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.