TEC rescued bile metabolic dysfunction in mice with intrahepatic cholestasis induced by ANIT or DDC
We then undertook experiments to verify bile metabolism after TEC intervention in the CLD experimental model. Consistent with previous studies[29], ANIT treatment alone resulted in the accumulation of bile acids in the liver and serum of mice, and reduced the content of bile acids in feces, which suggested that bile flow was mainly obstructed from the liver to the intestine (Figure 3A). CLD in mice was associated with reduced hepatic gene expression of canalicular transporters of bile salts (Bsep encoded by Abcb11), bilirubin (Mrp2, encoded by Abcc2), and phytosterols (the heterodimer sterolin1 and sterolin2, encoded by ABCG5 and ABCG8)[20, 29, 30]. Concomitant with biochemical cholestasis, mRNA expression of export transporters (Mrp2 and Bsep), phytosterol transporters (ABCG5 and ABCG8) and uptake transporters (Ntcp, Oatp) were significantly suppressed in mice with ANIT-induced CLD (Figure 3B). TEC significantly decreased the bile acid pool size in the liver and serum, and markedly increased the bile acid content in feces. Similarly, ANIT-induced inhibition of transporters was reversed after TEC intervention. FXR is the master regulator of bile acids transporters (Bsep, Mrp2 and Ntcp), while LXR is an important regulator of ABCG5 and ABCG8[20]. Therefore, western blotting analysis was performed, and the results showed that TEC intervention restored ANIT-induced decrease in FXR and LXR (Figure 3C).
Additionally, in 0.1% DDC-treated mice bile acids accumulated in the liver and serum, but decreased in feces (Figure 3D). Analysis of the expression of hepatic bile acid and phytosterol export transporters confirmed the expected reduction of the bile acid uptake transporters (Ntcp and Oatp), bile acid export transporters (Mrp2 and Bsep) and phytosterol export transporters (ABCG5 and ABCG8) after 0.1% DDC treatment (Figure 3E). Notably, TEC significantly rescued the bile acid pool size in the liver, serum and feces. In line with this finding, TEC improved the expression of bile acid uptake transporters (Oatp and Ntcp), bile acid export transporters (Mrp2 and Bsep) and phytosterol export transporters (ABCG5 and ABCG8) in 0.1% DDC fed mice. The expression of FXR and LXR was determined by western blot analysis, and the results showed that TEC restored DDC-induced downregulation of FXR and LXR in mouse liver (Figure 3F).
Taken together, these results showed that TEC attenuated ANIT-induced and DDC-induced bile metabolic dysfunction, and exerted a protective effect on CLD.