| 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.
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