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目的:研究染料木黄酮是否通过雌激素受体介导影响3T3-L1前脂肪细胞成脂分化,并探讨其可能的机制。方法:以不同浓度染料木黄酮处理3T3-L1细胞,四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法测定细胞存活率;在3T3-L1前脂肪细胞成脂分化过程中分别加入不同浓度染料木黄酮、染料木黄酮和ICI182780(一种雌激素受体抑制剂)混合物及不同浓度雌二醇,油红染色观察分化结果,测定细胞甘油三酯(triglyceride,TG)含量;染料木黄酮、染料木黄酮和ICI182780混合物及不同浓度雌二醇处理诱导成熟后的脂肪细胞,测定培养液甘油含量;分别用染料木黄酮(30μmol/L)、染料木黄酮(30μmol/L)和ICI182780混合物干预细胞成脂分化,Western blotting法检测细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、p-ERK、腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、p-AMPK、激素敏感性脂肪酶(hormone sensitive lipase,HSL)、脂肪酸合成酶(fatty acid synthetase,FAS)蛋白表达量。结果:3T3-L1细胞存活率随染料木黄酮浓度的升高而降低,50~200μmol/L染料木黄酮能抑制细胞生长(P<0.01);染料木黄酮能负向调节成脂分化后细胞胞浆内TG含量,在0~50μmol/L浓度范围内呈剂量依赖关系,高剂量雌二醇(100 nmol/L)能下调TG含量;染料木黄酮能促进成熟脂肪细胞脂解,提高细胞培养液甘油浓度;染料木黄酮(30μmol/L)能上调p-AMPK、HSL蛋白表达,下调FAS蛋白表达(P<0.01),对ERK、p-ERK、AMPK表达量无明显影响(P>0.05)。ICI182780(1μmol/L)能部分阻断染料木黄酮(30μmol/L)对p-AMPK的调节作用(P<0.05)。结论:染料木黄酮能抑制3T3-L1细胞成脂分化,其机制可能是染料木黄酮一方面下调FAS蛋白表达,抑制脂肪合成,另一方面激活AMPK-HSL途径促进脂肪分解;染料木黄酮还可能通过非雌激素受体途径抑制3T3-L1细胞成脂分化。
AIM: To investigate whether genistein can affect the adipogenic differentiation of 3T3-L1 preadipocytes by estrogen receptor and to explore its possible mechanism. Methods: The 3T3-L1 cells were treated with different concentrations of genistein, and the cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay. In the process of adipogenic differentiation of 3T3-L1 preadipocytes, Genistein, genistein, genistein and ICI182780 (an estrogen receptor inhibitor) mixture and different concentrations of estradiol, oil red staining were observed differentiation results, determination of cell triglyceride (triglyceride, TG) content; genistein, Genistein and ICI182780 mixture and different concentrations of estradiol were used to induce mature adipocytes. The glycerol content of the culture medium was determined. The cells were treated with genistein (30μmol / L), genistein (30μmol / L) and ICI182780 The expressions of p-ERK, p-ERK, AMPK, p-AMPK, hormone sensitivity The expression of lipoprotein (HSL) and fatty acid synthetase (FAS) protein were measured. Results: The survival rate of 3T3-L1 cells decreased with the increase of genistein concentration, and genistein at 50 ~ 200μmol / L inhibited cell growth (P <0.01). Genistein could negatively regulate the adipogenic differentiation The content of TG in plasma was dose-dependent in the range of 0-50 μmol / L, and the high dose of estradiol (100 nmol / L) could decrease the content of TG. Genistein could promote the lipolysis of mature adipocytes and increase the cell culture fluid (30μmol / L) could up-regulate the expression of p-AMPK and HSL protein and decrease the expression of FAS protein (P <0.01), but had no effect on the expression of ERK, p-ERK and AMPK (P> 0.05). ICI182780 (1μmol / L) partially blocked the regulation of p-AMPK by genistein (30μmol / L) (P <0.05). Conclusions: Genistein can inhibit the adipogenic differentiation of 3T3-L1 cells. The mechanism may be that genistein can down-regulate the expression of FAS protein, inhibit the synthesis of lipids, and activate the AMPK-HSL pathway to promote lipolysis. Genistein also may 3T3-L1 cells inhibit adipogenic differentiation through the non-estrogen receptor pathway.