叶下珠复方Ⅱ号对肝星状细胞凋亡和miR-16/Bcl-2表达的影响

Effect of Compound Phyllanthus urinaria Recipe Ⅱ on Apoptosis and miR-16/Bcl-2 Expression in Hepatic Stellate Cells

【目的】观察叶下珠复方Ⅱ号体外对肝星状细胞(HSC-T6)凋亡和miR-16/Bcl-2表达的影响,进一步探讨其抗肝纤维化的作用机制。【方法】体外培养HSC-T6细胞,分为空白对照组,叶下珠复方Ⅱ号高剂量组(3.6 g/L)、中剂量组(1.8 g/L)和低剂量组(0.9 g/L),采用四甲基偶氮唑盐(MTT)法检测药物对HSC-T6细胞增殖的影响,流式细胞仪检测细胞凋亡的变化,实时荧光定量PCR法检测药物处理后HSC-T6细胞miR-16和Bcl-2 mRNA表达的改变,Western Blot法测定Bcl-2蛋白的表达变化。【结果】叶下珠复方Ⅱ号处理HSC-T6细胞后,高、中、低各剂量组对HSC-T6细胞增殖均有明显的抑制作用,随着叶下珠复方Ⅱ号浓度的增加,细胞的凋亡率也逐渐增加;叶下珠复方Ⅱ号各处理组miR-16的表达显著增加,呈现明显的剂量—效应关系,且Bcl-2 mRNA和蛋白质表达显著减少,与空白对照组比较,差异均具有统计学意义(P<0.01)。【结论】叶下珠复方Ⅱ号能显著抑制HSC-T6细胞增殖和诱导其凋亡,其作用机制与提高miR-16的表达,抑制其靶基因Bcl-2的表达有关。

Objective To observe the effect of compound PhyUanthus urinaria Ⅱ （CPU Ⅱ ）, a Chinese herbal recipe mainly composed of Phyllanthus urinaria Linn, on apoptosis and miR-16/Bcl-2 expression in hepatic stellate cells （HSC-T6）, and to explore its anti-fibrosis mechanism. Methods HSC-T6 were cultured in vitro and were divided into 4 groups, including blank control group, and high-, middle- and low-dose CPU Ⅱ groups （in the dosage of 3.6, 1.8, 0.9 g/L, respectively） . Methyl thiazolyl tetrazolium （MTT） assay was used for evaluating the effect of CPU Ⅱ on inhibiting HSC-T6 proliferation. Flow cytometry assay was used for studying the action of CPUⅡ on inducing HSC-T6 cells apoptosis. Real-time fluorescence quantitative polymerase chain reaction （PCR） was used for detecting miR-16 and Bcl-2 mRNA expression levels. The expression level of Bcl-2 protein was analyzed by Western blotting method. Results HSC-T6 proliferation was obviously inhibited in the CPU II groups, and the apoptotic rate was increased gradually with the dosage, miR-16 expression level was elevated, which showed a significant dose-effect relationship. Bcl-2 mRNA and protein levels were decreased, the difference being significant compared with the blank control group （P〈0.01） Conclusion CPU II can effectively inhibit the proliferation of HSC-T6 and induce the apoptosis in vitro, and its therapeutic mechanism may be related with the increase of miR-16 expression and with the inhibition of the target gene Bcl-2 expression.