Upregulation of PDZK1 by Calculus Bovis Sativus May Play an Important Role in Restoring Biliary Transport Function in Intrahepatic Cholestasis

Abstract

Intrahepatic cholestasis is a main cause of hepatic accumulation of bile acids leading to liver injury, fibrosis, and liver failure. Our previous studies proved that Calculus Bovis Sativus (CBS) can restore biliary transport function through upregulating the multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) in 17α-ethynylestradiol- (EE-) induced intrahepatic cholestasis rats. The regulation mechanism of CBS on these transporters, however, remains unclear. This study was designed to evaluate the possible relationship between the effect of CBS on transport activities and the regulation of CBS on the expression of PDZK1, a mainly scaffold protein which can regulate MRP2 and BCRP. Intrahepatic cholestasis model was induced in rats with injection of EE for five consecutive days and then the biliary excretion rates and cumulative biliary excretions were measured. The mRNA and protein expression levels of PDZK1 were detected by reverse transcription-quantitative real-time polymerase chain reaction, western blot, and immunohistochemical analysis. When treated with CBS, cumulative biliary excretions and mRNA and protein expressions of PDZK1 were significantly increased in intrahepatic cholestasis rats. This study demonstrated that CBS exerted a beneficial effect on EE-induced intrahepatic cholestasis rats by restoring biliary transport function, which may result from the upregulation of PDZK1 expression.

Figure 1

Effect of CBS on biliary excretion of baicalin (n = 6). (a) Biliary excretion rate of baicalin. (b) Cumulative biliary excretion of baicalin in two hours. Data are represented as mean ± SD of six rats per group. ^∗P < 0.05 versus control group; ^#P < 0.05, ^##P < 0.01 versus EE group by one-way ANOVA and LSD post hoc test.

Figure 2

Effect of CBS on biliary excretion of mitoxantrone (n = 6). (a) Biliary excretion rate of mitoxantrone. (b) Cumulative biliary excretion of mitoxantrone in two hours. Data are represented as mean ± SD of six rats per group. ^∗∗P < 0.01 versus control group; ^##P < 0.01 versus EE group by one-way ANOVA and LSD post hoc test.

Figure 3

mRNA levels of PDZK1, measured by RT-PCR and normalized to β-actin mRNA, relative to the control set as 1. Data are represented as mean ± SD of six rats per group. ^∗∗P < 0.01 versus normal group; ^#P < 0.05, ^##P < 0.01 versus EE group by one-way ANOVA and LSD post hoc test.

Figure 4

Protein levels of PDZK1, measured by western blot and normalized to β-actin protein, relative to the control set as 1. Data are represented as mean ± SD of six rats per group. ^∗P < 0.05, ^∗∗P < 0.01 versus normal group; ^#P < 0.05, ^##P < 0.01 versus EE group by one-way ANOVA and LSD post hoc test.

Figure 5

Immunohistochemistry for PDZK1 expression in rat livers. (a) Control group. (b) EE group. (c) 50 mg/kg CBS group. (d) 150 mg/kg CBS group. The upper right images of each figure are enlargements of the square areas indicating the partial drawing of PDZK1 on the hepatocytes. Positive staining (brown staining) products are denoted by black arrows. Magnification: ×400.