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. Antioxid. Redox Signal. 22, 241–258.

Background: Cigarette smoking (CS) is a strong risk factor for idiopathic pulmonary fibrosis (IPF). It can activate lung fibroblasts (LF) by inducing redox imbalance. We previously showed that clearing mitochondrial reactive oxygen species (mtROS) protects against CS-induced pulmonary fibrosis. However, the precise mechanisms of mtROS in LF need further investigation. Here we focused on mtROS to elucidate how it was regulated by CS in LF and how it contributed to LF activation. Methods: We treated cells with 1% cigarette smoking extract (CSE) and examined mtROS level by MitoSOX^™ indicator. And the effect of CSE on expression of SIRT1, SOD2, mitochondrial NOX4 (mtNOX4), fatty acid oxidation (FAO)-related protein PPARα and CPT1a and LF activation marker Collagen I and α-SMA were detected. Nile Red staining was performed to show cellular lipid content. Then, lipid droplets, autophagosome and lysosome were marked by Bodipy 493/503, LC3 and LAMP1, respectively. And lipophagy was evaluated by the colocalization of lipid droplets with LC3 and LAMP1. The role of autophagy on lipid metabolism and LF activation were explored. Additionally, the effect of mitochondria-targeted ROS scavenger mitoquinone and SIRT1 activator SRT1720 on mitochondrial oxidative stress, autophagy flux, lipid metabolism and LF activation were investigated in vitro and in vivo. Results: We found that CS promoted mtROS production by increasing mtNOX4 and decreasing SOD2. Next, we proved mtROS inhibited the expression of PPARα and CPT1a. It also reduced lipophagy and upregulated cellular lipid content, suggesting lipid metabolism was disturbed by CS. In addition, we showed both insufficient FAO and lipophagy resulted from blocked autophagy flux caused by mtROS. Moreover, we uncovered decreased SIRT1 was responsible for mitochondrial redox imbalance. Furthermore, we proved that both SRT1720 and mitoquinone counteracted the effect of CS on NOX4, SOD2, PPARα and CPT1a in vivo. Conclusions: We demonstrated that CS decreased SIRT1 to activate LF through dysregulating lipid metabolism, which was due to increased mtROS and impaired autophagy flux. These events may serve as therapeutic targets for IPF patients.

Extracellular vesicles (EVs) have emerged as important vectors of intercellular dialogue. High mobility group box protein 1 (HMGB1) is a typical damage-associated molecular pattern (DAMP) molecule, which is cytotoxic and leads to cell death and tissue injury. Whether EVs are involved in the release of HMGB1 in lipopolysaccharide (LPS)-induced acute liver injuries need more investigation. EVs were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. The co-localization of HMGB1, RAGE (receptor for advanced glycation end-products), EEA1, Rab5, Rab7, Lamp1 and transferrin were detected by confocal microscopy. The interaction of HMGB1 and RAGE were investigated by co-immunoprecipitation. EVs were labeled with the PKH67 and used for uptake experiments. The pyroptotic cell death was determined by FLICA 660-YVAD-FMK. The expression of NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasomes were analyzed by western-blot or immunohistochemistry. Serum HMGB1, ALT (alanine aminotransferase), AST (aspartate aminotransferase), LDH (lactate dehydrogenase) and MPO (myeloperoxidase) were measured using a commercial kit. The extracellular vesicle HMGB1 was detected in the serums of sepsis patients. Macrophages were found to contribute to HMGB1 release through the EVs. HMGB1-RAGE interactions participated in the loading of HMGB1 into the EVs. These EVs shuttled HMGB1 to target cells by transferrin-mediated endocytosis leading to hepatocyte pyroptosis by the activation of NLRP3 inflammasomes. Moreover, a positive correlation was verified between the sepsis serum EVs-HMGB1 level and clinical liver damage. This finding provides insights for the development of novel diagnostic and therapeutic strategies for acute liver injuries.

Aims: Angiotensin II (AngII), a vasoconstrictive peptide of the renin–angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1–7) [Ang-(1–7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1β axis in liver fibrosis. Results:In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1–7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1–7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions:Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1–7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1–20.

Sepsis is a life-threatening organ dysfunction caused by a dysfunctional host response to infection. Neutrophils play a protective role by releasing antibacterial proteins or by phagocytizing bacteria. However, excess neutrophils can induce tissue damage. Recently, a novel intercellular communication pathway involving extracellular vesicles (EVs) has garnered considerable attention. However, whether EVs secreted by macrophages mediate neutrophil recruitment to infected sites has yet to be studied. In this study, we assessed the chemotactic effect of EVs isolated from mouse Raw264.7 macrophages on mouse neutrophils and found that CXCL2 was highly expressed in these EVs. By regulating CXCL2 in Raw264.7 macrophages, we found that CXCL2 on macrophage EVs recruited neutrophils in vitro and in vivo. The CXCL2 EVs activated the CXCR2/PKC/NOX4 pathway and induced tissue damage. This study provides information regarding the mechanisms underlying neutrophil recruitment to tissues and proposes innovative strategies and targets for the treatment of sepsis.