【作者】 周方；

【导师】 许红梅；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 硕士

【摘要】 背景:胆汁淤积是正常的胆汁流被损害或阻断,胆汁酸和其他有害物质在肝脏中过度蓄积而引发的肝损伤。它是多种疾病的组成部分,如:胆石症、妊娠肝内胆汁淤积症、原发性胆汁性肝硬变和原发性硬化性胆管炎等。近年来研究发现定位于肝细胞膜窦面和微胆管面上的转运体对维持正常的胆汁流和肝脏功能起着重要作用。大黄素即1,3,8-三羟基-6-甲基蒽醌,是从已广泛用于临床治疗淤胆型肝炎的传统中药大黄根茎提纯的一种蒽醌类衍生物,具有广泛的药理学作用,如:抗炎、抗病毒、抑菌、免疫调节和肝保护等。推测大黄素能缓解胆汁淤积可能与调节肝细胞膜上转运体基因的表达有关。第一部分:大黄素对肝内胆汁淤积大鼠的保护作用目的:探讨大黄素对α-萘异硫氰酸酯(ANIT)致急性肝内胆汁淤积的保护作用。方法:采用ANIT灌胃制备大鼠急性肝内胆汁淤积病理模型。将32只大鼠随机平均分为正常组、正常+大黄素组、模型组和模型+大黄素组。造模前,模型+大黄素和正常+大黄素组给予大黄素40mg/kg/d灌胃,正常组及模型组给予羧甲基纤维素钠处理,均持续到处死动物之前,于实验第5天给药后4小时,模型组和模型+大黄素组100mg/kg ANIT灌胃1次建立模型,其余两组予以橄榄油对照处理,建模后48小时处死动物,收集标本。观察各组实验动物肝功能各项生化指标TBiL, DBiL, ALT , AST , ALP和TBA的变化和肝脏组织病理学改变。结果:(1)模型组与正常组比较,TBiL,DBiL,ALT,AST,ALP和TBA浓度明显增高(P<0.01);光镜下可见肝细胞变性和坏死、中性粒细胞浸润。(2)模型+大黄素组与模型组比较,TBiL, DBiL, ALT, AST,ALP和TBA均降低(P<0.01或P<0.05);组织病理学改变轻微。(3)正常+大黄素组与正常组比较上述各指标均无显著变化(P>0.05)。结论:大黄素能明显降低胆汁淤积大鼠血清中TBiL,DBiL,ALT,AST,ALP和TBA浓度,显著降低胆汁酸的毒性作用,抑制肝损伤。第二部分:大黄素对大鼠肝内胆汁淤积相关转运体基因表达的研究目的:探讨大黄素治疗大鼠急性肝内胆汁淤积的作用机制。方法:采用ANIT灌胃制备大鼠急性肝内胆汁淤积病理模型,将32只大鼠随机分为正常组、正常+大黄素组、模型组和模型+大黄素组,采用实时荧光定量RT-PCR检测肝细胞膜转运蛋白基因胆盐输出泵(BSEP)、多药耐药相关蛋白2(MRP2)、钠-牛磺胆酸共转运蛋白(NTCP)、多药耐药蛋白2(MDR2)、多药耐药蛋白1a(Mdr1a)、多药耐药蛋白1b(Md1rb)、多药耐药相关蛋白3(MRP3)和核受体法尼酯衍生物X受体(FXR)mRNA水平的表达,Western blot方法检测肝脏P-糖蛋白(P-gp)的变化。结果:(1)模型组与正常组比较,在mRNA水平NTCP和FXR表达降低(P<0.01),MDR2, Mdr1b和MRP3表达增高(P<0.01或P<0.05),BSEP,Mdr1a和MRP2表达无显著差异(P>0.05);在蛋白水平P-gp表达增高(P<0.05)。(2)模型+大黄素组与模型组比较, NTCP,MDR2,Mdr1a,Mdr1b和MRP3 mRNA水平表达高于模型组(P<0.01或P<0.05),但BSEP, MRP2和FXR的表达无显著差异(P>0.05);P-gp表达同时也高于模型组(P<0.05)。(3)正常+大黄素组与正常组比较上述各指标均无显著变化(P>0.05)。结论:大黄素通过上调肝脏中与胆汁酸代谢相关的转运蛋白MRP3和P-gp的表达以减少胆汁酸及其他有毒化合物在肝脏中的蓄积可能为其退黄、恢复肝脏功能的作用机制之一更多还原

【Abstract】 Background: Cholestasis, an impairment or cessation in the flow of bile, causes hepatocoxicity due to the buildup of bile acids and other toxins in the liver. Cholestasis is a component of many liver diseases, including cholelithiasis, cholestasis of pregnancy, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis. The normal bile flow is fulfilled by the activity of a large panel of transporters located at the sinusoidal or at the apical pole of the plasma membrane (PM) of hepatocytes.Emodin, 1, 3, 8-trihydroxy-6-methyl-anthraquinone, is an anthraquinone derivative from the roots of Rheum officinale Baill that has been widely used for the treatment of cholestatic hepatitis. It had pharmacological activities such as inhibitory activity of inflammation, anti-virus, anti-bacteria, immunosuppression, hepatoprotection and so on. So emodin may regulate expression of the transporter gene to relieve cholestasis. PARTⅠProtective efect of emodin on intrahepatic cholestasis in ratsObjective: To investigate the protective effect on acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT) in rats.Methods: 32 SD rats were randomly divided into 4 groups: control group, emodin group without ANIT treatment, model group and emodin group with ANIT treatment. Before establishing the animal model, Emodin (40mg/kg/d) was intragastrically administrated to the rats in emodin group with and without ANIT treatment. Model and blank control were fed by sodium carboxymethylcellulose. All groups did not stop being administrated treating agent daily until executed. At the 5th day after administration and fasting for 4h, Model and emodin group with ANIT treatment were intragastrically administrated ANIT(100mg/kg) for modeling. Blank control and emodin group without ANIT treatment were fed by olive oil. At 48h after modeling, all rats were executed for taking specimens. Liver function and pathological changes of hepatic tissue were examined.Results: (1) In the model group compared with the normal control group,the concentrations of serum total bilirubin(TBiL), direct bilirubin (DBiL), alanine aminotransferase (ALT), aspartate aminotransferase (AST),alkaline phosphatase(ALP) and total bile acid(TBA) were increased(P<0.01); inflammatory cell infiltration,hepatic cellular change and necrosis could be observed by light microscop;(2) Compared to the model group, Emodin treatment resulted in significant reductions in TBiL, DBiL, ALT, AST, ALP and TBA (P<0.01 or P<0.05). By observing the liver pathology, it was found that hepatic cellular change and necrosis, inflammatory cell infiltration and bile duct proliferation were notably alleviated in emodin model with ANIT treatment. (3) In emodin group without ANIT treatment compared with the normal control group,the changes of Liver function and histopathology were not significant difference(P>0.05).Conclusion: Emodin remarkably could reduce the concentrations of serum TBiL, DBiL, ALT , AST , ALP and TBA,relieve the toxicity of bile acid and inhibit the hepatic injury. PARTⅡEffect of emodin on expression of transporter genes related to intrahepatic cholestasis of ratsObjective: To investigate the mechanism of emodin on acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT) in rats.Methods: Acute cholestatic model in rats was induced by ANIT. Normal control group, emodin group without ANIT treatment, model group and emodin group with ANIT treatment were set up. Real-time fluorescent quantitative RT-PCR was used to detect the mRNA levels of the hepatic transport protein genes bile salt export pump (BSEP) , multidrug resistance-associated protein 2 (MRP2),Na+/taurocholate cotransporting peptide (NTCP),multidrug resistance protein 2 (MDR2),multidrug resistance protein 1a (Mdr1a), multidrug resistance protein 1b (Mdr1b), multidrug resistance-associated protein 3 (MRP3) and the nuclear receptor farnesoid X receptor (FXR). The expression of P-gp (P-glycoprotein) was determined by Western blotting analysis.Results: (1) In the model group compared with the normal control group, the genes expression of NTCP and FXR were down-regulated(P<0.01),the MDR2 , Mdr1b and MRP3 were up-regulated(P<0.01 or P<0.05).but the expression of BSEP,Mdr1a and MRP2 could not be affected(P>0.05).The expression of P-gp was increased (P<0.05). (2) Compared to the model group, Analysis of gene expression in livers from emodin-treated cholestatic rats revealed that NTCP, MRP3, Mdr1a, Mdr1b and MDR2 could be up-regulated (P<0.01 or P<0.05), but the expression of BSEP, FXR and MRP2 could not be affected(P>0.05). The expression of P-gp was increased in accordance with its mRNA (P<0.05) .(3) In emodin group without ANIT treatment compared with the normal control group,the changes of transporter genes were not significant difference(P>0.05).Conclusion: Emodin had a protective effect on hepatocytes and a restoring activity on cholestatic hepatitis. Mechanism of Emodin action on intrahepatic cholestasis may be related to induce expression of the bile-metabolism-related transporter MRP3 and P-gp in the liver to prevent bile acids and other toxic compounds overaccumulation in hepatocytes and hepatic toxicity.更多还原