Carvedilol has been identified as a promising agent for the treatment of liver fibrosis. Meanwhile, autophagy and apoptosis have been reported to play key roles in the activation of hepatic stellate cells (HSCs), which can contribute to the progression of liver fibrosis. However, the effects of carvedilol on autophagy and apoptosis in HSCs remain unclear. Our study aimed to detect these effects and identify the underlying mechanisms by which carvedilol mediates HSC autophagy and apoptosis. For this purpose, the LX-2 cell line was used in this study, and the cells were exposed to various concentrations of carvedilol for specific times. First, we found that carvedilol increased autophagic marker levels, the number of GFP-LC3-containing puncta and LC3B-II levels in LX-2 cells. Interestingly, the addition of chloroquine (CQ) failed to enhance the effects on GFP-LC3 puncta and LC3B-II levels, and carvedilol treatment resulted in a significant increase in p62 protein levels. Moreover, carvedilol treatment led to the accumulation of yellow dots only in GFP-RFP-LC3-LX-2 cells, similar to the results following CQ treatment, indicating that carvedilol inhibited autophagic flux. Next, we found evidence that carvedilol inhibited autophagic flux by increasing lysosomal pH and not by impairing the fusion of autophagosomes with lysosomes. Moreover, carvedilol substantially reduced the viability of LX-2 cells and noticeably induced cell apoptosis, as observed by flow cytometry. In addition, increased levels of cleaved caspase-3, cleaved caspase-8 and cleaved PARP, increased Bax activity and decreased Bcl-2 expression were detected in LX-2 cells. Finally, the carvedilol treatment inhibited autophagy and subsequently induced apoptosis in vitro. In conclusion, carvedilol suppresses autophagy and promotes apoptosis in HSCs and the late-stage inhibition of autophagy preceded the induction of apoptosis.

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.

Aim: To investigate the effect of carvedilol on liver fibrosis and hepatic sinusoidal capillarization in mice with carbon tetrachloride (CCl₄)-induced fibrosis. Methods: A liver fibrosis mouse model was induced by intraperitoneal CCl₄ injection for 8 weeks. The mice were divided into five experimental groups: the normal group, the oil group, the CCl₄ group, the CCl₄+carvedilol (5 mg/kg/d) group, and the CCl₄+carvedilol (10 mg/kg/d) group. The extent of liver fibrosis was evaluated by histopathological staining, and the changes in fenestrations of hepatic sinus endothelial cells were observed by scanning electron microscope (SEM). The expression of α-smooth muscle actin (α-SMA) and vascular endothelial markers was detected by immunohistochemistry and Western blot assays. The effect of carvedilol on cell apoptosis was studied via Terminal deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) assay, and the serum levels of matrix metalloproteinase-8 (MMP-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 were detected through a Luminex assay. Results: Liver fibrosis in CCl₄-treated mice was attenuated by reduced accumulation of collagen and the reaction of inflammation with carvedilol treatment. Carvedilol reduced the activation of hepatic stellate cells (HSCs) and increased the number of apoptotic cells. The expression of α-SMA, CD31, CD34 and VWF (von Willebrand factor) was significantly decreased after carvedilol treatment. In addition, the number of fenestrae in the hepatic sinusoid showed notable differences between the groups, and the serum levels of MMP-8, VEGF and angiopoietin-2 were increased in the mice with liver fibrosis and reduced by carvedilol treatment. Conclusion: The study demonstrated that carvedilol could prevent further development of liver fibrosis and hepatic sinusoidal capillarization in mice with CCl₄-induced fibrosis.

Metformin attenuates motility, contraction, and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase

AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways. METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway. RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 μmol/L carvedilol for 24 h, respectively. Carvedilol (10 μmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 μmol/L and 10 μmol/L reduced cell invasion from 196.3% ± 18.76% to 114.0% ± 12.20% and 51.68% ± 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 μmol/L and 10 μmol/L carvedilol from 286.0 ± 36.72 to 135.7 ± 18.13 (P < 0.05) and 80.27 ± 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 μmol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% ± 8.54% to 52.67% ± 5.33% (P < 0.01). Additionally, 10 μmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% ± 15.37% to 53.57 ± 7.18% (P < 0.01) and 47.04% ± 9.74% (P < 0.01) at concentrations of 5 and 10 μmol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% ± 27.68% to 90.96% ± 17.16% (P < 0.01)]. CONCLUSION: Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.