Angiotensin-(1-7) Improves Liver Fibrosis by Regulating the NLRP3 Inflammasome via Redox Balance Modulation.

AIMS: 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. RESULTS: 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-1ß 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. INNOVATION AND CONCLUSIONS: 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.

The angiotensin-converting enzyme 2/angiotensin (1-7)/Mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the NOX4-derived ROS-mediated RhoA/Rho kinase pathway.

UNLABELLED: Reactive oxygen species (ROS) generated by NADPH oxidase-4 (NOX4) have been shown to initiate lung fibrosis. The migration of lung fibroblasts to the injured area is a crucial early step in lung fibrosis. The angiotensin-converting enzyme 2 (ACE2)/angiotensin (1-7) [Ang(1-7)]/Mas axis, which counteracts the ACE/angiotensin II (AngII)/angiotensin II type 1 receptor (AT1R) axis, has been shown to attenuate pulmonary fibrosis. Nevertheless, the exact molecular mechanism remains unclear. AIMS: To investigate the different effects of the two axes of the renin-angiotensin system (RAS) on lung fibroblast migration and extracellular matrix accumulation by regulating the NOX4-derived ROS-mediated RhoA/Rho kinase (Rock) pathway. RESULTS: In vitro, AngII significantly increased the NOX4 level and ROS production in lung fibroblasts, which stimulated cell migration and α-collagen I synthesis through the RhoA/Rock pathway. These effects were attenuated by N-acetylcysteine (NAC), diphenylene iodonium, and NOX4 RNA interference. Moreover, Ang(1-7) and lentivirus-mediated ACE2 (lentiACE2) suppressed AngII-induced migration and α-collagen I synthesis by inhibiting the NOX4-derived ROS-mediated RhoA/Rock pathway. However, Ang(1-7) alone exerted analogous effects on AngII. In vivo, constant infusion with Ang(1-7) or intratracheal instillation with lenti-ACE2 shifted the RAS balance toward the ACE2/Ang(1-7)/Mas axis, alleviated bleomycin-induced lung fibrosis, and inhibited the RhoA/Rock pathway by reducing NOX4-derived ROS. INNOVATION: This study suggests that the ACE2/Ang(1-7)/Mas axis may be targeted by novel pharmacological antioxidant strategies to treat lung fibrosis induced by AngII-mediated ROS. CONCLUSION: The ACE2/Ang(1-7)/Mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the NOX4-derived ROS-mediated RhoA/Rock pathway.

[Effect of angiotensin II type 1 receptor and angiotensin-converting enzyme gene silencing on nuclear factor-kappaB activity in hepatic stellate cells].

OBJECTIVE: To investigate the effect of angiotensin II (AngII) type 1 (AT-1) receptor and angiotensin-converting enzyme (ACE) gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic stellate cells (HSCs). METHODS: pSilencer/AT-1 alpha receptor siRNA and pSilencer/ACE siRNA plasmids were transfected into cultured HSC-T6 cells, which were subsequently stimulated by 10(-6) mol/L AngII or ACE inhibitor (ACEI). The DNA binding activity of NF-kappaB in the transfected cells was analyzed using electrophoretic gel mobility shift assay (EMSA). RESULTS: s Gel shift studies showed that stimulation of the HSCs by AngII markedly increased the DNA-binding activity of NF-kappaB, which was inhibited by the transfection with pSilencer/ AT-1 alpha receptor siRNA plasmid or pSilencer/ACE siRNA plasmid. CONCLUSION: AT-1 alpha receptor and ACE gene silencing result in inhibition of NF-kappaB activity in HSCs in vitro.

[Effect of AT-1 alpha receptor gene silencing on nuclear factor-kappaB activity in hepatic Kupffer cells].

OBJECTIVE: To investigate the effect of angiotensin II type-1 (AT-1) alpha receptor gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic Kupffer cells. METHODS: The expression of AT-1 alpha receptors in primary isolated cultured hepatic Kupffer cells was detected by immunohistochemistry. pSilencer/AT-1 alpha receptor siRNA plasmids were transfected into Kupffer cells, which were subsequently exposed to 10(-6) mol/L angiotensin II (Ang II) for 60 min. The changes in the DNA binding activity of NF-kappaB in the cells was assessed using electrophoretic gel mobility shift assay (EMSA). RESULTS: AT-1 alpha receptor expression was detected in Kupffer cells. NF-kappaB DNA binding activity was markedly increased in Kupffer cells after Ang II stimulation, and obviously inhibited by transfectiom with pSilencer/AT-1 alpha receptor siRNA plasmid. CONCLUSION: Ang II stimulation of Kupffer cell results in increased activation of NF-kappaB via AT-1 alpha receptor.

[Intrasplenic heterotransplantation of in vitro cultured human fetal hepatic stem cells for treatment of acute liver injury in mice with severe combined immunodeficiency].

OBJECTIVE: To observe the in vivo colonization, migration, and differentiation of in vitro cultured human fetal hepatic stem cells (HSCs) following intrasplenic transplantation for treatment of acute liver injury in mice with severe combined immunodeficiency (SCID). METHODS: Human fetal HSCs were isolated from the normal fetal liver (16-24 weeks) and purified, and the morphology of HSCs was observed under optical and transmission electron microscopes. The expressions of stem cell markers were examined in these HSCs by means of immunocytochemistry and flow cytometry. The passaged human fetal HSC suspension (0.2 ml) were injected into the spleen of SCID mice with acute liver injury induced by two-third partial hepatectomy, and 15, 30, 60, and 90 days after cell transplantation, immunohistochemistry was performed to examine the location and expressions of human hepatocytes, alpha1-AT and AFP antigen in the spleen and liver of the recipient SCID mice. PAS staining was used to examine the expression of glycogen and RT-PCR employed for detection of the expressions of AFP and albumin mRNA in the spleen of the mice on the scheduled time points. RESULTS: Under optical microscope and transmission electron microscope, most of the HSCs were small, about 1/6 to 1/3 of the size of the hepatocyte, with relatively large nucleus-cytoplasm ratio and only small quantities of endocytoplasmic reticulum, chondriosome, and ribosome. Immunohistochemistry and flow cytometry identified positive expressions of AFP, Thy-1, C-kit, CD34 and CK19 in the HSCs, and after cell transplantation, positive expressions of human hepatocyte, alpha1-AT, and AFP antigen occurred in the liver and spleen of the recipient SCID mice. PAS staining confirmed the presence of glycogenosome in the spleen of the mice following cell transplantation. RT-PCR on days 30, 60, and 90 showed positive expressions of human AFP and albumin mRNA in the spleen of the mice. CONCLUSION: Human fetal HSCs can survive and settle in the spleen and liver, and migrate to the damaged liver of the recipient mice after intrasplenic transplantation, with the capacity of proliferation and differentiation into hepatocytes in the recipient target organs.