Effects Of Berberine On Human Rheumatoid Arthritis Fibroblast-like Synoviocytes And Its Underlying Molecular

Background:Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease, characterized by the presence of chronic inflammation and synovial hyperphasia. Accumulating evidence indicates that, in addition to macrophages and T cells, activated RA fibroblast-like synoviocytes (FLSs) play a major role in both initiating and driving RA, including pannus formation and secretion of factors that promote inflammation, neovascularization and cartilage degradation. In animal models, histological examination of synovium during the chronic phase of RA reveals marked hyperplasia of the spindle-shaped synovial cell population. Transformed-appearing synovial cells with large pale nuclei and prominent nucleoli are abundant, particularly at sites of synovial invasion/erosion of surrounding periarticular bone. Microscopic examination of a proliferation marker, Ki67, demonstrates localized foci of proliferating FLSs in the lining layer of the synovium. Rapid proliferation and defective apoptosis of RAFLSs are two main mechanisms contributing to synovial hyperplasia. Moreover, RAFLSs have been reported to express altered levels of apoptotic signaling molecules such as p16, SENTRIN, and PTEN. Thus, RAFLSs resemble malignant cells in their potentially high proliferative capacity and in their expression of dysregulated intracellular molecules that can modulate cell cycle progression and apoptosis.Berberine (Ber) is the major constituent of Coptidis Rhizoma with multiple pharmacological activities, including antibiotic activity, anti-tumor and anti-platelet aggregation properties. Accumulated evidence indicates that berberine has anti-inflammatory activity in an animal model and other experimental systems. For example, in an experimental autoimmune tubulointerstitial nephritis model, berberine is found to be effective in decreasing the intensity of pathological injuries in mice and has an immunosuppressive effect in this model. It has been shown that berberine suppresses the delayed-type hypersensitivity (DTH) reaction. Interestingly, berberine is also shown to alleviate the clinical signs of adjuvant-induced arthritis (AIA) in rats. However, the mechanism of the suppressive effects of berberine on RA has not been completely elucidated yet. In the present study, we examined the effect of berberine on the proliferation, cell cycles and apoptosis of cultured human RAFLSs, Thus having potential therapeutic implications in treatment of RA.MethodsEffects of Berberine on RAFLSs(1) Primary cultured of RAFLSs:RAFLSs were obtained from 39 patients with RA at the time of total joint replacement as described previously. The diagnosis of RA conformed to the ACR 1987 revised criteria. Synovial tissue was chopped into fragments and digested with 1 mg/mL collagenase. The resulting cell suspension was seeded into tissue culture dishes and cultured. The purity of the cells was verified by flow cytometric analysis (>95% CD90+,<2% CD14+,<1% CD3+, and<1% CD19+).(2) The MTT assay was used to assess the effect of berberine on the cell proliferation of RAFLSs. RAFLSs were treated with varying concentrations of berberine for 12,24,48 or 72 h.2. the effect of berberine on cell cycle progression in human RAFLSs(1) The cell cycle phase was assayed by measuring DNA fragment staining with propidium iodide (PI). RAFLSs were treated with varying concentrations of berberine for 48 h.(2) Western blot analysis was performed to analyze the protein expression of cyclin-dependant kinase (CDK) inhibitors Cipl/p21 and Kipl/p27, and CDK2, CDK4, CDK6, and cyclins D1, D2, and E.3. Berberine induces apoptosis in human RAFLSs(1) The apoptotic status of RAFLSs with Berberine treatment was evaluated by measuring the exposure of phosphatidylserine on the cell membranes using AnnexinⅤ-fluorescein isothiocyanate (AnnexinⅤ-FITC) and PI staining. The BD Pharmingen AnnexinⅤ-FITC Apoptosis Detection Kit I was used for the apoptosis assay.(2) Western blot analysis was performed to analyze the protein expression of Bcl-2, Bcl-x1, Bax and RARP.(3) The loss of mitochondrial membrane potential was quantitatively determined by flow cytometry using the lipophilic cationic probe JC-1 dye Detection Kit, following the manufacturer’s instructions.(4) Intracellular caspase-3 and caspase-9 activity of the RAFLSs was assessed by caspase-3 and caspase-9 colorimetric assay kit.Results1. Berberine decreases cell proliferation in human RAFLSsThe MTT assay was used to assess the effect of berberine on the cell proliferation of RAFLSs. RAFLSs were treated with varying concentrations of berberine for 12, 24,48 or 72 h. The treatment with berberine resulted in a significant reduction in RAFLS proliferation/viability in a dose and time-dependent manner.2. Berberine induces G0/G1 phase cell cycle arrest in human RAFLSsTo determine the possible mechanism of anti-proliferative activity of berberine on RAFLSs, we assessed the effect of berberine on cell cycle progression in human RAFLSs. The dose-dependent effect of berberine on G0/G1 arrest in RAFLSs was largely at the expense of both S and G2-M phase cells compared with the non-berberine treated control cells. berberine inhibits human RAFLS proliferation may be through induction of G0/G1 arrest. RAFLSs with varying concentrations of berberine for 48 h resulted in a dose-dependent increase in protein expression of Kip1/p27 and Cip1/p21. Treatment of RAFLSs with berberine resulted in a marked decrease in the expression of Cdk2, Cdk4 and Cdk6 in a dose-dependent manner. Similarly, a marked reduction in the expression of cyclins D1, D2 and E was also observed dose-dependently on berberine. These results suggest that berberine exerts antiproliferative effects against RAFLSs, largely through modulation of cell cycle regulators.3. Berberine induces apoptosis in human RAFLSsTo investigate whether the inhibition of proliferation and the induction of G0/G1 arrest in human RAFLSs by berberine were due to the induction of apoptosis, we used AnnexinⅤ-FITC/PI double staining to assess the percentage of apoptotic cells induced by berberine. As expected, berberine induced RAFLSs apoptosis in a dose-dependent manner at 48 h. To further determine the possible mechanism of berberine-induced apoptosis in RAFLSs, western blot analysis was performed to analyze the protein expression of Bcl-2, Bcl-x1 or Bax. The treatment of RAFLSs with berberine resulted in a dose-dependent reduction of the anti-apoptotic protein Bcl-x1 and Bcl-2.We further determined the activation of procaspase 9, caspase 3 and PARP proteins in berberine-treated human RAFLSs. Loss of mitochondrial membrane potential can contributes to the activation of caspases and subsequent apoptotic cell death.Discussion1. Berberine significantly inhibited cell proliferation of human RAFLSs.2. Cell cycle analysis of berberine-treated RAFLSs indicated a cell cycle arrest at the G0/G1 phase. Mechanistically, the inhibitory effects of berberine were attributable to an induction of cyclin-dependant kinase (CDK) inhibitors Cip1/p21 and Kip1/p27 together with suppression of CDK2, CDK4, CDK6, and cyclins D1, D2, and E.3. Berberine treatment caused apoptotic death in RAFLSs, which was associated with an increased expression of pro-apoptotic protein Bax and decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-x1, disruption of mitochondrial membrane potential, and activation of caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP).Innovations and significancesThese results suggest that berberine exerts antiproliferative effects against RAFLSs, largely through modulation of cell cycle regulators and induction of apoptotic pathways, thus having potential therapeutic implications in treatment of RA.LimitationsThe effect of berberine on RAFLSs in vivo needs to be further investigated.