The Pharmacological Effects Of Berberine On Mouse Lymphocytes And Its Action Mechanisms

AIM: The present study is to investigate the pharmacological effects of berberine(Ben on the proliferation rates and the cell-cycle distribution of the mouse lymphocytes and its subsets, and on dinitrofluorobenzene (DNFB) -induced delayed type hypersensitivity (DTH). in order to elucidate the molecular and cellular mechanism of the immunosuppressive effect of Ber.METHODS:1. The lymphocytes were isolated from lymphoid nodes of C57BL/6 mouse and were stimulated with polyclonal activators, concanavalin (Con A) or phorbol 12,13-dibutyrate (PDB) plus Ion. WST-1 test was adopted to analyze the proliferation of mouse lymphocytes. The proliferation rate of mouse lymphocytes was also analyzed with CFDA-SE staining method. The distribution of the cell-cycle was analyzed by propidium iodide (PI) staining together with flow cytometry analysis. Annexin-V staining was used to detect the apoptotic cell death of lymphocytes.2. The lymphocytes were isolated from lymphoid nodes of C57BL/6 mouse. After the staining with CFDA-SE, mouse lymphocytes was stimulated with polyclonal activators. Con A or PDB plus Ion. After the staining with PE labeled anti-mouse CD3 and Cy-chrome labeled anti-mouse CD4, The proliferation rates of T lymphocytes and CD4~+ T lymphocytes were analyzed with flow cytometry.3. BALB/c mice were divided into three groups: one was control group, one was DTH group, and the other was Ber-treated DTH group. The control group was treated by the solvent without DNFB. The DTH group was sensitized to DNFB by painting the shaved abdomen with 20 u.L of 5 g/L DNFB on 0 day and 1. On 5 day DNFB-sensitized and control unsensitized mice were challenged by painting 10 uL of 2 g/L DNFB on one side of the left ears. The Ber-treated DTH group was injected (i.p.) daily for 7 consecutive days, with the total dose of 30 mg/kg. S he weight of two ears was weighted at 48 h after challenge. The change of local tissues of mouse auricles was observed under light microscopy. Violet crystal staining was used to determine the effect of Ber on adhesion of mouse splenic lymphocyte to extracellular matrices (ECM). Annexin-V staining was used to detect the apoptotic rate of the lymphocytes in the lymphoid nodes of BALB/c mice induced by DNFB.4. HeLa cells were transfected with expression vectors pPKCB-EGFP and pEGFP-Nl using Lipofectamine 2000. respectively. Their expression in the cells was analyzed by flow cytometry and subcellular localization by confocal microscopy. The effect of PDB and Ber on cell morphology was analyzed by confocal microscopy as well.RESULTS:1. WST-l assay showed that 100 umol/L, 30 umol/L and 10 umol/L Ber could significantly inhibit the proliferation of mouse lymphocytes induced by Con A or by PDB plus Ion. The inhibitory effect was dose-dependent. Flow cytometry analysis of the cell-cycle distribution revealed that the cell number in sub- G0/G1 peak (apoptotic peak) was increased, while the cell numbers in S and G2/M was decreased as the concentration of Ber increased. However the cell number in G0/G1 phase had no significant changes under the concentration range. Besides, the results of CFDA-SE staining were similar to that of WST-l assay.2. I he staining with CFDA-SE, PE labeled anti-mouse CD3 and Cy-chrome labeledanti-mouse CD4 showed that 100 umol/L and 30 umol/L Ber could significantly inhibit the proliferation of mouse CD4+ T lymphocytes and CD4~ T lymphocytes stimulated by Con A or by PDB plus Ion in vitro (P<0.05 ) .3. The results of weighting two ears showed that there were remarkable difference between the Ber-treated group and the DTH group (PO.05). Observation of the change of mouse auricles tissues under optical microscope indicated Ber could suppressed DTH and reduced significantly the infiltration of lymphocytes. The adhesion of T lymphocytes from Ber-treated group to ECM was siginificantly decreased (P<0.05). However, there was no obvious difference in apoptotic rate among the Ber-treated DTH group, DTH group and control group (P>0.05).4. ] he expression rates of green fluorescence in HeLa cells transfected with pHGFP-Nl and pPKCG-EGFP plasmids were 70.9±(4.4%) and 45.8(±2.3)% iP<0.0S) respectively at 48 h after transfection. PKC9-EGFP fusion protein was evenly distributed in the whole cells, which was similar to the distribution pattern of LGFP protein in HeLa cells. After 30 min stimulation of PDB, the shape of the HeLa cells expressed PKC9-EGFP was dramatically changed. From spindle to small round, and the green fluorescence intensity increased and concentrated in the central of the cells. In contrast, the cells expressing EGFP had no significant change both in the green fluorescence distribution and in cell morphology after PDB stimulation. Besides. Ber exerted unobvious effect on the PDB-induced changes in HeLa cells expressing PKC9-EGFP.CONCLUSION:1. Ber could inhibit the proliferation of the mouse lymphocytes and block the progression of mouse lymphocytes into S and G2/M phases. The inhibitory effect was cell-cycle specific. Moreover, Ber could induce mouse lymphocytes underlying apoptosis.2. Ber could remarkably inhibit the proliferation of the mouse CD4+ T lymphocytes and CD4 T lymphocytes in vitro.3. Ber could significantly inhibit the DNFB-induced DTH in mice. The distinctly reducd adhesion between mouse T lymphocytes and ECM probably was probably one mechanism of suppression of DTH by Ber.i The HeLa cells that expressed PKC9-EGFP had significant morphological change under PDB stimulation, while Ber had no effect on the change, suggesting that Ber might not affect lymphocytes through inhbiting the activity of PKC9.