染料木素对油酸诱导的HEⅡ4肝细胞脂质沉积的影响

Effect of genistein on lipid accumulation in H4Ⅱ E cell induced by oleic acid

目的观察染料木素（GEN）对油酸（oleicacid）诱导的HEⅡ4肝细胞脂质沉积细胞模型的作用，以及对腺苷酸活化蛋白激酶（AMPK）磷酸化的影响。方法采用MTT法检测不同浓度的GEN对细胞的毒性；以0．2mmoFL油酸诱导HEⅡ4肝细胞内脂质大量沉积，给予GEN干预后以GPO·DAOS法检测细胞内甘油三酯（TG）含量，以DCⅢ法检测细胞内总蛋白含量，用蛋白含量校正细胞内TG含量（TG／蛋白）来评估细胞内脂质沉积情况；Western blotting法检测肝细胞内AMPK及P．AMPK（Thr172）蛋白表达水平，以P-AMPK（Thr172）／AMPK表示AMPK的磷酸化水平。结果GEN可减少肝细胞内脂质沉积（分别为139．64±18．60、192．20±34．17，P〈0．05），并且提高细胞内AMPK的磷酸化水平（分别为1．93±0．36、0．78±0．27，P〈0．05），而GEN降低细胞内脂质沉积作用可被AMPK阻滞剂CompoundC所逆转。结论GEN可改善油酸诱导的HEⅡ4肝细胞内脂质沉积，其作用机制与增加AMPK磷酸化水平有关。

Objective To observe the effects of genistein（GEN）on oleic acid （OA） induced lipid accumulationin in H4ⅡE cells and to discuss the possible mechanism of GEN in the point of AMPK. Methods H4ⅡE cells were cultured in vitro. The control group （NOR）, OA treatment group （MOD group）and GEN treatment group were established according to the experimental requirements. The effects of GEN on the proliferation of H4ⅡE cells were measured with MTT assay. The intracellular TG mass was quantified spectrophotometrically using TG test kit. Cell protein was determined by DCTM Protein Assay kit. The intracellular TG concentration which was used to evaluate lipid accumulation was corrected using protein content as an internal standard. Western blotting was applied to determine the expression of AMPK and P-AMPK （Thr172）. Accordingly the phosphorylation levels of AMPK was by means of P-AMPK （Thr172）/AMPK. Results OA treatment can induce lipid accumulation in H4ⅡE cells while GEN treatment can decrease the intracellular TG concentration through up-regulate the phosphorylation levels of AMPK, though the effect was blocked by Compound C that is the inhibitor of AMPK. Conclusion GEN has anti-accumulation of lipid effect in H4ⅡE cell. The mechanism of GEN protective effect is partially due to AMPK.