Exosome-derived miR-142-5p from liver stem cells improves the progression of liver fibrosis by regulating macrophage polarization through CTSB

Abstract

Background: This study aims to explore the effect of liver stem cells (LSCs)-derived exosomes and the miR-142a-5p carried by them on the process of fibrosis by regulating macrophages polarization.

Methods: In this study, CCL₄ was used to establish liver 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 liver 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 liver fibrosis model were constructed to verify the expression of miR-142a-5p and ctsb.

Results: Immunofluorescence of LSCs markers CK-18, epithelial cell adhesion molecule (EpCam), and AFP showed that these markers were up-regulated in LSCs. In addition, we evaluated the ability of LSCs to excrete EVs by labeling LSCs-EVs with PKH67. We found that CCL₄ and EVs were simultaneously treated at 50 and 100 μg doses, and both doses of EVs could reduce the degree of liver fibrosis in mice. We tested markers of M1 or M2 macrophage polarization and found that EVs reduced M1 marker expression and promoted M2 marker expression. Further, ELISA was used to detect the secreted factors related to M1 and M2 in tissue lysates, which also verified the above views. Further analysis showed that the expression of miR-142a-5p increased significantly with the increase of EVs treatment concentration and time. Further, in vitro and in vivo LSCs-EVs regulate macrophage polarization through miR-142a-5p/ctsb pathway and affect the process of liver fibrosis.

Conclusion: Our data suggest that EVs-derived miR-142-5p from LSCs improves the progression of liver fibrosis by regulating macrophage polarization through ctsb.

Keywords: exosome; liver fibrosis; liver stem cells; macrophage polarization; miRNA.