染料木黄酮对HepG2细胞胆固醇代谢和SREBP-2通路的调控作用

目的探讨染料木黄酮（Gen）对HepG2人肝癌细胞胆固醇代谢和SREBP-2通路的调节作用。方法体外培养HepG2细胞，将HepG2细胞在含有0、0．01、0．10、1．00、10．00、50．00、100．00μmol／LGen的培养液内分别培养24、48、72h后，用MTT法检测细胞增殖活性。用分别含0、0．01、1．00、10．00、50．00μmol／LGen的完全培养液培养HepG2细胞24h，收集细胞，分别进行细胞内总胆固醇（TC）含量检测、实时荧光定量PCR检测细胞低密度脂蛋白受体（ldlr）、3-羟基-3-甲基戊二酸单酰辅酶A还原酶（hmgcr）基因的mRNA表达和蛋白印迹法检测核转录因子SREBP-2的表达。结果0．01、0．10、1．00、10．00、50．00、100．00μmol／LGen分别作用于HepG2细胞24h，0．01、0．10、1．00μmol／LGen组细胞增殖率高于溶剂对照组（均P〈0．05）；作用48、72h，1．00、10．00、50．00、100．00μmol／LGen组细胞增殖率均低于溶剂对照组（均P〈0．05）。1.00、10．00、50．00μmoL／LGen与HepG2细胞共同孵育24h，HepG2细胞内TC水平分别为（1．81±0．15）、（2．29±0．17）、（2．88±0．08）mmol／L，均高于对照组[（1．44±0．17）mmol／L]，且随着Gen浓度升高，呈增加趋势（R2=0．48，P〈0．01）；各Gen实验组的hmgcr和ldlr基因的mRNA表达水平随着Gen浓度升高而升高（R2分别为0．53、0．79，均P〈0．01）。1．00、10．00、50．00μmol／LGen组蛋白表达灰度值均高于对照组（均P〈0．01）。结论染料木黄酮能够调节细胞内胆固醇的代谢，其作用机制可能与调控SREBP-2通路有关。

Effect of genistein on cholesterol metabolism and expressions of gene or protein involved in SREBP- 2 pathway in HepG2 cells

Objective To investigate the effect of genistein on cholesterol metabolism and SREBP- 2 pathway in HepG2 cell. Methods HepG2 cells were cultured with 0, 0.01, 0. 10, 1.00, 10. 00, 50.00, and 100.00 μmol/L of genistein for 24, 48, and 72 hours, respectively. Then, the cell prolifera- tive activity was detected by MTT. HepG2 cells were incubated with 0, 0.01, 1.00, 10.00, and 50. 00 μmol/L genistein for 24 hours and then collected for the cholesterol determination. The gene expression levels of ldlr and hmgcr were measured by real-time quantitative PCR. The expression of nuclear transcrip- tion factor SREBP-2 was determined by Western blotting. Results After incubation with genistein for 24 hours, the proliferative rates of ceils treated with 0.01,0. 10, and 1.00 μmol/L genistein were higher than that of the control （P 〈 0. 05 for all）. However, after incubation with genistein for 48 or 72 hours, the pro- liferative rates of cells treated with 1.00, 10. 00, 50. 00, and 100. 00 μmol/L genistein were lower than that of the control （P 〈0.05 for all）. Cholesterol levels in HepG2 cells incubated with 1, 00, 10. 00, and 50. 00 μmol/L genistein for 24 hours were [ 1.81 ± 0. 15 ], [ 2. 29 ±0. 17 ], and [ 2. 88 ± 0. 08 ] mmoL/L,respectively, which were higher than that of the control cells （ [ 1.44 ±0. 17 ] mmol/L）. There was a rising trend as the genistein concentration increased （R2 = 0.48 ,P 〈 0. 01 ）. Meanwhile, mRNA levels of hmgcr and Idlr were also increased as the genistein concentration increased （R2 = 0. 53 or 0. 79, P 〈 0. 01 for all）. SREBP-2 protein levels in HepG2 cells incubated with 1.00, 10. 00, and 50. 00 μmol/L genistein were much higher than that in control cells （P 〈 0. 01 for all）. Conclusion Genistein could regulate the cholesterol metabolism in HepG2 cells and its mechanism may be related to regulating gene or protein ex- pressions involved in the SREBP-2 pathway.