The α-1 Adrenergic Receptor Antagonist Doxazosin Attenuates Liver Fibrosis by Alleviating Sinusoidal Capillarization and Liver Angiogenesis.

Liver fibrosis and cirrhosis, which are caused by chronic liver injury, represent common and intractable clinical challenges of global importance. However, effective therapeutics are lacking. Therefore, the study examines the effect of doxazosin on liver fibrosis. Carbon tetrachloride (CCl4) is injected into mice to establish a liver fibrosis model. Doxazosin (5 and 10 mg/kg) is administered daily by gavage. HE staining, Masson staining, Sirius Red staining, scanning electron microscopy, western blotting, real-time PCR, and immunofluorescence analysis are performed to estimate liver fibrosis and sinusoidal capillarization in mice. Cell Counting Kit-8 assays, western blotting, immunofluorescence analysis, tube formation, and transwell migration assays are performed on human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs) to elucidate the potential mechanism of doxazosin. Doxazosin alleviates liver fibrosis and sinusoidal capillarization in CCl4-induced mice. Angiogenesis is attenuated by doxazosin in HUVECs and HHSECs. This study demonstrates that doxazosin attenuated liver fibrosis by alleviating sinusoidal capillarization and liver angiogenesis.

Upregulation of ATG9b by propranolol promotes autophagic cell death of hepatic stellate cells to improve liver fibrosis.

Proranolol has long been recommended to prevent variceal bleeding in patients with cirrhosis. However, the mechanisms of propranolol in liver fibrosis have not yet been thoroughly elucidated. Autophagic cell death (ACD) of activated hepatic stellate cells (HSCs) is important in the alleviation of liver fibrosis. Our study aims to assess the mechanisms of propranolol regulating HSC ACD and liver fibrosis. ACD of HSCs was investigated using lentivirus transfection. The molecular mechanism was determined using a PCR profiler array. The role of autophagy-related protein 9b (ATG9b) in HSC ACD was detected using co-immunoprecipitation and co-localization of immunofluorescence. Changes in the signalling pathway were detected by the Phospho Explorer antibody microarray. Propranolol induces ACD and apoptosis in HSCs. ATG9b upregulation was detected in propranolol-treated HSCs. ATG9b upregulation promoted ACD of HSCs and alleviated liver fibrosis in vivo. ATG9b enhanced the P62 recruitment to ATG5-ATG12-LC3 compartments and increased the co-localization of P62 with ubiquitinated proteins. The PI3K/AKT/mTOR pathway is responsible for ATG9b-induced ACD in activated HSCs, whereas the p38/JNK pathway is involved in apoptosis. This study provides evidence for ATG9b as a new target gene and propranolol as an agent to alleviate liver fibrosis by regulating ACD of activated HSCs.

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

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.

Partial splenic embolization combined with endoscopic therapies and NSBB decreases the variceal rebleeding rate in cirrhosis patients with hypersplenism: a multicenter randomized controlled trial.

BACKGROUND: Global research on endoscopic therapies in combination with partial splenic embolization (PSE) for variceal hemorrhage (VH) is limited. Therefore, we aimed to evaluate the efficacy and safety of endoscopy plus PSE (EP) treatment in comparison to endoscopic (E) treatment for the secondary prophylaxis of VH in cirrhosis patients with hypersplenism. METHODS: Cirrhosis patients with hypersplenism (platelet count < 100, 000/µL) and those who had recovered from an episode of VH were enrolled in a multicenter randomized controlled trial. The participants were randomly assigned into EP and E groups in a 1:1 ratio. The primary endpoint was variceal rebleeding, and the secondary endpoints were severe variceal recurrence and mortality during the 2-year follow-up. Hematological indices, serum biochemical parameters, and the Child-Pugh score were measured at each time point. RESULTS: From June 2016 to December 2019, 108 patients were enrolled in the study, among which 102 patients completed the protocol (51 in EP and 51 in E group). The rebleeding rate of the varices was significantly reduced in the EP group compared to that in the E group during the 2 years (16% vs. 31%, p < 0.001). The EP group showed a significantly lower variceal recurrence rate than the E group (22% vs. 67%, p < 0.001). The COX proportional hazard models revealed that grouping was an independent predictor for variceal rebleeding (H = 0.122, 95% CI 0.055-0.270, p < 0.001) and variceal recurrence (hazard ratio, H = 0.160, 95% CI 0.077-0.332, p < 0.001). The peripheral blood cell count, Child-Pugh class/score, albumin concentration, and coagulation function in the EP group improved significantly compared to the values observed in the E group at any time point (p < 0.05). CONCLUSIONS: The EP treatment was more effective in preventing variceal rebleeding and variceal recurrence than the conventional E treatment during the secondary prophylaxis of VH in cirrhosis patients with hypersplenism. Furthermore, the EP treatment could significantly increase the peripheral blood cell count and albumin concentration and also improved the coagulation function and the Child-Pugh score. CLINICAL TRIALS REGISTRATION: Trial registration number ClincialTrials.gov: NCT02778425. The URL of the clinical trial: https://clinicaltrials.gov/.

Carvedilol Inhibits Angiotensin II-Induced Proliferation and Contraction in Hepatic Stellate Cells through the RhoA/Rho-Kinase Pathway.

AIM: Carvedilol is a nonselective beta-blocker used to reduce portal hypertension. This study investigated the effects and potential mechanisms of carvedilol in angiotensin II- (Ang II-) induced hepatic stellate cell (HSC) proliferation and contraction. METHODS: The effect of carvedilol on HSC proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell cycle progression and apoptosis in HSCs were determined by flow cytometry. A collagen gel assay was used to confirm HSC contraction. The extent of liver fibrosis in mice was evaluated by hematoxylin-eosin (H&E) and Sirius Red staining. Western blot analyses were performed to detect the expression of collagen I, collagen III, α-smooth muscle actin (α-SMA), Ang II type I receptor (AT1R), RhoA, Rho-kinase 2 (ROCK2), and others. RESULTS: The results showed that carvedilol inhibited HSC proliferation and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Carvedilol also modulated Bcl-2 family proteins and increased apoptosis in Ang II-treated HSCs. Furthermore, carvedilol inhibited HSC contraction induced by Ang II, an effect that was associated with AT1R-mediated RhoA/ROCK2 pathway interference. In addition, carvedilol reduced α-SMA expression and collagen deposition and attenuated liver fibrosis in carbon tetrachloride (CCl4)-treated mice. The in vivo data further confirmed that carvedilol inhibited the expression of angiotensin-converting enzyme (ACE), AT1R, RhoA, and ROCK2. CONCLUSIONS: The results indicated that carvedilol dose-dependently inhibited Ang II-induced HSC proliferation by impeding cell cycle progression, thus alleviating hepatic fibrosis. Furthermore, carvedilol could inhibit Ang II-induced HSC contraction by interfering with the AT1R-mediated RhoA/ROCK2 pathway.

Carvedilol attenuates carbon tetrachloride-induced liver fibrosis and hepatic sinusoidal capillarization in mice.

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.

Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma.

AIM: To clarify the role of proteinase-activated receptor 2 (PAR2) in hepatocellular carcinoma, especially in the process of metastasis. METHODS: PAR2 expression levels were assessed by qRT-PCR and immunohistochemistry (IHC) in patient tissues and in hepatocellular carcinoma cell lines SMMC-7721 and HepG2. Cell proliferation and metastasis were assessed both in vitro and in vitro. Immunoblotting was carried out to monitor the levels of mitogen-activated protein kinase (MAPK) and epithelial-mesenchymal transition markers. RESULTS: The prognosis was significantly poorer in patients with high PAR2 levels than in those with low PAR2 levels. Patients with high PAR2 levels had advanced tumor stage (P = 0.001, chi-square test), larger tumor size (P = 0.032, chi-square test), and high microvascular invasion rate (P = 0.037, chi-square test). The proliferation and metastasis ability of SMMC-7721 and HepG2 cells was increased after PAR2 overexpression, while knockdown of PAR2 decreased the proliferation and metastasis ability of SMMC-7721 and HepG2 cells. Knockdown of PAR2 also inhibited hepatocellular carcinoma tumor cell growth and liver metastasis in nude mice. Mechanistically, PAR2 increased the proliferation ability of SMMC-7721 and HepG2 cells via ERK activation. Activated ERK further promoted the epithelial-mesenchymal transition of these cells, which endowed them with enhanced migration and invasion ability. CONCLUSION: These data suggest that PAR2 plays an important role in the proliferation and metastasis of hepatocellular carcinoma. Therefore, targeting PAR2 may present a favorable target for treatment of this malignancy.