Doxazosin Attenuates Liver Fibrosis by Inhibiting Autophagy in Hepatic Stellate Cells via Activation of the PI3K/Akt/mTOR Signaling Pathway

Abstract

Purpose

To investigate the effect of doxazosin on autophagy and the activation of hepatic stellate cells (HSCs) in vivo and in vitro and determine the underlying mechanism.

Methods

In vivo, a mouse liver fibrosis model was induced by the intraperitoneal injection of carbon tetrachloride (CCl₄). Doxazosin was administered at doses of 2.5, 5 and 10 mg/(kg*day) by gavage. After 20 weeks, blood and liver tissues were collected for serological and histological analysis, respectively. Blood analysis, hematoxylin and eosin (HE) staining, Masson’s trichrome staining, immunohistochemistry and immunofluorescence staining were used to measure the extent of liver fibrosis in model and control mice. In vitro, the human HSC cell line LX-2 was cultured and treated with different doses of doxazosin for the indicated times. The effects of doxazosin on LX-2 cell proliferation and migration were examined by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The number of autophagosomes in LX-2 cells was observed by transmission electron microscopy (TEM). Infection with green fluorescent protein (GFP)-LC3B adenovirus, GFP-red fluorescent protein (RFP)-LC3B adenovirus and mCherry-EGFP-LC3 adeno-associated virus was performed to examine changes in autophagic flux in vitro and in vivo. Cell apoptosis was measured by flow cytometry in vitro and by TUNEL assays both in vitro and in vivo. Immunoblotting was performed to evaluate the expression levels of proteins related to fibrosis, autophagy, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR).

Results

Doxazosin inhibited HSC proliferation and migration. HSC activation was attenuated by doxazosin in a concentration-dependent manner in vivo and in vitro. Doxazosin also blocked autophagic flux and induced apoptosis in HSCs. In addition, the PI3K/Akt/mTOR pathway was activated by doxazosin and regulated fibrosis, autophagy and apoptosis in HSCs.

Conclusion

The study confirmed that doxazosin could inhibit autophagy by activating the PI3K/Akt/mTOR signaling pathway and attenuate liver fibrosis.

Keywords:

• doxazosin
• liver fibrosis
• autophagy
• apoptosis
• mTOR

Acknowledgments

This research was supported by grants from the National Nature Foundation of China (81770606 and 81970533).

Statement of Ethics

This study was carried out in strict accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Utilization Committee of Shandong Provincial Hospital affiliated with Shandong University (NSFC: no. 2019-251).

Disclosure

The authors have no conflicts of interest to declare in relation to this study.