Change Of Expression And Localization Of Canalicular Bsep And Mrp2 In Rifampicin–induced Cholestasis In Mice

Objective Based on the mice model of rifampicin (RIF) -induced liver injury, we detect the molecule mechanism of rifampicin-induced intrahepatic cholestasis. Methods To observe liver injury induced by is rifampicin in mice, eighty females mice were randomly divided into 4 group: Rifampicin (RIF) 1w group and RIF 6 h group: Cholestasis was induced by administration of RIF (200 mg/kg·d) for 7 consecutive days or treatment with a single dose of rifampicin (200 mg/kg) by gastric intubation. Control 1w and 6h group: mice were administered with the equal volume of physiological saline by gastric intubation. After the last administration 6 h, mice were killed to collect blood and liver specimens. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), conjugated bilirubin (DB), total bile acid (TBA); liver TBA; histological analysis was carried out to assess the injury of the liver.To study the effects of the gene expression and integrity of the hepatic bile acid transporters in rifampicin-induced intrahepatic cholestasis, 48 female ICR mice were randomly divided into 4 group: Rifampicin (RIF) 1w group and RIF 6 h group: Cholestasis was induced by administration of RIF (200 mg/kg·d)for 1w or treatment with a single dose of RIF (200 mg/kg) by gastric intubation. Control 1w and 6 h group: mice were administered with the equal volume of physiological saline by gastric intubation. After the last administration 6h, mice were killed to collect liver specimens. RT-PCR was used to detect the expression of the hepatic Besp and Mrp2 mRNA. Immunofluorescence was used to investigate the effects of rifampicin on the localization and integrity of the hepatic Besp and rp2. Results In the RIF 1w group, serum ALT and AST were slightly increased. Importantly, the levels of serum TB, DB, ALP and TBA were increased about 70-fold, 80-fold, 1.5-fold and 5-fold, respectively. The level of TBA in liver tissue was also increased about 2-fold. Hepatic histology showed a steatosis associated with mild necrosis and inflammation. After a single dose of RIF the levels of TB, DB and TBA in serum were significantly increased, but caused a slight increase in ALT and AST and little pathological damage in mouse liver.Results showed that whether given with a single dose of rifampicin (200 mg/kg) 6h, or administration of RIF (200 mg/kg·d) for 7 consecutive days, the expression bile acid transporter Bsep and Mrp2 mRNA levels were not significantly impact by rifampicin.The effects of a seven-day treatment with rifampicin on hepatic bile acid transporter protein localization were characterized. The staining of Bsep and Mrp2 in control liver tissue was predominantly limited to focal regions of hepatocyte canalicular membranes. Bsep and Mrp2 staining in rifampicin-treated mice was tortuous and discontinuous. Densitometric analysis of Bsep and Mrp2 fluorescence profile showed that an obvious decrease in occludin staining intensity had an increased immunostaining of Bsep and Mrp2 in the intracellular area. A significant broadening of Bsep and Mrp2 peak was also observed after a week of treatment with rifampicin. In a single dose of rifampicin-treated mice, Bsep and Mrp2 staining was tortuous, discontinuous in liver specimens. In addition, the intensity of staining was significantly decreased after a single dose of rifampicin 6h.Conclusions These results suggest that the altered integrity and internalization of hepatocyte bile acid transporters are associated with rifampicin-induced cholestasis.