The Effects And Mechanism Of Cholinergic Anti-inflammatory Pathway On Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a systemic inflammatory autoimmune disease,with high rate of disability.Although the pathogenesis and therapy strategy of RA have been studied extensively for many years,but its etiopathogenisis is not clear and the therapeutic outcome is not satisfied.About half of RA patients manifest with joint destruction in two years.It is one of major diseases which cause loss of labor force and disability in China.Proinflmmatory cytokines net have play an important role on the synonitis and joint destruction of RA,and it is the key factor of leading to the lesions of RA continued existence and persistent progress.Tumor necrosis factor alpha(TNF-α)and interleukin (IL)-1 is a central component in the cascade of cytokines,stimulating the production of additional inflammatory mediators and the further recruitment of immune and inflammatory cells into the joint.The resent study found:there is a pathway of control inflammation between the nervous system and immune system.The pathway is composed of the vagus nerve,acetylcholine and acetylcholine receptor,so it was called cholinergic anti-inflammatory pathway.Resent many studies indicate that cholinergic anti-inflammatory pathway plays an important role in acute inflammation such as endotoxemia,sepsis and trauma.In these studies,it can inhibit the production of pro-inflammatory cytokines such as TNF-α, IL-1 and high mobility group box chromosomal protein 1(HMGB1) through the inhibition of NF-κB pathway However,it is unknown whether it involves in chronic inflammations such as RA which is characterized by chronic swelling and inflammation of the synovial membrane that lines the joints.So the effects of nicotine by intraperitoneal injection on joint swollen,inflammation of the synovial membrane,joint destruction and inflammatory cytokines in serum and joint tissue were investigated in adjuvant induced arthritis rats in our study.The fibroblast-like synoviocytes were isolated from synovium of RA patients and stimulated by TNF-αin order to investigate the role of cholinergic agonists and receptors in cholinergic anti-inflammatory pathway.These studies may illuminate the effects of cholinergic anti-inflammatory pathway on rheumatoid arthritis and its molecular mechanism,which can offer a new idea and strategy for the treatment of RA and other inflammatory diseases. of the first part: The protective effects of nicotine on rat adjuvant induced arthritis and the role on the pro-inflammatory cytokines.Objective and design:Nicotine is an important part of cholinergic anti-inflammatory pathway and it inhibits tumor necrosis factor alpha (TNF-α)and interleukin-1 beta(IL-1β)production in inflammatory cells. In this study we investigated the effects of nicotine on paw inflammation, joint destruction and the role on the pro-inflammation cytokines such as TNF-α,IL-1βand high mobility group protein 1(HMGB1)protein in adjuvant -induced arthritis(AA)in rats.Methods:Adjuvant -induced arthritis was induced by immunization of male SD rats with complete Freud's adjuvant.Sixty four rats randomly divided into four groups:Normal control group,AA group,treated with vehicle(CIA-V),low dose of nicotine group,AA rats treated with 0.05mg/kg nicotine,twice a day.High dose of group,AA rats treated with 0.2mg/kg nicotine,twice a day.Nicotine and its placebo formulation was intraperitoneal injection administered to rats for 36 days starting on the day of injection of complete Freud's adjuvant.Foot swelling was evaluating every other day.Lesions of bone and cartilage were evaluated on the basis of histological change in ankle joint,radiographic analysis in hind paw with Larsen score.Systemic TNF-αand IL-1βlevels were measured by ELISA.The expression of HMGB1 in synovium and joints was assessed by immunohistochemical analysis.The levels of HMGB1 protein in serum was determined by Western blot.Results:High dose of nicotine can prevent the onset and progression of joint swollen and reduce the rate of arthritis of AA rats.High dose of nicotine significantly suppressed paw swelling(P＜0.01).The mean radiographic scores were significantly lower in the nicotine group than in the AA rats without treatment.Histological analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after treatment of nicotine(0.4mg/kg/day).Systemic TNF-α,IL-1βand HMGB1 levels were significantly reduced after treatment of 0.4mg/kg/day nicotine.HMGB1 positive was found in many cells in bone erosion areas in CIA-V group.The level of expression of HMGB1 in cartilage and bone was higher in CIA-V group than that of control group.Nicotine(0.4mg/kg/day)significantly suppressed HMGB1 expression in the synovium and joint(p＜0.01).Conclusions:These results show that nicotine is effective in suppressing inflammation,and damage to bone and cartilage in AA rats, and may be useful in the treatment of rheumatoid arthritis.Systemic TNF-α,IL-1βand HMGB1 was reduced and fewer synovial HMGB1 expression was detected after nicotine treatment.Nicotine maybe suppresses the inflammation and joint destruction through the inhibition of the production and expression of TNF-α,IL-1βand the inhibition on the expression and the release of HMGB1.Our data strongly indicate that nicotine could provide therapeutic strategy for RA.The second part: The expression ofα7 nicotinic acetyicholine receptor on fibroblast-like synoviocytes and the role of nicotine on the inflammation of fibroblast-like synoviocytesObjectives:To investigate the role of cholinergic anti-inflammatory pathway the inflammation of fibroblast-like synoviocytes induced by TNF-α.Methods:Primary RA fibroblast-like synoviocytes(FLS)was cultured,and the mRNA expression ofα7 nicotinic acetylcholine receptor (α7nAChR)on FLS was detected by reverse transcription chain reaction(RT-PCR),the protein expression ofα7nAChR on FLS was examined by cell indirect immunofluorescence and Western blot.The inflammation of FLS was induced by tumor necrosis factor alpha (TNF-α).The effects of nicotine on interleukin(IL)-6 and IL-8 were detected by ELISA and RT-PCR.The effects nicotine on NF-κB pathway of FLS induced by TNF-αwas assessed by Cell indirect immunofluorescence and Western blot,the effects of nicotine on MAPK pathway of FLS induced by TNF-αwas evaluated by Western blot.Results:The mRNA expression ofα7nAChR on FLS was confirmed by RT-PCR.An antibody specific for theα7nAChR subunit recognized a clear band with an apparent relative molecular mass of 55KD by Western blot and clustered on the surface of FLS by Cell indirect immunofluorescence.Low dose of nicotine(10^-7-10^-4)mol/l has no damage to FLS by MTT anaysis and lactate dehydrogenase(LDH) release.Nicotine can reduced the production and the mRNA expression of IL-6 and IL-8 induced by TNF-α.Nicotinic stimulation can inhibit NF-κB(p65)transferred from cytoplasm to nuclear.Nicotine has no effects on the phosphorylation of p38.Conclusion:Theα7 nicotinic acetylcholine receptor is expressed in RA,and nicotine can inhibit the inflammatory reaction by TNF-αinduced on synoviocytes.Nicotine maybe inhibit inflammatory through the prevention of activation of the NF-kappa B pathway.