【作者】 陈熙；

【导师】 许建明；

【作者基本信息】 安徽医科大学 ， 老年医学， 2009， 博士

【摘要】 利福平是临床上广泛使用的一线抗结核药物,也是我国急性药物性肝损伤的主要病因。本文首次系统观察了体内条件下利福平引起的肝内胆汁淤积及其动态过程,并在成功建立利福平致小鼠肝内胆汁淤积动物模型的基础上,从肝细胞紧密连接蛋白基因表达和完整性改变、肝细胞胆汁酸合成与分解代谢以及胆汁酸的转运过程等不同途径,初步探讨了利福平致肝内胆汁淤积的部分分子机理。1.利福平引起的小鼠肝内胆汁淤积为研究利福平致小鼠肝内胆汁淤积的剂量-效应关系,分别给予小鼠利福平(100 mg/kg·d,150 mg/kg·d,200 mg/kg·d)灌胃,连续1周;为研究不同给药时间对小鼠肝生化指标的影响,分别给予小鼠利福平(200 mg/kg·d)灌胃,连续1周或两周,末次给药后6 h取材。血清生化检测发现,利福平(200 mg/kg·d)1周所致的胆汁淤积性肝损伤最明显。因此,本研究着重探讨连续1周给予利福平(200 mg/kg·d)引起的小鼠肝内胆汁淤积及其分子机理。经口给予利福平(200 mg/kg·d)1周后,小鼠血清总胆红素(TB)上升近70倍,结合胆红素(DB)上升约82倍,碱性磷酸酶(ALP)上升约1.5倍,血清总胆汁酸(TBA)上升约8倍并伴有小鼠血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)轻度升高,这些结果与临床胆汁淤积性肝损伤病人的肝生化指标一致。此外,小鼠肝脏组织TBA上升约2.5倍,肝脏组织HE染色显示肝细胞出现脂肪变性、轻度坏死和炎症。为探讨胆汁淤积发生与发展的时间过程,单次给予利福平(200 mg/kg)后于不同时点(0.5 h,2 h,6 h,12 h)取材,血清生化检测发现,单次给予利福平0.5 h后血清ALT、AST和ALP即开始轻度上升,2-6 h后达到高峰,12 h开始呈下降趋势;而单次给予利福平后血清TB、DB和TBA的变化趋势与血清ALT、AST和ALP一致,但是上升幅度更加明显;单次给予利福平所有时点均未见小鼠肝脏组织病理学损害。这些研究结果提示,单次或连续1周给予利福平(200 mg/kg)均引起小鼠肝内胆汁淤积,并造成轻度的肝细胞损伤。2.肝细胞紧密连接完整性破坏在利福平致小鼠肝内胆汁淤积中的作用为探讨肝细胞紧密连接蛋白相关基因表达的改变在利福平致小鼠肝内胆汁淤积中的作用,采用RT-PCR技术检测肝细胞紧密连接蛋白(ZO)-1、2、3,occludin和claudin-1 mRNA水平。结果显示,尽管单次给予利福平(200 mg/kg)引起ZO-1和ZO-2 mRNA水平短暂下调,但连续给予利福平(200 mg/kg·d)1周对ZO-1、2、3,occludin和claudin-1 mRNA表达水平无明显影响。为探讨肝细胞紧密连接完整性改变在利福平致肝内胆汁淤积中的作用,采用免疫荧光技术检测利福平对ZO-1和occludin定位及完整性的影响。结果显示,给予利福平(200 mg/kg·d)1周后,ZO-1和occludin蛋白条带完整性受损,出现扭曲、断裂和不完整的荧光条带,胞浆内ZO-1和occludin荧光强度明显增强,提示ZO-1和occludin发生细胞内陷;单次给予利福平(200 mg/kg)后0.5 h,ZO-1和occludin荧光条带即出现变形、扭曲和不完整等改变,这些改变至少持续12 h。免疫荧光强度定量分析显示,给予利福平(200 mg/kg·d)1周后, ZO-1荧光强度无明显变化,而occludin荧光强度明显下降;进一步观察发现,单次给予利福平(200 mg/kg)后0.5 h,ZO-1和occludin荧光强度即明显下降,这种改变也至少持续12 h。这些研究结果提示,利福平通过改变肝细胞紧密连接完整性引起小鼠肝内胆汁淤积,未见利福平引起的肝内胆汁淤积与肝细胞紧密连接蛋白基因表达有明显的关联。3.胆汁酸合成和代谢过程在利福平致小鼠肝内胆汁淤积中的作用胆固醇7α羟化酶(CYP7A1)是肝细胞胆汁酸合成关键酶,而细胞色素P450 3A (CYP3A)是肝细胞胆汁酸分解关键酶。为探讨胆汁酸合成和代谢过程在利福平致小鼠肝内胆汁淤积中的作用,用RT-PCR技术检测肝脏cyp7a1和cyp3a11 mRNA水平;用Western blotting技术检测CYP7A1和CYP3A蛋白表达水平。结果显示,给予利福平(200 mg/kg·d)1周后,肝脏cyp7a1 mRNA水平和CYP7A1蛋白水平均明显下降,而cyp3a11 mRNA和CYP3A蛋白水平显著升高。进一步研究显示,cyp7a1 mRNA和蛋白表达水平在给予单剂量利福平(200 mg/kg)后0.5 h出现短暂升高但迅速下降;而cyp3a11 mRNA和CYP3A蛋白表达水平在给予单剂量利福平(200 mg/kg)后表现为迅速而持久的上升。这些研究结果提示,利福平引起的小鼠肝内胆汁淤积与胆汁酸合成及代谢无关。4.利福平对肝细胞胆汁酸转运体的影响为探讨胆汁酸转运体在利福平致小鼠肝内胆汁淤积中的作用,用RT-PCR技术检测了小鼠肝细胞转运体bsep、mrp2、mrp3、ntcp、oatp1和oatp2的mRNA表达水平。初步研究结果显示,给予利福平(200 mg/kg·d)1周后,肝细胞毛细胆管膜侧的转运体bsep、mrp2和mrp3 mRNA表达水平无明显变化;肝细胞基底膜侧转运体ntcp mRNA表达水平轻度下调,而oatp1和oatp2 mRNA表达水平无明显变化。进一步观察发现,给予单剂量利福平(200 mg/kg)后,ntcp mRNA表达水平表现为快速而持久的下降;而bsep、mrp2、mrp3、oatp1和oatp2 mRNA水平均无明显变化。根据以上研究结果,本文得出以下结论:(1)连续多次及单次给予利福平均引起小鼠肝内胆汁淤积;(2)利福平所致的小鼠肝内胆汁淤积与肝细胞紧密连接蛋白ZO-1和occludin的完整性破坏有关;(3)利福平引起的小鼠肝内胆汁淤积与胆汁酸合成及代谢过程无关;(4)利福平对小鼠肝细胞胆汁酸转运体基因表达无明显影响,利福平是否通过改变胆汁酸转运体在肝细胞膜的定位和功能引起胆汁淤积尚需在下一步研究中进一步探讨。更多还原

【Abstract】 Rifampicin, one of the most commonly used anti-tubercular drugs, has been known to be hepatotoxic, but little is known about the mechanism of rifampicin-induced hepatotoxicity. Rifampicin is the main reason of acute drug-induced liver injury in China. Based on establishing a mouse model of rifampicin-induced intrahepatic cholestasis, the present study investigated molecular mechanism of rifampicin-induced cholestasis.1. Rifampicin induced intrahepatic cholestasis in miceTo investigate dose-effect relationships on rifampicin-induced intrahepatic cholestasis, mice were administered with different doses of rifampicin (100 mg/kg·d, 150 mg/kg·d, 200 mg/kg·d) by gastric intubation for 7 consecutive days. To investigate effects of different time of rifampicin on biochemical indicators, mice were administered with rifampicin (200 mg/kg·d) for 7 or 14 consecutive days. All mice were sacrificed at 6 h after the last administration to collect sera and liver tissues. Results showed that serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), conjugated bilirubin (DB), total bile acid (TBA) and alkaline phosphatase (ALP) were significantly increased in a dose - dependent manners. In the rifampicin (200 mg/kg·d for a week) group, serum ALT and AST were increased about 1.5-fold. Importantly, the levels of serum TB, DB, TBA and ALP were increased about 70-fold, 82-fold, 8-fold and 1.5-fold, respectively. Hepatic histology showed a steatosis associated with mild necrosis and inflammation. These results are in agreement with clinical signs of cholestasis. To investigate time-course on rifampicin-induced intrahepatic cholestasis, mice were administered with a single dose of rifampicin (200 mg/kg) by gastric intubation. Serum ALT, AST, ALP, TB, DB and TBA were measured at 0.5, 2, 6 and 12 h after administration of rifampicin. Results showed that all biochemical indicators were significantly increased, beginning at 30 min, being at the highest level from 2 h to 6 h and returning to basal level at 12 h after a single dose of rifampicin. In addition, the level of TBA in liver tissue was also significantly increased after a single dose of rifampicin, whereas a single dose of rifampicin did not result in any pathological damage in mouse liver. These results suggest that either a single dose of rifampicin or a seven-day treatment with rifampicin causes cholestatic liver damage.2. Role of hepatic tight junction on rifampicin-induced cholestasisHepatocyte tight junctions are the only intercellular barriers between the sinusoidal and the canalicular spaces, whose integrity is essential for holding back diffusion of bile constituents from the canalicular spaces to the sinusoidal spaces. To study the effects of rifampicin on the hepatic tight junction, mice were orally administered with either rifampicin (200 mg/kg·d) for 7 consecutive days or a single dose of rifampicin (200 mg/kg). RT-PCR was used to detect the expression of the hepatic ZO-1, 2, 3, occludin and claudin-1 mRNA. Immunofluorescence was used to investigate the effects of rifampicin on the localization and integrity of the hepatic ZO-1 and occludin. Results showed that administration of rifampicin for 7 consecutive days had no effect on the expression of hepatic tight junction mRNA, whereas a single dose of rifampicin induced a slight decrease in hepatic ZO-1 and ZO-2 mRNA, beginning at 30 min, being at the lowest level at 2 h and returning to basal level 12 h after rifampicin administration. Immunofluorescence showed that staining of ZO-1 and occludin in control liver tissue was predominantly limited to focal regions of hepatocyte-hepatocyte contact. ZO-1 and occludin staining in rifampicin-treated mice was tortuous and discontinuous. Densitometric analysis of ZO-1 fluorescence profile showed that although there was no difference on ZO-1 staining intensity in mice treated with rifampicin for 7 consecutive days, a significant broadening of ZO-1 peak was observed in mice treated with rifampicin. In addition, rifampicin treatment for 7 consecutive days induced an obvious decrease in occludin staining intensity with an increased immunostaining of occludin in the intracellular area. A significant broadening of occludin peak was also observed after a week of treatment with rifampicin. In a single dose of rifampicin-treated mice, ZO-1 and occludin staining was tortuous, discontinuous in liver specimens. In addition, the intensity of staining was significantly decreased, beginning at 30 min, and remaining a lower level 12 h after a single dose of rifampicin. These results suggest that rifampicin-induced cholestasis is associated with the altered integrity of hepatocyte tight junctions.3. Role of bile acid synthesis and metabolism on rifampicin-induced choleatasisIn order to explore the effects of rifampicin on bile acid synthesis and metabolic enzyme, RT-PCR and Western blotting were used to detect the levels of cholesterol- 7α-hydroxylase (cyp7a1) and cytochrome P450 3a (cyp3a) mRNA and protein. The results showed that administration of rifampicin for 7 days significantly reduced the level of cyp7a1 mRNA and CYP7A1 protein. By contrary, the expressions of cyp3a11 mRNA and CYP3A protein were obviously up-regulated after a week of treatment with rifampicin. To investigate the effects of a single dose of rifampicin on the expressions of cyp7a1 and cyp3a11, mice were orally administered with 200 mg/kg of rifampicin. Results showed that the levels of cyp7a1 mRNA and protein were slightly increased at 0.5 h, and then decreased quickly. By contrary, the levels of cyp3a11 mRNA and protein were increased, beganing at 0.5 h, and remaining a higher level at 12 h after rifampicin administration. Taken together, these results indicate that rifampicin-induced cholestasis is independent of bile acid synthesis and metabolism in mouse liver.4. Effects of rifampicin on hepatobiliary transportersTo study the role of hepatobiliary transporters in rifampicin-induced intrahepatic cholestasis, mice were administered with rifampicin (200 mg/kg·d) by gastric intubation for 7 consecutive days. The levels of bsep, mrp2, mrp3, ntcp, oatp1 and oatp2 mRNA in mouse liver were determined using RT-PCR. Results showed that no significant difference was observed on the expression of bsep, mrp2 and mrp3 mRNA between rifampicin-treated mice and controls. Although mRNA expression of hepatic ntcp, a basolateral hepatobiliary transporter, was slightly downregulated in rifampicin-treated mice, but rifampicin treatment had no effect on mRNA level of other basolateral hepatobiliary transporters including oatp1 and oatp2. To investigate the effects of a single dose of rifampicin on hepatobiliary transporters, mice were administered with rifampicin (200 mg/kg) by gastric intubation. Results showed that ntcp mRNA level was slightly downregulated, whereas a single dose of rifampicin had no effect on the levels of bsep, mrp2, mrp3, oatp1 and oatp2 mRNA in mouse liver.In summary, we concluded that (1) a single dose of rifampicin or a seven-day administration of rifampicin can induce cholestatic liver damage in mice; (2) rifampicin-induced cholestasis is associated with the altered integrity of hepatocyte tight junctions; (3) rifampicin-induced cholestasis is independent of bile acid synthesis and metabolism in mouse liver; (4) the expression of ntcp mRNA was slightly downregulated in rifampicin-treated mice, whereas rifampicin treatment had no effect on the levels of bsep, mrp2, mrp3, oatp1 and oatp2 mRNA. Further studies are necessary for the role of hepatobiliary transporters in rifampicin-induced choleatasis.更多还原