Figure 1. TEC alleviates ANIT-induced intrahepatic cholestasis.C57BL/6J mice were pre-treated with TEC for 3 days followed by ANIT treatment for 48 h. (A) Serum level of AST, ALT, γ-GT, and AP were measured by ELISA kits, n=6. (B) Representative images of hepatic HE staining were shown. (C) Representative images of hepatic TUNEL staining (left) and quantitative statistics (right) are shown in the figure. (D) The hepatic activity of caspase-3 was determined by following the kit instructions, n=6. (E-F) E: liver-isolated immune cells were stained for CD11b and F4/80 and analyzed by flow cytometry, representative dot plots are shown. F: further quantification of flow cytometry. (G) ELISA kits were used to detect inflammatory factors (IL-1β) in serum, n=6. (H) The mRNA levels of TNF-α, CCR2 and MCP-1 were measured by qRT-PCR, n=6. *p<0.05. The data represent the mean ± SD.
Further experiments in mice fed with TEC in a diet containing 0.1% DDC for 2 weeks confirmed hepatocyte protection by TEC in cholestatic liver. DDC-fed mice showed marked liver injury compared to wild-type mice fed normal chow, as shown by increased AP, aspartate transaminase (AST), alanine transaminase (ALT) and γ-glutamyltransferase (γ-GT) (Figure 2A). The results of HE staining and Sirius red staining also supported these findings, as seen by decreased areas of tissue damage and collagen deposition in mouse liver after TEC intervention (Figure 2B and 2C). Flow cytometry analysis showed that the 0.1% DDC diet increased the recruitment of macrophages in the liver of mice (Figure 2D and 2E). In accordance with this result, the mRNA level of inflammatory and profibrogenesis factors were also dramatically increased in 0.1% DDC-fed mice (Figure 2F and 2G). As expected, TEC significantly reduced hepatic macrophage recruitment, and the expression of inflammatory and profibrogenesis factors in the liver.
Overall, our results show that TEC treatment attenuated liver injury induced by ANIT or DDC.