基于FXR-SULT1E1通路探究盐补骨脂致小鼠胆汁淤积性肝损伤的性别差异

Gender difference of cholestatic liver injury induced by salt-processed Psoraleae Fructus in mice based on FXR-SULT1E1 pathway

目的从法尼醇受体(farnesoid X receptor,FXR)-硫酸基转移酶1E1(sulfotransferase 1E1,SULT1E1)通路探讨盐补骨脂致小鼠胆汁淤积性肝损伤性别差异的潜在机制。方法ICR雌性和雄性小鼠连续ig不同剂量(5、10、20 g/kg)的盐补骨脂水煎液30 d,采用HPLC仪检测补骨脂炮制后化学成分含量;采用全自动生化仪检测肝损伤相关指标;取肝脏、脾脏、肾脏、肺脏以及卵巢、睾丸,称定质量并计算脏器指数;采用苏木素-伊红(HE)染色法进行肝脏病理组织学检查;采用透射电镜观察盐补骨脂致胆汁淤积性肝损伤的显微结构变化;采用ELISA法检测血清中雌二醇(estradiol,E_(2))、促卵泡激素(follicle-stimulating hormone,FSH)、促黄体生成素(luteinizing hormone,LH)、白细胞介素-6(interleukin-6,IL-6)、对氧磷酶1(paraoxonase 1,PON1)及FXR含量;采用免疫荧光染色法检测肝脏胆盐输出泵(bile salt export pump,Bsep)、多药耐药相关蛋白2(multidrug resistance protein 2,Mrp2)和SULT1E1的表达;采用Western blotting检测肝脏FXR、Mrp2、胆固醇7α-羟化酶(cholesterol 7α-hydroxylase,CYP7a1)、Bsep、SULT1E1、肝细胞核因子4α(hepatocyte nuclear factor 4α,HNF4α)和核因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)蛋白表达。结果盐补骨脂给药后小鼠肝脏出现明显的肝细胞肿胀、胆管扩张、血清生化指标升高等病理变化,其中雌鼠更为严重(P<0.05、0.01、0.001)。免疫荧光实验显示给药后肝脏Bsep和Mrp2荧光强度均显著降低(P<0.05、0.001),但雌鼠更为明显;雌鼠肝脏中SULT1E1表达水平显著降低(P<0.001),而在雄鼠的肝脏中没有差异。ELISA结果显示雌鼠和雄鼠血清中E2、FSH和LH水平均显著升高(P<0.05、0.01、0.001),雌鼠更为明显。Western blotting结果显示,雌鼠给药后肝脏FXR、Mrp2、Bsep、SULT1E1、HNF4α和Nrf2表达明显降低(P<0.05、0.01、0.001),CYP7a1表达显著升高(P<0.01);雄鼠给药后FXR和Bsep表达明显降低(P<0.05、0.01)。结论盐补骨脂通过HNF4α调控FXR-SULT1E1信号通路,补骨脂下调了胆固醇和胆汁酸生物合成的关键酶SULT1E1,增加了胆汁淤积性肝损伤的风险,这可能是肝损伤性别差异的主要原因。

Objective To explore the potential mechanism of salt-processed Buguzhi(Psoraleae Fructus)induced gender difference of cholestatic liver injury in mice through farnesoid X receptor(FXR)-sulfotransferase 1E1(SULT1E1)pathway.Methods ICR female and male mice were subjected to continuous ig of different dosages(5,10,20 g/kg)of salt-processed Psoraleae Fructus water decoction for 30 d,and chemical composition content of processed Psoraleae Fructus was detected by HPLC;Fully automated biochemical analyzer was used to detect liver injury related indicators;Liver,spleen,kidney,lung,ovary and testis were taken and weighed,organ index was calculated.Hematoxylin-eosin(HE)staining method was used for histopathological examination of liver.The microscopic structure of cholestatic liver injury induced by salt-processed Psoraleae Fructus was observed by transmission electron microscopy.ELISA was used to detect the levels of estradiol(E_(2)),follicle stimulating hormone(FSH),luteinizing hormone(LH),interleukin-6(IL-6),paraoxonase 1(PON1)and FXR in serum;The expressions of bile salt export pump(Bsep),multiple drug resistance protein 2(Mrp2)and SULT1E1 in liver were detected by immunofluorescence staining;Western blotting was used to detect FXR,Mrp2,cholesterol 7α-hydroxylase(CYP7a1),Bsep,SULT1E1,hepatocyte nuclear factor 4α(HNF4α)and nuclear factor E2 related factor 2(Nrf2)protein expressions in liver.Results After the administration of salt-processed Psoraleae Fructus,liver of mice showed significant liver cell swelling,bile duct dilation,and elevated serum biochemical indicators,with female mice showing more severe pathological changes(P<0.05,0.01,0.001).The immunofluorescence experiment showed that fluorescence intensity of Bsep and Mrp2 in liver were significantly decreased after administration(P<0.05,0.001),but it was more pronounced in female mice;The expression level of SULT1E1 was significantly reduced in liver of female mice(P<0.001),while there was no difference in liver of male mice.ELISA results showed a significant increase in E2,FSH and LH levels in serum of both female and male mice(P<0.05,0.01,0.001),with female mice being more pronounced.Western blotting results showed that FXR,Mrp2,Bsep,SULT1E1,HNF4αand Nrf2 expressions in liver of female mice after administration were significantly reduced(P<0.05,0.01,0.001),while the expression of CYP7a1 was significantly increased(P<0.01);The expressions of FXR and Bsep in male mice were significantly decreased after administration(P<0.05,0.01).Conclusion Salt-processed Psoraleae Fructus regulates the FXR-SULT1E signaling pathway through HNF4α,reduces the key enzyme SULT1E1 for cholesterol and bile acid biosynthesis,increases the risk of cholestatic liver injury,which may be the main reason for gender differences in liver injury.