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目的研究白花丹醌对瘦素刺激的体外培养人肝星状细胞(HSC-LX2)细胞周期及周期相关蛋白表达的影响,探讨白花丹醌抗肝纤维化的作用机制。方法 ELISA法检测HSC-LX2细胞培养上清液中自分泌瘦素水平。将HSC-LX2细胞分成对照组,瘦素组,白花丹醌2、8μmol/L组,秋水仙碱6.25μg/mL阳性对照组。除对照组外,其余各组细胞用瘦素100μg/mL刺激24 h、再与药物共同培养24 h后,流式细胞仪检测HSC-LX2细胞周期,Western blotting法检测细胞周期相关蛋白cyclin D1、cyclin E1、p21的表达。结果 HSC-LX2细胞自分泌瘦素的浓度先随着培养时间的延长而递减,24 h达到低谷值,48 h后又升至6 h水平,每个时间点分泌的瘦素量无显著差异(P>0.05)。与对照组相比,瘦素组HSC-LX2细胞增殖能力显著增强,S期和G2/M期细胞比例显著增加;白花丹醌2、8μmol/L干预HSC-LX2细胞后,G0/G1期细胞的比例明显提高,而S期和G2/M期细胞比例明显降低,且呈明显的浓度相关性。与对照组比较,瘦素组HSC-LX2细胞经瘦素刺激后细胞周期相关蛋白cyclin D1、cyclin E1的量显著增加,p21蛋白的表达受到抑制;白花丹醌2、8μmol/L和秋水仙碱明显降低cyclin Dl、cyclin E1蛋白水平,增强p21蛋白表达。结论白花丹醌可明显抑制瘦素诱导的HSC-LX2细胞增殖,该作用主要是通过抑制细胞由G0/G1期向S期转变而产生的,作用机制可能与其降低cyclin Dl、cyclin E1蛋白水平,增加p21蛋白表达相关。
Objective To study the effect of plumbagin on cell cycle and related protein expression of leptin stimulated human hepatic stellate cells (HSC-LX2) in vitro and to explore the mechanism of action of plumbagin on hepatic fibrosis. Methods The level of autocrine leptin in culture supernatant of HSC-LX2 cells was detected by ELISA. HSC-LX2 cells were divided into control group, leptin group, plumbagin 2,8μmol / L group, colchicine 6.25μg / mL positive control group. Except the control group, the other groups of cells were stimulated with leptin 100μg / mL for 24 hours and then incubated with drugs for 24 hours. The cell cycle of HSC-LX2 was detected by flow cytometry. The expressions of cyclin D1, cyclin E1, p21 expression. Results The concentration of autocrine leptin in HSC-LX2 cells decreased firstly with the prolongation of incubation time, reached a low value at 24 h, then increased to 6 h after 48 h, and there was no significant difference in the amount of leptin secreted at each time point P> 0.05). The proliferation of HSC-LX2 cells was significantly increased in leptin group and the proportion of cells in S phase and G2 / M phase was significantly increased compared with control group. After HSC-LX2 cells were treated with plumbagin 2 and 8μmol / L, the cells in G0 / G1 phase The proportion of cells in S phase and G2 / M phase was significantly decreased, and the concentration was significantly correlated. Compared with the control group, the expressions of cyclin D1 and cyclin E1 in leptin-treated HSC-LX2 cells were significantly increased after stimulation with leptin, and the expression of p21 protein was inhibited. Compared with leucoindine, the concentrations of 2, 8 μmol / L and colchicine Significantly reduce cyclin D1, cyclin E1 protein levels, enhance p21 protein expression. Conclusions Plumbagin can significantly inhibit leptin-induced proliferation of HSC-LX2 cells, which is mainly caused by the inhibition of cell transformation from G0 / G1 phase to S phase, which may be related to the decrease of cyclin D1 and cyclin E1 protein levels, Increased p21 protein expression.