Department of Gastroenterology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, People's Republic of China.
Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, People's Republic of China.
Drug Des Devel Ther. 2021 Aug 21;15:3643-3659. doi: 10.2147/DDDT.S317701. eCollection 2021.
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.
In vivo, a mouse liver fibrosis model was induced by the intraperitoneal injection of carbon tetrachloride (CCl). 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).
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.
The study confirmed that doxazosin could inhibit autophagy by activating the PI3K/Akt/mTOR signaling pathway and attenuate liver fibrosis.
研究多沙唑嗪在体内和体外对肝星状细胞（HSCs）自噬和活化的影响，并探讨其潜在机制。
体内，通过腹腔注射四氯化碳（CCl）诱导小鼠肝纤维化模型。通过灌胃给予多沙唑嗪 2.5、5 和 10mg/（kg·天）。20 周后，采集血液和肝脏组织进行血清学和组织学分析。采用血液分析、苏木精和伊红（HE）染色、马松三色染色、免疫组织化学和免疫荧光染色来测量模型和对照小鼠的肝纤维化程度。体外，培养人 HSC 细胞系 LX-2，并给予不同剂量的多沙唑嗪处理指定时间。通过细胞计数试剂盒-8（CCK-8）和 Transwell 测定分别检测多沙唑嗪对 LX-2 细胞增殖和迁移的影响。透射电子显微镜（TEM）观察 LX-2 细胞中自噬体的数量。通过感染绿色荧光蛋白（GFP）-LC3B 腺病毒、GFP-红色荧光蛋白（RFP）-LC3B 腺病毒和 mCherry-EGFP-LC3 腺相关病毒，检测体外和体内自噬流的变化。体外通过流式细胞术和体内通过 TUNEL 测定检测细胞凋亡。通过免疫印迹法评估与纤维化、自噬、凋亡和磷脂酰肌醇 3-激酶（PI3K）/蛋白激酶 B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）相关的蛋白表达水平。
多沙唑嗪抑制 HSC 增殖和迁移。多沙唑嗪在体内和体外以浓度依赖的方式减弱 HSC 的激活。多沙唑嗪还阻断 HSCs 的自噬流并诱导其凋亡。此外，多沙唑嗪激活 PI3K/Akt/mTOR 通路，并调节 HSCs 的纤维化、自噬和凋亡。
该研究证实，多沙唑嗪通过激活 PI3K/Akt/mTOR 信号通路抑制自噬，从而减轻肝纤维化。
