around its anti-inflammatory target effect, our research concentrate on JAK-STAT signaling pathways as the main line and SOCS1、PTPN22、PIAS1as the breakthrough point to using RNAi、RT-PCR、Westernblot for revealing underling mechanism of moxibustion on negative feedback of JAK-STAT signaling pathways. The study has important scientific value for revealing the mechanism of moxibustion in the treatment of RA more comprehensive and in-depth.Methods:36Japanese white rabbits,which divided into six group--normal group,model group,moxibustion group, SOCS1RNAi group, PTPN22RNAi group and PIAS1RNAi group.Each group has6rabbits. Just injectd the Freund’s Complete Adjuvant (FCA) into the joint cavity of two hind legs of model group and all moxibustion groups by0.5ml/Kg. Using lentiviral-mediated SOCS1, PTPN22, PIAS1RNAi as means of intervention, Apply treatment of Ai tablets moxibustion on "shenshu","Zusanli" to treat experimental RA (5Zhuang/point/day,6days a course of treatment, three courses, one day rest between treatments).1.Observed the anti-inflammatory effect of moxibustion treatment on Experimental RA rabbit:the overall state, knee circumference, synovial tissue inflammation pathological;2. studies on synovial cell secretory function of experimental RAby moxibustion: Detected IL-1β, TNF-α, EGF, FGF content of synovial fluid with Elisa assay;3.Research on SOCS1, PTPN22, PIAS1expression of moxibustion on experimental RA with synovial cell in JAK-STAT signaling pathway:western blot and RT-PCR were used to detect the expression of SOCS1, PTPN22, PIAS1protein and mRNA with synovial tissue;4.Study on effect of SOCS1, PTPN22, PIAS1regulation in the JAK-STAT pathway of RA synovial cells by moxibustion:detection of JAK-STAT signaling pathway in the expression of related molecules JAK1, JAK2, STAT1, STAT3, STAT4, STAT5, STAT6with RT-PCR.Results:1. moxibustion on "shenshu","Zusanli" of experimental RA rabbit can significantly improve the general condition of RA, knee swelling, joint synovitis pathological changes, and significantly reduce its role in the case of SOCS1, PTPN22, PIAS1disturbed.2. test on cytokine levels of Synovial fluid showed that:Compared with the normal group, IL-1β, TNF-a, EGF, FGF levels of model group were significantly higher (P<0.01); compared with the model group, IL-1β, TNF-a, EGF, FGF levels of moxibustion group were significantly lower (P<0.01); compared with the moxibustion group, the content of IL-1β, TNF-a, EGF, FGF with PTPN22RNAi group, PIAS1RNAi group and SOCS1RNAi group reduced is less than the effect of moxibustion group (P<0.05).3. SOCS1, PTPN22, PIAS1expression test results of synovial tissue showed that: compared with the normal group, gene and protein expression of SOCS1、PIAS1、 PTPN22in SOCS1、PIAS1、PTPN22RNAi group were significantly reduced (P <0.05),which meaned the success of lentivirus-mediated SOCS1, PTPN22, PIAS1RNAi model; compared with the normal group, gene and protein expression of SOCS1, PTPN22, PIAS1with model group wrer decreased significantly (P<0.01); compared with the model group, gene and protein expression of SOCS1, PTPN22, PIAS1with moxibustion group significantly increased (P<0.01), which prompted moxibustion therapy can significantly improve the level of expression in synovial tissue of SOCS1, PTPN22and PI AS1.4. Analysis of signaling molecules expression in JAK-STAT signal pathway in synovial tissue showed that:(1) Compared with mormal group, gene expression of JAK1, JAK2, STAT1, STAT4in model group had significantly increased (P<0.05), gene expression of STAT3, STAT5, STAT6was significantly reduced (P<0.05).(2) Compared with model group, gene expression of JAK1, JAK2, STAT1, STAT4in model group had significantly lower (P<0.05), the gene expression of STATS, STAT5, STAT6gene were significantly increased (P<0.05).(3) compared with moxibustion group, gene expression of JAK2, STAT1,STAT4were increased significantly (P<0.01) and gene expression of STAT3was significantly decreased (P<0.05) in SOCS1RNAi group; gene expression of JAK1, STAT1, STAT4were significantly increased (P<0.05) and gene expression of STAT3was significantly decreased (P<0.05) in PTPN22RNAi group; gene expression of JAK1, STAT1were significantly increased (P O.05) and gene expression of STAT3, STAT5, STAT6were significantly decreased (P<0.05) in PIAS1RNAi group.Conclusions:1. moxibustion on "shenshu","Zusanli" can significantly improve the general condition of experimental rabbits with RA, knee swelling, joint synovitis pathological changes, which indicated that moxibustion has a clear anti-inflammatory effects, it can effectively control synovial inflammation of RA, but in the condition of SOCS1, PTPN22, PIAS1RNAi, its anti-inflammatory effect was significantly reduced, that is to say negative feedback regulation of SOCS1, PTPN22, PIAS1in JAK-STAT signaling pathway maybe closely associated with the anti-inflammatory effects of moxibustion treatment of RA.2. moxibustion on "shenshu","Zusanli" can significantly reduced the content levels of inflammatory cytokines IL-ip, TNF-a, EGF and FGF in synovial fluid of experimental RA rabbits. But in the condition of SOCS1, PTPN22, PIAS1RNAi, its anti-inflammatory effect was significantly reduced, which suggested that negative feedback regulation of SOCS1, PTPN22, PIAS1in JAK-STAT signaling pathway may be an important mechanism of inhibition of cell secretory function by moxibustion in RA synovial.3. moxibustion "shenshu","Zusanli" can enhance protein and gene expression of SOCS1, PTPN22, PIAS1in experimental rabbits of RA synovial tissue, which suggested that the negative feedback regulation of SOCS1, PTPN22, PIAS1in JAK-STAT signaling pathway maybe closely associated with the anti-inflammatory effects of moxibustion treatment of RA.4. Moxibustion "Shenshu","Zusanli" can reduced gene expression of JAK1, JAK2, STAT1, STAT4and enhance gene expression of STAT3, STAT5, STAT6in experimental RA rabbits, in the condition of SOCS1, PTPN22, PIAS1was interferenced, different signaling molecules have different expression, which were affected by different ways.It suggested that SOCS1, PTPN22, PIAS1benign regulae JAK-STAT signaling pathway may be one possible way to achieve anti-inflammatory effects of moxibustion treatment of RA...