| BackgroundRheumatoid arthritis (RA), the most frequent autoimmune diseases, is characterized by chronic joint inflammation. However, the precise mechanism of RA remains unclear.Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, not only inhibits nitric oxide production but also is a proinflammatory mediator. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for ADMA metabolism. Elevated ADMA was reported in RA patients. A positive correlation between plasma ADMA levels and anticitrulinated protein antibodies, highly specific for RA, has been observed in RA patients. These results suggest that DDAH/ADMA may be involved in the pathogenesis of RA.Cortistatin (CST), a member of somatostatin neuropeptide family, is widely distributed in central nervous system, peripheral organs and immune organs. It was reported that CST treatment significantly reduced the severity of established collagen-induced arthritis (CIA) and abrogated joint swelling and destruction of cartilage and bone. The aim of this study was to investigate the potential role of DDAH/ADMA on inflammation response of collagen-induced arthritis, and whether the effects of DDAH/ ADMA is mediated by CST.MaterialsCollagen-induced arthritic model was evaluated by arthritis score, arthritis circumference and histological analysis. The plasma, hindpaw and knew joint synovium were collected for Enzyme-linked immunosorbent assay (ELISA), histological observation, immunohistochemistry and Real-time PCR. Fibroblast-like synoviocytes (FLS) were isolated from knee of rats and cultured. The levels of ADMA in plasma and medium were measured by high-performance liquid chromatography. The levels of TNF-a, IL-1β and IL-6in plasma and medium were measured by ELISA. The expression of TNF-a, IL-1β, IL-6, DDHA-1, DDAH-2and CST mRNA was analyzed by Real-time PCR. Protein expression of DDAH-2and CST in CIA was detected by immunohistochemistry. Protein expression of DDAH-1and DDAH-2in FLS was detected by Western blotting. Protein expression of CST in FLS was detected by immunofluorescence. To study the regulatory effect of DDAH-2on CST, cells were transfected by PEGFP-N1-DDAH-2or PEGFP-N1-CST.ResultsIn CIA rats, alteralation the plasma levels of ADMA was not showed, while the expression of DDAH-2was markedly reduced in inflamed joint synovium. Plasma levels of inflammatory cytokines TNF-a, IL-1β and IL-6were markedly increased. The expression of anti-inflammatory factor CST was markedly decreased in joint synovium of CIA rats. Treatment of cultured FLS with TNF-a significantly increased the levels of ADMA, and decreased the expression of DDAH-2accompany with an increase in the levels of IL-1β and IL-6and a reduction in the expression of CST. Treatment of FLS with ADMA also significantly increased the levels of IL-1β and IL-6, and reduced the expression of CST. Overexpression of DDAH-2markedly inhibited the increased levels of IL-1β and IL-6and the reduction of CST induced by TNF-a. Overexpression of CST markedly inhibited the increased levels of IL-1β and IL-6induced by TNF-a, but had no effect on the levels of ADMA and DDAH-2.ConclusionDDAH/ADMA participates in the pathogenesis of collagen-induced arthritis; the isoform of DDAH mediated in inflammatory response is DDAH-2not DDAH-1; the effect of DDAH/ADMA is mediated by CST. BackgroundResveratrol, a natural phytoalexin, possesses a wide range of pharmacological activities including cardiovascular protection, cancer prevention and antioxidant properties. A recent study has shown that resveratrol treatment significantly reduced the severity of established CIA and abrogated joint swelling and destruction of cartilage and bone. However, the mechanism of pharmacologic effect of resveratrol is still unknown.(E)-3,5,4’-trimethoxyilbene (BTM-0512) is a methylated derivative of resveratrol. Its stability, lipid solubility and bioavailability were better than those of resveratrol. BTM-0512treatment significantly reduced the severity of surgery-induced osteoarthritis, but its mechanism remains unknown.Based on the fact that the first part of our study proved the pathological process of collagen-induced arthritis was involved in DDAH/ADMA/CST pathway, and resveratrol and BTM-012could upregulate the expression of DDAH and reduce ADMA levels, we in the present study investigated the potential role of BTM-0512on rheumatoid arthritis in CIA rats and cultured FLS. MaterialsCollagen-induced arthritic model was evaluated by arthritis score, arthritis circumference and histological analysis. Animals were randomly assigned seven groups:control rat; CIA rat; CIA rats treated with sodium carboxymethylcellulose; CIA rats treated with BTM-0512(20mg/kg.d); CIA rats treated with BTM-0512(60mg/kg.d); CIA rats reated with resveratrol (60mg/kg.d) and CIA rats treated with leflunomide (6mg/kg.d). The plasma, hindpaw and knew joint synovium were collected for Enzyme-linked immunosorbent assay (ELISA), histological observation, immunohistochemistry and Real-time PCR. Fibroblast-like synoviocytes (FLS) were isolated from knee of rats and cultured. The levels of ADMA in plasma and medium were measured by high-performance liquid chromatography. The levels of TNF-a, IL-1β and IL-6in plasma and medium were measured by ELISA. The expression of TNF-a, IL-1β, IL-6, DDHA-1, DDAH-2and CST mRNA was analyzed by Real-time PCR. Protein expression of DDAH-2and CST in CIA was detected by immunohistochemistry. Protein expression of DDAH-1and DDAH-2in FLS was detected by Western blotting. Protein expression of CST in FLS was detected by immunofluorescence. To study the role of BTM-0512, cells were transfected by DDAH-2siRNA or CST siRNA. ResultsThe plasma levels of ADMA were not increased, while the expression of DDAH-2was markedly reduced in inflamed joint synovium of CIA rats. In CIA rats, the plasma levels of inflammatory cytokines TNF-a, IL-1β and IL-6were markedly increased. Moreover, the expression of anti-inflammatory factor CST was markedly decreased in joint synovium of CIA rats. BTM-0512significantly reduced inflammatory cells infiltration, synovial hyperplasia, cartilage destruction and bone destruction, and reduced the levels of TNF-a, IL-1β and IL-6. BTM-0512also markedly inhibited the reduction of DDAH-2and CST expression. Treatment of cultured FLS with TNF-a significantly increased the levels of ADMA, and decreased the expression of DDAH-2accompany with an increase in the levels of IL-1β and IL-6and a reduction in the expression of CST, while the effects of TNF-a were abolished by BTM-0512. Treatment of FLS with ADMA also significantly increased the levels of IL-1β and IL-6, and reduced the expression of CST, while the effects of TNF-a were abolished by BTM-0512. DDAH-2siRNA markedly inhibited the effects of BTM-0512showed by reduction of levels of IL-1β and IL-6and upregulation of the expression of CST in cultured FLS pretreated with TNF-a. Moreover, CST siRNA also markedly inhibited the effect of BTM-0512on inflammatory cytokines in cultured FLS pretreated with TNF-a, but had no effect on the levels of ADMA and DDAH-2.ConclusionBTM-0512had anti-arthritic effect in CIA rats, and the effect of BTM-0512may be mediated by DDAH/ADMA/CST pathway. |
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