Therapeutic Effect Of Berberine On Adjuvant-Induced Arthritis In Rats And Regulation Of Berberine On Macrophage Polarization

Objective:1.To observe the therapeutic effect of berberine(BBR)on adjuvant-induced arthritis(AA)rats;2.To clarify the effect and mechanism of BBR in attenuating arthritis is associated with regulating the balance of M1/M2 macrophages.Methods:Complete Freund adjuvant(CFA)was used to establish AA model.The experiment was divided into the normal group,the AA group,the BBR(40 mg/kg,80mg/kg,160 mg/kg)dose groups and the methotrexate(MTX,0.5 mg/kg)dose groups.The arthritis global assessment,arthritis index score and arthritis swollen joint count were evaluated by animal arthritis score system.Arthritis paw swelling was measured by paw volume plethysmograph.The damage of bone was examined by X-ray in animal vivo imaging.The thymus and spleens were weighed and the thymus and spleen index were calculated.T cell proliferation was evaluated by cell counting kit-8(CCK-8).The histopathology was assayed in joints and spleens by hematoxylin-eosin staining.The percentage changes of T subsets in spleen were assayed by flow cytometry.The phagocytosis of macrophages was detected by neutral red phagocytosis experiment.The concentrations of cytokines were measured using enzyme-linked immunosorbent assay.Nitric oxide synthase(iNOS)and arginase 1(Arg1)were detected by Immunofluorescence identification.The protein expressions of iNOS,Arg1,adenosine 5’-monophosphate-activated protein kinase(AMPK),p-AMPK,p65(RelA),p-p65,NF-κB inhibitor alpha(IκBα),p-IκBαand cyclooxygenase(COX)-2 were detected by Western Blot.Results:1.The therapeutic effect of BBR on AA ratsThe inflammatory response of AA rats was significantly improved after BBR treatment.BBR decreased arthritis global assessment and alleviated arthritis paw swelling.BBR also reduced arthritis index and arthritis swollen joint count.BBR was significantly relieved bone destruction in AA rats observed by X-ray.The results of histopathology showed that BBR significantly alleviated synovial cell proliferation,pannus generation,inflammatory infiltration and cartilage erosion.As the same time,BBR improved congestion of red pulp,and attenuated hyperplasia of white pulp and marginal zone in spleen.BBR obviously decreased the thymus and spleen index.BBR obviously inhibited the expressions of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-2,IL-17A,interferon-γ(IFN-γ),monocyte chemotactic protein 1(MCP-1),while the levels of transforming growth factor-β1(TGF-β1),IL-4 and IL-10were notably increased in serum.2.The effect of BBR on the functions and polarization of macrophages in AA ratsBBR distinctly inhibited the levels of TNF-α,IL-1β,IL-6,MCP-1,while increased the secretion of TGF-β1 and IL-10 in macrophage supernatant.BBR significantly inhibited the phagocytosis of macrophages.BBR downregulated the expressions of iNOS and COX-2,while distinctly upregulated the expression of Arg1.The results show that BBR reduced M1 macrophage polarization and promoted the polarization of M2 macrophages.3.The effect of BBR on T lymphocytesT cell proliferation was significantly suppressed after treatment with BBR.The proportion of CD3~+CD4~+total T cells and CD4~+CD44~+memory T cells were no significant changed.BBR upregulated the percentage of CD4~+CD62L~+nav?e T cells.BBR(80 mg/kg,160 mg/kg)downregulated the percentage of CD4~+CD25~+activated T cells.Further studies showed that BBR significantly reduced the proportion of Th17cells,and BBR(80 mg/kg,160 mg/kg)significantly increased the proportion of Treg cells.4.BBR activated AMPK and inhibited the activation of NF-κB in macrophages in AA ratsThe expression of p-AMPK was distinctly decreased,while the expressions of p-p65 and p-IκBαwere markedly increased in macrophages in AA rats.BBR(80mg/kg)obviously upregulated the expression of p-AMPK while suppressed the levels of p-p65 and p-IκBα.Conclusions:1.BBR had a good therapeutic effect by alleviating arthritis and bone destruction in AA rats;2.BBR inhibited M1 macrophage polarization and promoted the polarization of M2 macrophages in AA rats;3.BBR inhibited the proliferation and activation of T lymphocytes and regulated the balance of Th17/Treg cells;4.BBR regulated the polarization of macrophages through AMPK/NF-κB pathway.