Abstract
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.
Highlights
Liver fibrosis and cirrhosis are worldwide public health problems with severe complications including portal hypertension and hepatic failure [1]. ey are chronic inflammation and tissue repair processes in which excess extracellular matrix (ECM) deposition occurs [2, 3]
AT1R is associated with the BioMed Research International stimulation and activation of several signaling pathways involved in cell contraction and ECM production. e RhoA/Rho-kinase pathway is one of the pathways that participate in the development of hepatic fibrosis and portal hypertension [12, 13]
After the cells were treated with Angiotensin II (Ang II) and carvedilol at the indicated concentrations for 24 hours, 10 μL of Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) reagent was added to each well. e plates were incubated at 37°C for 1 hour, and spectro-photometric absorbance was measured at 450 nm using a scanning multiwell spectrophotometer (BioRad Model 550, CA, USA). e results are based on triplicate experiments
Summary
Liver fibrosis and cirrhosis are worldwide public health problems with severe complications including portal hypertension and hepatic failure [1]. ey are chronic inflammation and tissue repair processes in which excess extracellular matrix (ECM) deposition occurs [2, 3]. HSC contraction can increase hepatic sinusoidal pressure, which is important in the development of portal hypertension [4,5,6]. Activation of the RAS resulting in Ang II type I receptor (AT1R) stimulation plays a crucial role in HSC activation and fibrogenesis [11]. E RhoA/Rho-kinase pathway is one of the pathways that participate in the development of hepatic fibrosis and portal hypertension [12, 13]. After activation of AT1R by Ang II, RhoA activates Rho-kinase, which increases myosin light chain (MLC) phosphorylation and related contraction [12]. Erefore, we infer that Ang II may activate the AT1R-mediated RhoA/ Rho-kinase pathway to participate in the activation, proliferation, and contraction of HSCs Kitamura et al [14] demonstrated that the Rho/ Rho-kinase pathway is partly involved in the RAS and affects the processes of liver fibrosis and steatosis. erefore, we infer that Ang II may activate the AT1R-mediated RhoA/ Rho-kinase pathway to participate in the activation, proliferation, and contraction of HSCs
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