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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/L Gen的培养液内分别培养24、48、72 h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/L Gen的完全培养液培养HepG2细胞24 h,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/L Gen分别作用于HepG2细胞24 h,0.01、0.10、1.00μmol/L Gen组细胞增殖率高于溶剂对照组(均P<0.05);作用48、72 h,1.00、10.00、50.00、100.00μmol/L Gen组细胞增殖率均低于溶剂对照组(均P<0.05)。1.00、10.00、50.00μmol/L Gen与HepG2细胞共同孵育24 h,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/L Gen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。
Objective To investigate the effect of genistein on the regulation of cholesterol metabolism and SREBP-2 pathway in HepG2 human hepatoma cells. Methods HepG2 cells were cultured in vitro. HepG2 cells were cultured for 24, 48 and 72 h in medium containing 0, 0.01, 0.10, 1.00, 1.00, 50.00 and 100.00 μmol / L Gen, respectively. Cell proliferation was measured by MTT assay. HepG2 cells were cultured in complete medium containing 0, 0.01, 1.00, 10.00 and 50.00 μmol / L Gen for 24 h respectively, and the cells were harvested for total cholesterol (TC) detection. Real-time fluorescence quantitative PCR Protein receptor (ldlr), 3-hydroxy-3-methyl glutaryl coenzyme A reductase (hmgcr) gene mRNA expression and Western blotting assay nuclear transcription factor SREBP-2 expression. Results Genotoxicity of 0.01, 0.10, 1.00, 1.00, 50.00 and 100.00 μmol / L Gen for 24 h in HepG2 cells was significantly higher than that in the control group (0.01, 0.10 and 1.00 μmol / L, respectively) The cell proliferation rate of 48,72 h, 1.00,10.00,50.00,100.00μmol / L Gen group were lower than that of the solvent control group (all P <0.05). The levels of TC in HepG2 cells were (1.81 ± 0.15), (2.29 ± 0.17) and (2.88 ± 0.08) mmol / L, respectively, when incubated with HepG2 cells at a concentration of 1.00, 10.00 and 50.00 μmol / L for 24 h [(1.44 ± 0.17) mmol / L], and increased with Gen concentration (R2 = 0.48, P <0.01). The mRNA expression levels of hmgcr and ldlr genes in Gen groups increased with Gen concentration High and increased (R2 0.53, 0.79, P <0.01 respectively). The gray value of the expression of GenBank in 1.00, 10.00 and 50.00μmol / L Gen group was higher than that in the control group (all P <0.01). Conclusion Genistein can regulate intracellular cholesterol metabolism and its mechanism may be related to the regulation of SREBP-2 pathway.