Molecular Mechanism Of Curcumin Inhibits B Lymphocyte Stimulator Expression And The Significance Of The Inhibition In Anti-rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation targeting the synovial membrane, cartilage, and bone. Both B cells and T cells form aggregates in the synovium of inflamed joints and mediate the pathogenesis of RA. B cells play a critical role in the pathogenesis of RA. Recently, a number of biologic agents that target B cells have been tested as therapies for these conditions. Overexpression of B lymphocyte stimulator (BLyS) is closely involved in the pathogenesis and progression of RA. Curcumin is a diferuloylmethane present in Curcuma longa, an anti-inflammatory and anti-oxygen agent used in traditional medicine, has been shown to suppress cellular transformation, proliferation, and invasion. Because of its ability to modulate immune cells, curcumin has been shown to benefit on several autoimmune diseases. However, it is not clear whether curcumin could affect the expression of BLyS.Part I Curcumin inhibits the expression of BLyS on basal levelObjective: to evaluate the molecular mechanism of curcumin inhibits the basal level of BLyS expression in Namalwa and Daudi cells.Methods and Results:1. RT-PCR, Real-time PCR and Western blot analyses demonstrated that curcumin inhibits BLyS expression in a dose- and time-dependent manner in Namalwa and Daudi cells.2. We amplified BLyS promoter region from genomic DNA of Namalwa cells and constructed luciferase reporter expression plasmids (pBLyS/1082, pBLyS/571). Report assay showed that curcumin reduces the BLyS promoter activity and the DNA fragment (-1082?-571) in the BLyS promoter region might contain the binding site of transcriptional factor(s) in response to curcumin. 3. EMSA confirmed that the transcriptional factor NF-?B specifically bind to the binding site which found in the BLyS promoter region (?874 to ?858). Furthermore, curcumin reduces the DNA-binding activity of NF-?B to the BLyS promoter region in a dose- and time-dependent manner in the nuclear extracts of Namalwa and Daudi cells.4. ChIP assay indicated that curcumin reduces the p65 binding to the BLyS promoter region in Namalwa and Daudi cells.5. Immunocytochemistry and Western blot demonstrated that curcumin blocked the nuclear translocation of p65 in Namalwa and Daudi cells. Furthermore, curcumin inhibits the phosphorylation of I?B?Conclusion: Curcumin suppressed BLyS expression at both transcriptional and translational levels by reducing the NF-?B activity through inhibition of DNA binding of NF-?B, nuclear translocation of p65 and phosphorylation of I?B?.. Our findings suggest that suppression of NF-?B by curcumin is one of the mechanisms of curcumin to inhibit the BLyS expression.Part?Curcumin inhibits IFN-?-induced BLyS expressionObjective: to evaluate the molecular mechanism of curcumin inhibits IFN-?-induced BLyS expression in Namalwa and Daudi cells.Methods and Results:1. RT-PCR, Real-time PCR and Western blot analyses demonstrated that curcumin inhibits IFN-?-induced BLyS expression in a dose-dependent manner in Namalwa and Daudi cells.2. Report assay showed that curcumin reduces IFN-?-induced BLyS luciferase reporter expression plasmids activity (pBLyS/1082).3. RT-PCR, Real-time PCR and Western blot analyses demonstrated that AG 490 blocks IFN-?-induced BLyS expression at both transcriptional and translational levels in Namalwa and Daudi cells.4. Western blot analyses confirmed that curcumin inhibits the phosphorylation of STAT1 in a dose-dependent manner in Namalwa and Daudi cells. Conclusion: Curcumin suppressed BLyS expression at both transcriptional and translational levels by inhibiting the JAK2-STAT1 signal pathway. Part?Curcumin attenuates inflammatory response and suppresses BLyS expression in collagen-induced arthritisObjective: to evaluate the preventive effects of curcumin treatment on the inflammatory response and suppresses BLyS expression in a murine collagen-induced arthritis (CIA) model.Methods and Results:1. RT-PCR and Western blot analyses demonstrated that curcumin inhibits mBLyS expression in a dose-dependent manner in spleen lymphocyte.2. Arthritis was induced in male DBA/1 mice at 7–8 weeks of age. On day 1, bovine type II collagen (CII) was diluted with Freund's complete adjuvant (CFA). Mice were injected intradermally at the base of the tail with 100?g of the bovine CII/CFA emulsion. On day 21, 100?g bovine CII/IFA was injected intraperitoneally (IP).3. Mice were monitored for clinical signs of arthritis on a daily basis. Curcumin treatment suppressed the evolution of CIA in a time-dependent manner, demonstrated by suppressed arthritis onset and reduced arthritis severity.4. The pro-inflammatory cytokine such as BLyS, IFN-?, and IL-6 levels in serum from CIA mice were determined by specific ELISA. Compared with vehicle-treated mice, curcumin-treated mice had lower serum BLyS, IFN-?, and IL-6 levels.5. RT-PCR and Western blot analyses demonstrated that the expression of mBLyS was down-regulated in the spleen of CIA mice treatment with curcumin. Conclusion: Curcumin suppresses the onset of CIA and attenuates its severity in CIA mice. Curcumin reduced the inflammatory response by inhibiting the expression of BLyS mediators. The data could suggest that curcumin treatment is an effective prophylactic approach to suppress the evolution of arthritis in RA.