| Rheumatoid arthritis (RA) is a systemic autoimmune disease that primary targets thesynovial membrane, cartilage and bone. Although the precise pathogenesis of RA isunclear, the presence of germinal centers, plasma cells in synovium suggest that B cellsactivation are involved in disease pathogenesis. B cell-activating factor belonging to theTNF family (BAFF) is a B cell survival and maturation factor. Excessive BAFF candestroy B cell self tolerance mechanisms, resulting in systemic autoimmune disease.BAFF-R (B cell-activating factor receptor) was one of three receptors of BAFF.BAFF-R appears as a most crucial receptor mature B cells survival and homeostasis inperipheral lymphoid organs. BAFF/BAFF-R activate anti-apoptotic signal by inducingNF-κB and Bcl-2, resulting in anti-apoptotic protein production. Recent year, someresearch indicate that the phosphatidylinositol3-kinase (PI3K) catalytic subunit P110δpromote the survival and growth of B cell mediated by BAFF, and the PI3K/Akt/mTORpathway is likely to be important for BAFF response. Natural productEpigallo-catechin-3-gallate (EGCG) is a potent antioxidant, antiinflammatory, andantioncogenic compound, which is extracted from green tea. EGCG has been shown toselectively inhibit cell proliferation and induce cancer cells apoptosis without affectingnormal cells. Recent year, EGCG has attracted attention for its emerging anti-arthritic andimmune modulating properties. Collagen-induced arthritis (CIA) is an establishedexperimental model of polyarthritis with many histopathological features similar to RA.In this study, we investigated whether PI3K/Akt/mTOR signaling mediated by BAFF involved in upregulating the function of B lymphocytes and evaluated regulatoryfunction of EGCG on this signaling pathway, as well as the influence of EGCG on Blymphocytes function on the establishment of CIA model.Objective:To investigate the role of PI3K/Akt/mTOR signaling mediated by BAFF involved inupregulating the function of B lymphocytes in rats with collagen-induced arthritis (CIA)and the regulatory function of EGCG on this signaling pathway, as well as the influenceof EGCG on B lymphocytes function. SD rats were immunized to induce CIA.Methods:SD rats were immunized to induce CIA. CIA rats were randomly separated into differentgroups and treated with EGCG at the concentration of40and80mg/kg, Pae at the100mg/kg respectively from day18to day38after immunization. Arthritis scores wereevaluated. Histopathological examination and pathology scores of joints and spleenswere assayed; Serum BAFF, anti-CII antibody, IgA, IgG and IgM were assayed byELISA. The expressions of BAFF-R, PI3K, p-Akt, mTORC1, Bcl-xL and Bim wereassayed by immunohistochemistry and western blot; B lymphocyte proliferations wereanalyzed by the MTT assay. Apoptosis of B lymphocyte were assayed by flow cytometry.Results:1. The therapeutic effects of EGCG on CIA ratsCompared with normal rats, fore-paws of CIA rats were swelled firstly at day18afterimmunization, the hind-paws swelled slowly. Arthritis scores and the swelling reachedto peak on day34. In the ankle joint of CIA rats, synoviocytes proliferated tomultilayers, and articular cartilages were destructed and infiltrated with inflammatorycells. In the spleen of CIA rats, white pulp proliferated, germinal centers emerged, and red pulp hyperemia. EGCG (40,80mg/kg) could reduce the arthritis scores and pawswelling of CIA rats, reduce the histological pathology scores of synovium hyperplasia,cell infiltration, pannus, cartilage and bone erosion of joints in CIA rats. EGCG (80mg/kg) reduce the histological pathology scores of cell density lymphatic sheath,lymphoid follicular hyperplasia, marginal zone and red pulp, as well as the number ofgerminal center in spleens of CIA rats. EGCG (40mg/kg) only diminished the scores ofcell density lymphatic sheath and lymphoid follicular hyperplasia scores. These resultssuggested that EGCG had therapeutic effects on CIA rats.2. EGCG decreased the concentrations of serum anti-CII antibody, IgA, IgG andIgM levels in CIA rats.The concentrations of serum anti-CII antibody, IgG, IgM, and IgA were significantlyincreased in CIA rats. EGCG (80mg/kg) could decrease anti-CII antibody, IgA, IgG andIgM levels, and EGCG (40mg/kg) only reduced IgA, IgG and IgM levels. These resultssuggested that EGCG had therapeutic effects on CIA rats, which might be closely relativeto its regulation on the function of B lymphocytes.3. EGCG decreased the concentrations of serum BAFF levels, down-regulatedBAFF-R expression in the spleens of CIA rats.In CIA rats, the concentrations of serum BAFF were significantly increased, theexpression of BAFF-R were also increased greatly in CIA spleens. BAFF-R were mainlyexpressed in the mantle zone and marginal zone in the spleen but were lower in thegerminal center. EGCG (40,80mg/kg) could decrease BAFF levels and the expressionsof BAFF-R. These results suggested that BAFF and BAFF-R involved in the abnormalactivation of B lymphocytes. EGCG had regulation effects on B lymphocytes, whichmight be relative to its down-regulation on BAFF and BAFF-R expression. 4. EGCG reduced P110δ, p-AKT1/2/3, mTORc1expressions in the spleens of CIArats.In the spleens of CIA rats, the expressions of P110δ, p-AKT1/2/3and mTORC1wereincreased greatly. EGCG (40,80mg/kg) significantly decreased the expressions of P110δ,p-AKT1/2/3and mTORC1in CIA rats. In short, the enhancement of PI3K/Akt/mTORpathway was closely relative to B lymphocytes proliferation and survival in CIA rats;EGCG could down-regulate PI3K/Akt/mTOR pathway, inhibit the function of Blymphocytes proliferation and survival in CIA rats futher.5. EGCG up-regulated pro-apoptotic protein, down-regulated anti-apoptoticprotein expression and induced B lymphocytes apoptosis.In the spleens of CIA rats, the expression of Bcl-xL was increased greatly, whilepro-apoptotic protein Bim was decreased. EGCG (40,80mg/kg) significantly decreasedthe expression of Bcl-xL in CIA rats, while increased Bim expression.B lymphocyte proliferations stimulated by BAFF or IGF-1(PI3K agonists) wereincreased compared with untreated cells, while the percentages of B lymphocytesapoptosis were significantly reduced. BAFF/IGF-1+EGCG (10-5,10-6,10-7mol/L) couldreduce B lymphocyte proliferation. BAFF+EGCG (10-5,10-6mol/L) and IGF-1+EGCG(10-5mol/L) could induce B lymphocyte apoptosis.The results suggested that the unbalance between apoptosis proteins played an importantrole in B lymphocyte proliferation and survival in CIA rats. EGCG might regulate thebalance between apoptosis proteins, then induced B lymphocytes apoptosis in futher.Conclusion:1. EGCG had therapeutic effects on CIA rats2. EGCG had therapeutic effects on CIA rats, which might be closely relative to itsregulation on the function of B lymphocytes. 3. EGCG had regulation effects on B lymphocytes, which might be relative to itsdown-regulation on BAFF and BAFF-R expression.4. EGCG inhibited the function of B lymphocytes proliferation and survival bydown-regulating PI3K/Akt/mTOR pathway in CIA rats.5. EGCG induced B lymphocytes apoptosis by regulating the balance between apoptosisproteins in CIA rats. |
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