双酚A不同昼夜节律时间点暴露对小鼠肝脏脂质代谢的影响

Effects of bisphenol A exposure at different circadian time on hepatic lipid metabolism in mice

[背景]肝脏脂质代谢呈现昼夜变化,昼夜节律影响环境化学物的肝毒性。[目的]观察不同昼夜节律时间点双酚A(BPA)暴露对小鼠肝脏、血液脂质代谢指标影响的差异,探索昼夜节律核心基因在BPA干扰肝脏脂质代谢中的作用机制。[方法]首先选用35只雌性C57BL/6J小鼠在24 h光照周期内(光照/黑暗为12 h/12 h)每隔4 h处死5只,收集肝脏组织,检测昼夜节律核心基因Rev-erbα、Bmal1、Clock及调控脂质代谢关键转录因子基因Srebp1c、Chrebp在24 h内不同昼夜节律时间点的表达。随后,将30只雌性C57BL/6J小鼠随机分为6组,每个时间点设置对照组、BPA 50μg·kg^(−1)·d^(−1)和BPA 500μg·kg^(−1)·d^(−1)组。分别于开灯3 h后(ZT3,光照期)和关灯3 h后(ZT15,黑暗期)两个时间点经口灌胃BPA,每日灌胃1次,连续4周。实验期间小鼠自由饮食,每周测量体重。实验结束后在ZT3收集血浆,分离肝组织,测定生化指标,检测肝昼夜节律核心基因和脂质代谢基因mRNA表达水平。[结果]小鼠肝脏Rev-erbα、Bmal1和Clock以及Srebp1c、Chrebp转录水平呈现24 h昼夜节律。ZT3和ZT15 BPA染毒4周均未引起小鼠体重、血浆葡萄糖、血浆总胆固醇、血浆低密度脂蛋白胆固醇和血浆甘油三酯明显改变(P>0.05)。ZT3染毒时,50μg·kg^(−1)·d^(−1)BPA组小鼠血浆高密度脂蛋白胆固醇含量较对照组降低了14.56%(P<0.05)。ZT15染毒时,50μg·kg^(−1)·d^(−1)BPA组小鼠肝脏甘油三酯含量较对照组增加了115.20%(P<0.05)。与对照组相比,ZT3染毒时,BPA组Srebp1c mRNA表达下调;ZT15染毒时,BPA组Chrebp、Srebp1c、Acc1 mRNA表达上调(P<0.05)。ZT3染毒时,BPA组Bmal1 mRNA表达上调,Rev-erba mRNA表达下调;而ZT15染毒时,BPA组Bmal1 mRNA表达下调,Rev-erba mRNA表达上调(P<0.05)。[结论]不同昼夜节律时间点BPA暴露对小鼠肝脏脂质代谢的影响不同。黑暗期染毒引起肝脏脂质沉积,可能的机制是干扰Rev-erbα和Bmal1构成的昼夜节律反馈环路,上调调控脂质合成的核受体Srebp1c和Chrebp及其靶基因Acc1表达水平,促进肝脏脂质沉积。

[Background]Lipid metabolism in liver shows circadian-dependent profiles.The hepatotoxicity of environmental chemicals is dependent on circadian time.[Objective]To observe the effects of bisphenol A(BPA)exposure at different zeitgeber time(ZT)on hepatic and blood lipid metabolism and decipher the underlying mechanisms related to circadian rhythm in mice.[Methods]Thirty-five female C57BL/6J mice were sacrificed every 4 h in a light-dark cycle(12 h/12 h).The liver tissues were collected to describe the circadian profiles of hepatic Rev-erba,Bmal1,Clock,Srebp1c,and Chrebp mRNA expression levels within 24 h.Thirty female mice were divided into 6 groups by the timing(ZT3 represents the 3 h after light on,ZT15 represents the 3 h after light off)and dose(50 or 500μg·kg^(−1)·d^(−1))of BPA exposure to observe hepatotoxicity.Mice were gavaged with designed doses of BPA once per day for 4 weeks.Mice were maintained with ad libitum access to food and water and measured body weight weekly.After the experiment,mice were euthanatized and liver tissues were separated to determine the biochemical indicators of lipid metabolism and lipid metabolism-and circadian-related gene mRNA expressions.[Results]Hepatic Rev-erba,Bmal1,Clock,Srebp1c,and Chrebp mRNA expression levels were rhythmic during a 24 h period in mice.At ZT3 and ZT15,BPA did not alter body weight,plasma glucose,plasma total cholesterol,plasma low density lipoprotein cholesterol,and plasma triglycerides(P>0.05).The plasma high density lipoprotein cholesterol decreased in the 50μg·kg^(−1)·d^(−1)BPA group at ZT3 by 14.56%compared with the control group(P<0.05).The liver triglycerides increased in the 50μg·kg^(−1)·d^(−1)BPA group at ZT15 by 115.20%compared with the control group(P<0.05).BPA decreased Srebp1c mRNA expression level when dosing at ZT3 and increased Chrebp,Srebp1c,and Acc1 mRNA expression levels when dosing at ZT15 compared with the control group(P<0.05).BPA increased Bmal1 mRNA expression level and decreased Rev-erbαmRNA expression level at ZT3 exposure and decreased Bmal1 and increased Rev-erbαmRNA expression level at ZT15 exposure(P<0.05).[Conclusion]BPA exposure at light or dark period has different effects on hepatic lipid metabolism in mice.Hepatic lipid deposit appears when BPA is dosed at dark period.Rev-erbα-Bmal1 regulation circuits and the subsequent upregulation of Srebp1c and Chrebp and the target gene Acc1 may be involved.