金雀异黄素在去卵巢非酒精性脂肪性肝病小鼠模型中的保护作用及机制

Protective effect of Genistein against nonalcoholic fatty liver disease in ovariectomized mice and its mechanism

目的探讨金雀异黄素(Genistein)在去卵巢(OVX)小鼠非酒精性脂肪性肝病(NAFLD)中的保护作用及机制。方法取40只6周龄雌性C57BL/6小鼠建立OVX小鼠模型,随机分为5组,每组8只,分别为对照组、模型4周组、模型6周组、模型8周组、模型10周组。相同环境下,采用高脂饮食对5组OVX小鼠进行饮食造模,病理学检查显示,经10周高脂饮食诱导NAFLD成功。另取40只6周龄雌性C57BL/6小鼠随机分为5组:空白组、假手术(Sham)组、OVX组、OVX+L-Genistein(4 mg/kg体质量)组、OVX+H-Genistein(8 mg/kg体质量)组。Sham组进行相同的OVX手术过程,但不结扎卵巢动脉和切除卵巢。空白组小鼠正常饮食喂养,其余各组供给高脂饮食。将Genistein溶解在DMSO中,Sham组和OVX组的动物仅用溶媒溶液处理,所有小鼠每天灌胃给药1次,持续10周。记录小鼠体质量及脏器指数,处死小鼠并收集血清及肝组织。试剂盒检测血清ALT、AST活性及TG、TC水平。HE和油红O染色观察肝组织病理。通过Western Blot分析肝组织中脂质代谢相关的固醇调节元件结合蛋白1(SREBP-1c)、过氧化物酶体增殖物激活受体α(PPARα)的蛋白表达。计量资料多组间比较采用单因素方差分析,进一步两组间比较采用Dunnett-t检验。结果高脂饮食10周后,Genistein低、高剂量组的体质量、肝指数和肝质量均低于OVX组(P值均<0.05)。此外,Genistein显著下调了血清TC、TG水平(P值均<0.05),降低了血清中AST和ALT活性(P值均<0.05)。HE和油红O染色结果表明,Genistein低、高剂量组较OVX组脂滴累积明显减少。Western Blot分析结果显示,给予Genistein干预后,SREBP-1c的蛋白表达量明显下降,PPARα的蛋白表达量明显升高(P值均<0.05)。结论Genistein对OVX小鼠NAFLD具有保护作用,其部分作用机制可能与调节SREBP-1c、PPARα的表达有关,从而促进脂肪酸氧化和抑制肝脏脂质合成。

Objective To investigate the protective effect of Genistein against nonalcoholic fatty liver disease(NAFLD)in ovariectomized(OVX)mice and its mechanism.Methods A total of 40 female C57BL/6 mice,aged 6 weeks,were used to establish an OVX mouse model,and then they were randomly divided into blank group,4-week model group,6-week model group,8-week model group,and 10-week model group,with 8 mice in each group.Under the same environmental conditions,the mice were given high-fat diet for modeling,and pathological examination showed that NAFLD was successfully induced by 10-week high-fat diet.Another 40 female C57BL/6 mice,aged 6 weeks,were randomly divided into blank group,sham operation group(Sham group),OVX group,OVX+L-Genistein(4 mg/kg body weight)group,and OVX+H-Genistein(8 mg/kg body weight)group.The mice in the Sham group were given the same procedure of OVX,without the ligation of the ovarian artery and the resection of the ovary.The mice in the blank group were given normal diet,and those in the other groups were given high-fat diet.Genistein was dissolved in DMSO,and the mice in the Sham group and the OVX group were treated with solvent solution alone by gavage,once a day for 10 consecutive weeks.Body weight and visceral index were recorded,and the mice were sacrificed to collect serum and liver tissue.Kits were used to measure the activity of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)and the serum levels of triglyceride(TG)and total cholesterol(TC),and HE staining and oil red O staining were used to observe liver histopathology;Western blot was used to measure the protein expression levels of sterol regulatory element-binding protein 1c(SREBP-1c)and peroxisome proliferator-activated receptor alpha(PPARα)associated with lipid metabolism in liver tissue.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the Dunnett-t test was used for further comparison between two groups.Results After 10 weeks of high-fat diet,the OVX+L-Genistein group and the OVX+H-Genistein group had significantly lower body weight,liver index,and liver tissue weight(all P<0.05).In addition,Genistein significantly downregulated the serum levels of TC and TG(P<0.05)and reduced the activities of serum AST and ALT(P<0.05).HE and oil red O staining showed that compared with the OVX group,the OVX+L-Genistein group and the OVX+HGenistein group had a significant reduction in the accumulation of lipid droplets.Western blot showed that after Genistein intervention,there was a significant reduction in the protein expression level of SREBP-1c and a significant increase in the protein expression level of PPARα(P<0.05).Conclusion Genistein exerts a protective effect against NAFLD in OVX mice possibly by regulating the expression of SREBP-1c and PPARα,thereby promoting fatty acid oxidation and inhibiting liver lipid synthesis.