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Angiotensin II (Ang II) aggravates hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Angiotensin-(1-7) [Ang-(1-7)], which counter-regulates Ang II, has been evidenced to protect against hepatic fibrosis. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of liver fibrosis. However, whether the NLRP3 inflammasome involves in regulation of Ang II-induced hepatic fibrosis remains unclear. This study investigates the different effects of the Ang II and Ang-(1-7) on collagen synthesis by regulating the NLRP3 inflammasome/Smad pathway via redox balance modulation.In vivo, Ang-(1-7) improved bile duct ligation-induced hepatic fibrosis, reduced H2O2 content, protein levels of NOX4, and the NLRP3 inflammasome, whereas it increased glutathione (GSH) and nuclear erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE). In vitro, Ang II treatment elevated NOX4 protein expression and ROS production in hepatic stellate cells (HSCs), whereas it inhibited GSH and Nrf2-ARE, resulting in the activation of the NLRP3 inflammasome in the mitochondria of HSCs. NLRP3 depletion inhibited Ang II-induced collagen synthesis. Furthermore, Ang II increased NLRP3 and pro-IL-1β levels by activating the Toll-like receptor 4 (TLR4)/MyD88/NF-κB pathway. Treatment with antioxidants, NOX4 small interference RNA (siRNA), or Nrf2 activator inhibited Ang II-induced NLRP3 inflammasome activation and collagen synthesis. In contrast, the action of Ang-(1-7) opposed the effects of Ang II.Ang-(1-7) improved liver fibrosis by regulating NLRP3 inflammasome activation induced by Ang II-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 24, 795-812.

To establish a method for simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells.By combining in situ perfusion, in vitro perfusion, density gradient centrifugation and differential adhesion, primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells were obtained. The purity of these cells were assessed with morphological observation, immunofluorescent staining and ink phagocytosis assay.We successfully obtained the 4 primary cells simultaneously by combining in situ perfusion with in vitro perfusion, density gradient centrifugation, and differential attachment. The cell yield rate, cell viability and purity all met requirements for the subsequent cell experiment.The combined cell isolation and culture method is feasible to isolate primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells simultaneously.

To investigate the inhibitory effects of spironolactone against hepatic sinusoid angiogenesis in rats with hepatic fibrosis.Twenty-four male Wistar rats were randomly divided into sham-operated group, bile duct ligation (BDL) group, and BDL+SP group in which the rats received daily spironolactone injection (20 mg/kg) the day after BDL. Four weeks after the operation, the rats were sacrificed for examination of liver histology using Masson staining and the expression of vascular endothelial growth factor A (VEGF-A) mRNA in the liver using real-time quantitative PCR. Immunohistochemistry was used to detect the expression of von Willebrand factor (vWF) in the hepatic tissues.Spironolactone significantly inhibited liver fibrogenesis in rats after BDL (METAVIR liver fibrosis scores 2.84∓0.44 vs 19.73∓3.54, P=0.00). Real-time PCR and immunohistochemistry showed that compared with BDL group, spironolactone treatment significantly inhibited the expression of VEGF-A mRNA (0.71∓0.12 vs 1.75∓0.15, P=0.00) and vWF (1.15∓0.09 vs 3.08∓0.17, P=0.00) in the liver. The expression of VEGF-A mRNA was highly correlated with the expression of vWF (r=0.890, P=0.000).Spironolactone can inhibit hepatic sinusoid angiogenesis in rats with BDL-induced hepatic fibrosis by inhibiting the expression of VEGF-A.