Hepatocytic AP-1 and STAT3 contribute to chemotaxis in alphanaphthylisothiocyanate-induced cholestatic liver injury

by Selleckchem | Sep 13 2023

The development of cholestatic liver injury (CLI) involves inflammation, but the dominant pathway mediating the chemotaxis is not yet established. This work explored key signaling pathway mediating chemotaxis in CLI and the role of Kupffer cells in the inflammatory liver injury. Probe inhibitors T-5224 (100 mg/kg) for AP-1 and C188-9 (100 mg/kg) for STAT3 were used to validate key inflammatory pathways in alpha-naphthylisothiocyanate (ANIT, 100 mg/kg)-induced CLI. Two doses of GdCl3 (10 mg/kg and 40 mg/kg) were used to delete Kupffer cells and explore their role in CLI. Serum and liver samples were collected for biochemical and mechanism analysis. The liver injury in ANIT-treated mice were significantly increased supported by biochemical and histopathological changes, and neutrophils gathering around the necrotic loci. Inhibitor treatments down-regulated liver injury biomarkers except the level of total bile acid. The chemokine Ccl2 increased by 170-fold and to a less degree Cxcl2 by 45-fold after the ANIT treatment. p-c-Jun and p-STAT3 were activated in the group A but inhibited by the inhibitors in western blot analysis. The immunofluorescence results showed AP-1 not STAT3 responded to inhibitors in ANIT-induced CLI. With or without GdCl3, there was no significant difference in liver injury among the CLI groups. In necrotic loci in CLI, CXCL2 colocalized with hepatocyte biomarker Albumin, not with the F4/80 in Kupffer cells. Conclusively, AP-1 played a more critical role in the inflammation cascade than STAT3 in ANIT-induced CLI. Hepatocytes, not the Kupffer cells released chemotactic factors mediating the chemotaxis in CLI.

Comments:

The study you mentioned investigated the key signaling pathways involved in the development of cholestatic liver injury (CLI) and the role of Kupffer cells in inflammatory liver injury. The researchers used probe inhibitors T-5224 and C188-9 to target specific pathways and two doses of GdCl3 to delete Kupffer cells. They collected serum and liver samples for analysis.

The results of the study showed that the liver injury in mice treated with alpha-naphthylisothiocyanate (ANIT) was significantly increased, as evidenced by biochemical and histopathological changes, as well as the presence of neutrophils around necrotic areas. However, the inhibitor treatments were able to down-regulate liver injury biomarkers, except for the level of total bile acid.

The chemokines Ccl2 and Cxcl2 were found to be significantly increased after ANIT treatment. Western blot analysis revealed the activation of p-c-Jun and p-STAT3 in the group treated with ANIT, but these activations were inhibited by the inhibitor treatments. Immunofluorescence results indicated that AP-1, rather than STAT3, responded to the inhibitors in ANIT-induced CLI.

Interestingly, the study found that there was no significant difference in liver injury among the CLI groups with or without GdCl3 treatment, suggesting that Kupffer cells may not play a major role in CLI. In the necrotic areas of CLI, CXCL2 was found to colocalize with the hepatocyte biomarker Albumin, rather than the F4/80 marker associated with Kupffer cells.

In conclusion, the study suggests that AP-1 signaling pathway plays a more critical role than STAT3 in the inflammatory cascade of ANIT-induced CLI. Furthermore, hepatocytes, rather than Kupffer cells, seem to release chemotactic factors that mediate chemotaxis in CLI.