亚慢性氟虫腈暴露致小鼠肝脏脂质代谢紊乱的风险及机制研究

目的 研究氟虫腈(Fipronil, FPN)亚慢性暴露对不同营养条件下成年雄鼠肝脏脂质代谢稳态的影响与分子机制。方法 雄性C57BL/6J小鼠随机分为8组,每组7只,4组喂常规饲料饮食(normal-chow diet,ND),4组喂养高脂饲料饮食(high-fat diet,HFD),每种喂养条件下按FPN处理剂量分为对照组,0.25、1和4 mg/kg组,每日经口灌胃,于染毒5周后处死,称取体重、肝脏重量,并计算肝脏脏器系数;HE染色观察肝脏组织学形态变化,生化分析法检测甘油三酯(triglycerides, TG)、总胆固醇(total cholesterol, TC)、游离脂肪酸(free fatty acids, FFA)水平;Western Blot和qPCR法检测肝脏脂质代谢相关蛋白和基因的表达情况。结果 ND、HFD小鼠肝脏重量和体重均无明显变化。ND小鼠:HE染色未见显著病理性变化,血清TG升高(P<0.05),肝脏TG、TC在0.25 mg/kg组升高(P<0.05),在4 mg/kg组降低(P<0.05),肝脏FFA降低(P<0.05),过氧化物体增殖物激活受体α(peroxisome proliferator activated receptor alpha, PPARα)表达增加(P<0.05),乙酰辅酶A羧化酶(acetyl-coa carboxylase, Acc)在0.25 mg/kg组表达增加(P<0.05),4 mg/kg组表达下调 (P<0.05)。HFD小鼠:HE染色表明FPN暴露后有显著病理变化和脂质沉积。血清TG、TC水平下降(P<0.05),肝脏TG、TC、FFA水平升高(P<0.05)。蛋白PPARα表达随FPN染毒剂量增大而降低(P<0.05),蛋白Acc、胆固醇调节元件结合蛋白-1c (sterol regulatory element-binding protein-1c, Srebp-1c)表达下降(P<0.05)。结论 FPN慢性暴露可导致普通饮食小鼠肝脏脂质代谢紊乱,增加非酒精性脂肪肝(nonalcoholic fatty liver disease, NAFLD)发生风险,低剂量的FPN诱导NAFLD效应更明显;高脂饮食条件下,FPN暴露致小鼠肝脏出现明显病理性损害,且FPN对肝脏脂质代谢的干扰效应呈剂量依赖式变化,FPN暴露剂量越高,发生肝脏脂质异常积累和NAFLD的风险也越高。

Risk and mechanism of hepatic lipid metabolism disorders induced by subchronic exposure to fipronil in mice

Objective To study the effects and molecular mechanisms of subchronic exposure to fipronil (FPN) on the homeostasis of hepatic lipid metabolism in adult male mice under different nutritional conditions. Methods C57BL/6J male mice were randomly divided into 8 groups, each group consisting of 7 mice. 4 groups were fed with a normal-chow diet (ND), while other 4 groups with a high-fat diet (HFD). ND-fed and HFD-fed mice were subgrouped by FPN dosage respectively, including the control group and FPN groups of 0.25 mg/kg, 1 mg/kg and 4 mg/kg. They experienced daily oral gavage, and were sacrificed after 5 weeks of exposure to FPN. Body weight and liver weight were measured, and the liver coefficient was calculated. HE staining was performed to observe hepatic morphological changes. The levels of triglycerides (TG), total cholesterol (TC) and free fatty acids (FFA) were detected by biochemical analysis methods. Western Blot and qPCR were used to determine protein and gene expression related to liver lipid metabolism. Results No obvious alterations in body weight and liver weight were observed in ND-fed or HFD-fed mice after FPN treatment. As for ND-fed mice, no significant hepatic pathological changes were found by HE staining, and serum level of TG increased (P<0.05). Hepatic TG and TC levels increased in 0.25 mg/kg FPN group (P<0.05), but decreased in 4 mg/kg FPN group (P<0.05). Hepatic FFA level declined (P<0.05). PPARα protein expression increased (P<0.05). Acc protein expression increased in 0.25 mg/kg FPN group (P<0.05), but was down-regulated in 4 mg/kg FPN group (P<0.05). As for HFD-fed mice, HE staining revealed that there were significant pathological changes in liver tissue with accumulated lipid deposition. Serum TG and TC levels decreased (P<0.05), while hepatic TG, TC and FFA levels increased (P<0.05). PPARα protein expression decreased with the increasing dosage of FPN (P<0.05), but Acc and Srebp-1c proteinexpression decreased (P<0.05). Conclusion Subchronic exposure to FPN can disturb the homeostasis of hepatic lipid metabolism in ND-fed mice, which may further initiate the development of nonalcoholic fatty liver disease (NAFLD), especially in exposure to low doses of FPN. Under HFD-fed condition, exposure to FPN induces obvious hepatic pathological changes in mice. And there is a dose-response relationship between FPN exposure level and the risk to develop abnormal hepatic lipid accumulation and even NAFLD.