葫芦素B对小鼠淋巴细胞活化、增殖和凋亡的影响

Effects of cucurbitacin B on the activation,proliferation and apoptosis of mouse lymphocytes

目的分析葫芦素B(CuB)对多克隆丝裂原激活的小鼠淋巴细胞活化、增殖、凋亡和γ-干扰素(IFN-γ)表达的影响,探讨其免疫调节作用的机制。方法经CuB处理的小鼠淋巴细胞,在刀豆蛋白(ConA,5μg/ml)或者佛波醇酯(PDB,10-7 mol/L)和离子霉素(Ion,0.5μg/ml)刺激后,细胞计数观察CuB对小鼠淋巴细胞增殖的抑制作用;流式细胞术分析CD69和IFN-γ的表达及亚二倍体峰和线粒体膜电位的变化;免疫印迹分析Caspase 3的活化水平。结果细胞计数分析显示,CuB对小鼠淋巴细胞增殖的抑制呈剂量依赖关系。对细胞DNA含量分析表明,随着CuB浓度的增加,处于亚二倍体峰(即凋亡峰)的细胞数量增加。同样,经CuB处理后,早期活化标志CD69和IFN-γ在CD3+T细胞中的表达均受剂量依赖性抑制。JC-1染色结果表明,10μmol/L CuB能显著使小鼠淋巴细胞的线粒体膜电位发生下降(P<0.01)。免疫印迹分析表明,10μmol/L CuB处理24 h后,Caspase 3明显活化。结论 CuB对小鼠淋巴细胞的增殖和活化具有明显抑制作,可以诱导体外培养的小鼠淋巴细胞发生凋亡,具有潜在的抗炎免疫调节活性。

To elucidate the mechanism of the immunomodulatory effects of cucurbitacin B（CuB）,we inves-tigated the effects of CuB on the activation,proliferation,apoptosis and interferon-γ（IFN-γ） synthesis of mouse lymphocytes.We stimulated lymphocytes with concanavalin A（ConA,5 μg/ml） or phorbol ester（PDB,10-7 mol/L） plus ionomycin（Ion,0.5 μg/ml） in the presence or absence of different concentrations CuB,and then determined the proliferation,IFN-γ and CD69 expressions,sub-G0/G1 peaks mitochondrial membrane potential（ΔΨm）,and caspase 3 activation in activated lymphocytes.Analysis by counting the total numbers of cells revealed that CuB inhibited the proliferation of the activated lymphocytes in a dose-dependent manner;DNA content analysis showed that cell numbers in the apoptotic peak（sub-G0/G1） was elevated with the increase of CuB concentration.Moreover,in the CuB-treated groups,syntheses of CD69（the marker of early activation） and IFN-γ in T lymphocytes were inhibited in a dose-dependent manner.The JC-1 staining revealed that CuB（10 μmol/L） could significantly reduce the ΔΨm of lymphocytes（P 〈 0.01）.Western blotting analysis showed that CuB（10 μmol/L） treatment for 24 h resulted in the activation of caspase-3.These data indicated that CuB could inhibit the proliferation and activation of mouse lymphocytes and promote apoptosis in mouse lymphocytes,thus promising to be a potential anti-inflammatory agent.