不同性激素诱导的SD大鼠妊娠期肝内胆汁淤积症模型的肝脏病理生理研究

Liver pathophysiology of Sprague-Dawley rat models of intrahepatic cholestasis of pregnancy induced by different sex hormones

摘要:

目的 通过分析雌二醇、孕酮诱导的大鼠妊娠期肝内胆汁淤积症（intrahepatic cholestasis of pregnancy，ICP）模型的肝脏损伤水平、胆汁酸谱以及炎症因子、氧化应激水平，探索妊娠期肝内胆汁淤积的发病机制。方法 妊娠期Sprague-Dawley（SD）大鼠颈部皮下注射雌二醇或孕酮构建大鼠ICP模型。通过检测血清总胆汁酸（total bile acid，TBA）、丙氨酸氨基转移酶（alanine amiotransferase，ALT）、门冬氨酸氨基转移酶（aspartate aminotransferase，AST）、碱性磷酸酶（alkaline phosphatase，ALP）水平评价模型是否成功；通过肝脏病理切片评价肝脏损伤情况；采用液相色谱-质谱联用仪分析肝脏胆汁酸谱；使用RT-qPCR检测肝脏中炎症因子肿瘤坏死因子a（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、白细胞介素-6（interleukin-6，IL-6）的转录水平；使用试剂盒检测肝脏氧化和抗氧化标志物丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）和谷胱甘肽（glutathione，GSH）的水平；采用RT-qPCR和Western blotting检测肝脏抗氧化应激标志物核因子红系2相关因子2（nuclear factor erythroid 2-related factor 2，NRF2）、谷氨酸-半胱氨酸连接酶修饰亚基（glutamate-cysteine ligase modifier subunit，GCLM）、谷氨酸-半胱氨酸连接酶催化亚基（glutamate-cysteine ligase catalytic subunit，GCLC）、 NAD（P）H醌氧化还原酶-1［NAD（P）H quinone oxidoreductase-1，NQO1］、血红素氧合酶-1（heme oxygenase-1，HO-1）的mRNA和蛋白质水平。结果 雌激素组、孕激素组大鼠血清TBA、ALT、AST、ALP水平均较对照组显著升高。HE染色显示雌激素组大鼠肝血窦变窄，胆汁淤积；孕激素组部分肝细胞水肿，少量脂肪变性细胞出现。胆汁酸谱在各组间有较大差异。雌激素组肝脏中TNF-α、IL-1β、IL-6、MDA水平上升，而GSH、SOD和NRF2、GCLM、GCLC、NQO1的mRNA和蛋白质水平下降，而孕激素组均无明显变化。结论 雌激素、孕激素均能诱导大鼠发生ICP，其中雌激素可抑制NRF2-GCLM、NRF2-GCLC通路并导致肝脏损伤加重，而孕激素则无以上作用。

Abstract:

Objective To explore the pathogenesis of intrahepatic cholestasis of pregnancy（ICP） in rats induced by either estradiol or progesterone，through determining the levels of liver damage，bile acid profiles，inflammatory factors，and oxidative stress.Methods ICP models were established by subcutaneous injection of either estradiol or progesterone in the neck of Sprague-Dawley（SD） rats during pregnancy. The models were evaluated by measuring the levels of TBA（serum total bile acid），ALT（alanine aminotransferase），AST（aspartate aminotransferase），and ALP（alkaline phosphatase）. Liver injury was evaluated through pathological section observation. The bile acid profiles of the liver were analyzed by liquid chromatography-mass spectrometry. The transcription levels of inflammatory cytokines（tumor necrosis factor-α[TNF-α]，interleukin[IL]-1β，and IL-6） in the liver were measured using RT-qPCR. The levels of oxidative and antioxidative markers（malondialdehyde[MDA]，glutathione[GSH]，and superoxide dismutase[SOD]） were determined using commercial kits. The mRNA and protein levels of antioxidative stress markers（nuclear factor erythroid 2-related factor 2[NRF2]，glutamate-cysteine ligase modifier subunit[GCLM]，glutamate-cysteine ligase catalytic subunit[GCLC]，NAD（P）H quinone oxidoreductase-1[NQO1]，and heme oxygenase-1[HO-1]） in the liver were determined using RT-qPCR and Western blotting，respectively.Results The levels of serum total bile acid，ALT，AST，and ALP in the estradiol group and progesterone group were significantly higher than those in the control group. HE staining showed narrower hepatic sinusoids and cholestasis in the estradiol group and hepatocyte swelling and mild hepatic steatosis in the progesterone group. The bile acid profiles were significantly different between the groups. In the estradiol group，the levels of TNF-α，IL-1β，IL-6，and MDA in the liver were significantly increased，while the levels of GSH and SOD as well as the mRNA and protein levels of NRF2，GCLM，GCLC and NQO1 were significantly decreased，with no significant changes in the progesterone group.Conclusion Both estrogen and progesterone induce ICP in rats. Estrogen can inhibit the NRF2-GCLM and NRF2-GCLC pathways to aggravate liver injury，while progesterone has no such effect.