目的 探讨妊娠期给予槲皮素（quercetin,Que）对双酚A（bisphenol A,BPA）诱导的仔鼠肝脏脂质累积的影响及其机制。方法 15只雌性SD大鼠（12 w）随机分为三组：对照组（生理盐水灌胃）、BPA[1μg/(kg·d) BPA溶液灌胃]、BPA+Que组[1μg/(kg·d) BPA溶液+120 mg/（kg·d）槲皮素溶液灌胃]。自母鼠孕前2 w分别进行灌胃处理,持续干预至哺乳期结束（产后3 w）。鉴别子代性别,实验期间每隔1日测量母鼠及子代雄鼠体重,断乳后将子代雄鼠麻醉处死后取血清和肝脏组织,Western blot检测腺苷酸活化蛋白激酶（adenosine 5'-monophosphate （AMP）-activated protein kinase,AMPK）-固醇调节元件结合蛋白（sterol-regulatory element binding protein 1 ,SREBP1）-脂肪酸合成酶(fatty acid synthase, FAS)/ 乙酰辅酶a羧化酶（acetyl-coenzyme A carboxylase , ACC）等蛋白的表达,q-PCR检测AMPK、SREBP1、FAS、ACC mRNA的表达水平。结果 与对照组相比,BPA组子代雄鼠出生第17 d后体重明显增加,子鼠肝脏重量、肝体比以及血清中ALT、AST、TC、TG含量明显增高（P<0.05）;与BPA组相比,BPA+Que组从第7 d起体重有所下降,断乳后第3 w, BPA+Que组肝脏重量、肝体比以及血清中ALT、AST、TG、TC含量有所下降（P<0.05）。相比与对照组,BPA组油红O结果表明子代雄鼠的肝脏组织中脂滴增多且变大,H&E染色结果显示子代雄鼠肝细胞内脂肪空泡增多增大;与BPA组相比,经槲皮素处理后BPA+Que组脂滴累积情况以及脂肪空泡情况有所减轻。Western blot及q-PCR结果显示,与对照组相比,孕期暴露BPA会导致BPA组p-AMPK/AMPK蛋白及AMPK mRNA表达降低,SREBP1、FAS、ACC蛋白及mRNA表达升高（P<0.05）;与BPA组相比,槲皮素上调了BPA+Que组BPA引起的子代雄鼠肝脏中p-AMPK/AMPK蛋白及AMPK mRNA的表达,同时逆转了SREBP1、FAS、ACC蛋白及mRNA的上调(P<0.05)。结论 孕期及哺乳期低剂量 BPA暴露可导致子代雄鼠的体重增大以及脂肪合成相关调控因子表达增加,而槲皮素对BPA诱导的肝脏脂质累积有一定的改善作用。这对探索槲皮素预防产前BPA暴露对子代肝脏脂质累积的发生有重要的意义。
Abstract
Objective To investigate the effect of quercetin (Que) administration during pregnancy on bisphenol A (BPA)-induced hepatic lipid accumulation in littermates and its mechanism. Methods Fifteen female SD rats (12 w) were randomly divided into three groups: control group (saline gavage), BPA [1 μg/(kg·d) BPA solution by gavage], and BPA+Que group [1 μg/(kg·d) BPA solution + 120 mg/(kg·d) quercetin solution by gavage]. Mothers were treated by gavage separately from two weeks before conception, and the intervention was continued until the end of lactation (3 w after delivery). The sex of the offspring was identified, and the body weight of the female and male offspring was measured every 1 day during the experiment. After weaning, the male offspring were anesthetized and killed, and the serum and liver tissues were collected, and Western blot was used to detect adenosine 5'-monophosphate-activated protein kinase (AMPK), sterol-regulatory element binding protein 1 (SREBP1), fatty acid synthase (FAS), and fatty acid synthase (FAS). AMPK- SREBP1- FAS/acetyl-coenzyme A carboxylase (ACC) were detected by Western blot. The expression of AMPK, SREBP1, FAS and ACC mRNA was detected by q-PCR. Results Compared with the control group, the offspring male rats in the BPA group had a significant increase in body weight at the 17th d of birth, and the offspring rats had a significant increase in liver weight, liver-to-body ratio, and serum ALT, AST, TC, and TG contents (P<0.05). Compared with the BPA group, the BPA+Que group had a decrease in body weight at the 7th d, and in the third week after weaning, the BPA+Que group had a decrease in liver weight, liver-to-body ratio, and serum ALT, AST, TG, and TC contents (P<0.05). Compared with the control group, the oil red O staining in the BPA group showed an increase in the number of lipid droplets in the liver tissues of the offspring male rats. The HE staining showed an increase in the number of fat vacuoles in the hepatocytes of the offspring male rats, while the accumulation of lipid droplets as well as fat vacuoles was reduced in the BPA+Que group compared with the BPA group after quercetin treatment. Western blot and q-PCR results showed that exposure to BPA during pregnancy resulted in decreased expression of p-AMPK/AMPK protein and AMPK mRNA and increased expression of SREBP1, FAS, and ACC proteins and mRNA in the BPA group compared to the control group (P<0.05). Quercetin up-regulated the expression of p-AMPK/AMPK protein and AMPK mRNA in the livers of offspring male rats compared to the BPA group, while reversing the up-regulated levels of SREBP1, FAS, ACC protein and mRNA (P<0.05). Conclusion Low-dose BPA exposure during pregnancy and lactation resulted in body weight gain and increased expression of fat synthesis-related regulatory factors in offspring male rats, and quercetin ameliorated BPA-induced hepatic lipid accumulation. It provides an important implication for exploring the prevention of the hepatic lipid accumulation in the offspring by quercetin in response to prenatal BPA exposure.
关键词
双酚A /
槲皮素 /
肝脏脂质累积 /
子代雄鼠
Key words
bisphenol A /
quercetin /
liver lipid accumulation /
male rat offspring
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基金
军队医学科技青年培育计划拔尖项目（No.20QNY059）; 陕西省自然科学基金面上项目(No.2021JM-240); 陕西省重点研发计划（No.2018SF-061）
