Constitutive androstane receptor agonist, TCPOBOP, attenuates steatohepatitis in the methionine choline-deficient diet-fed mouse

Abstract

Aim: To ascertain whether constitutive androstane receptor (CAR) activation by 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP) modulates steatohepatitis in the methionine choline-deficient (MCD) diet-fed animal.

Methods: C57/BL6 wild-type mice were fed the MCD or standard diet for 2 wk and were treated with either the CAR agonist, TCPOBOP, or the CAR inverse agonist, androstanol.

Results: Expression of CYP2B10 and CYP3A11, known CAR target genes, increased 30-fold and 45-fold, respectively, in TCPOBOP-treated mice fed the MCD diet. TCPOBOP treatment reduced hepatic steatosis (44.6 +/- 5.4% vs 30.4 +/- 4.5%, P < 0.05) and serum triglyceride levels (48 +/- 8 vs 20 +/- 1 mg/dL, P < 0.05) in MCD diet-fed mice as compared with the standard diet-fed mice. This reduction in hepatic steatosis was accompanied by an increase in enzymes involved in fatty acid microsomal omega-oxidation and peroxisomal beta-oxidation, namely CYP4A10, LPBE, and 3-ketoacyl-CoA thiolase. The reduction in steatosis was also accompanied by a reduction in liver cell apoptosis and inflammation. In contrast, androstanol was without effect on any of the above parameters.

Conclusion: CAR activation stimulates induction of genes involved in fatty acid oxidation, and ameliorates hepatic steatosis, apoptosis and inflammation.

Figure 1

Over-expression of CAR target genes in MCD diet-fed mice treated with TCPOBOP. CAR activation was assessed by measuring CYP2B10 and CYP3A11 (CAR target genes) expression in whole liver from vehicle-treated and TCPOBOP-treated chow-fed and MCD diet-fed mice. Expression was measured by real-time PCR and normalized as a ratio using 18S mRNA as housekeeping genes. A value of 1 for this ratio was arbitrarily assigned to the data obtained from vehicle-treated CAR^+/+ mice. (A) CYP2B10 and (B) CYP3A11 mRNA expressions were increased in TCPOBOP-treated (3 mg/kg ip for 3 d) chow-fed mice (^bP < 0.01 for CYP2B10 and CYP3A11 compared to control) and MCD diet-fed mice (^dP < 0.01 for CYP2B10 and CYP3A11 compared to untreated). This phenomenon was abated by treatment with a CAR inhibitor androstanol (100 mg/kg ip) daily for 3 d at the starting of weeks one and two of the MCD diet (total of 6 injections), (n = 5 in each group).

Figure 2

Hepatic and serum fat content reduced by TCPOBOP treatment in the MCD diet-fed mice. (A) Top panel, from left to right: Fixed liver specimens from vehicle-treated chow-fed mice, TCPOBOP-treated (3 mg/kg ip for 3 d) chow-fed mice, vehicle-treated (corn oil) MCD diet-fed diet mice for 2 wk, TCPOBOP-treated MCD diet-fed mice for 2 wk; CAR inhibitor, androstanol-treated MCD diet-fed mice for 2 wk were stained by conventional H&E. Lower panel: Quantitation of hepatic fat content by Oil red O staining. Note the marked reduction of hepatic fat in the TCPOBOP-treated MCD diet-fed mice (^bP = 0.001); (B) Serum triglyceride levels from mice fed the MCD diet for 2 wk with either TCPOBOP or androstanol treatment. Treatment with TCPOBOP reduced serum triglyceride levels by half (^aP = 0.03).

Figure 3

Hepatocyte apoptosis is attenuated by TCPOBOP in the MCD-diet fed animal. (A and B) Fixed liver specimens were analyzed by TUNEL and immunofluorescence for active caspase 3/7 to identify apoptotic liver cells. The percent/field area of TUNEL- (^bP = 0.01) and active caspase 3/7-positive cells (^aP = 0.03) were significantly higher in vehicle-treated MCD diet-fed mice than TCPOBOP-treated (3 mg/kg ip for 3d) MCD diet-fed mice (n = 5 in each group).

Figure 4

Hepatic inflammation attenuated by TCPOBOP treatment in the MCD diet-fed mice. Top panel: Representative photomicrographs of immunohistochemistry for CD68, a marker of Kupffer cells; Lower panel: The percentage of CD68-positive areas in the liver sections was quantitated using digital image analysis. Immunoreactivity for CD68 was significantly reduced in TCPOBOP-treated MCD diet-fed mice as compared with vehicle- and androstanol-treated mice (^bP = 0.002) (n = 5 in each group).