Autophagy affects hepatic fibrosis progression by regulating macrophage polarization and exosome secretion

Abstract

Background: In this study, the role of autophagy in hepatic fibrosis and its effects on macrophage polarization and exosomes (EVs) were verified by establishing hepatic fibrosis model and co-culture model, providing evidence for treatment.

Methods: In this study, CCL₄ was used to establish hepatic fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB), and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect hepatic fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and hepatic fibrosis model were constructed to verify the expression of miR-423-5p.

Results: Hepatic fibrosis model showed that CCL₄ promoted early autophagy increase but inhibited autophagy flux in liver. mRFP-GFP-LC3 detection showed that both LPS group and Baf group inhibited autophagy flux. This inhibitory effect was reversed by Rap combination therapy. The M1/M2 markers of macrophage polarization were further tested, and it was found that LPS and Baf could promote M1 polarization and inhibit M2 polarization. Rap processing reverses this phenomenon. These data suggest that autophagy can regulate the polarization process of liver macrophages. WB and NTA showed that LPS induced EVs generation. In addition, LPS-induced EVs could promote HSC proliferation, cell cycle, migration, and the expression of fibrosis markers. Macrophage-EVs could affect the fibrosis process of stellate cells through the secretion of miR-423a-5p expression. The hepatic fibrosis model was further established to verify the regulation of autophagy and EVs on the fibrosis process.

Conclusion: This study was showed that autophagy could regulate fibrosis by promoting HSC activation by regulating macrophage polarization and exosome secretion.

Keywords: autophagy; exosome; hepatic fibrosis; hepatic stellate cells; macrophage.