In vivo, autophagy and the NLRP3 inflammasome were activated in fibrotic patients and positively correlated with oxidation. Treatment with rapamycin promoted autophagy but inhibited oxidation, NLRP3 inflammasome, and lung fibrosis after bleomycin (BLM) infusion. The autophagy inhibitor 3-methyladenine reduced BLM-induced lung fibrosis and concurrently facilitated NLRP3 inflammasome activation and oxidation in fibroblasts. In vitro, AngII promoted intercellular ROS, hydrogen peroxide, and NADPH oxidase 4 (NOX4) protein levels and reduced the glutathione concentration, thereby leading to NLRP3 inflammasome activation and consequent collagen synthesis. AngII induced autophagy, while VAS2870, NOX4, small-interfering RNA (siRNA), and compound C eliminated AngII-induced LC3B augmentation. Moreover, blocking autophagy with bafilomycin A1 or LC3B siRNA resulted in oxidant accumulation, NLRP3 inflammasome hyperactivation, and collagen deposition. Finally, AngII induced P62/SQSTM1, targeting ubiquitinated apoptosis-associated speck-like protein containing a CARD for degradation, thereby contributing to NLRP3 inflammasome inactivation. Innovation and Conclusion: Autophagy attenuates pulmonary fibrosis by regulating NLRP3 inflammasome activation induced by AngII-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 00, 000-000.
MicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1–7) [ACE2/Ang(1–7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1–7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study’s aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1–7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1–7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1–7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1–7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.
Cigarette smoking is acknowledged as the major risk factor of pulmonary fibrosis. Angiotensin (Ang) II has been reported to aggravate smoking-induced lung fibrosis, whereas the effect of Ang-(1-7) on smoking-related lung fibrosis remains unknown. The autophagy, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of pulmonary fibrosis. However, whether autophagy is involved in regulation of smoking-induced lung fibrosis still needs investigation. Here, we aim to investigate the effect of Ang-(1-7) on smoking-related lung fibrosis by the regulation of autophagy and ROS. In vivo, Ang-(1-7) was constantly infused into passive smoking rats for 8 weeks. In vitro, primary lung fibroblasts were pretreated with antioxidant, nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) 4 siRNA, or light chain (LC) 3B siRNA before exposure to cigarette smoke extract (CSE). GFP-mCherry red fluorescent protein-LC3 advenovirus was introduced to evaluate the autophagic flux in cells. We found that Ang-(1-7) reduced hydrogen peroxide (HO) concentration, protein levels of NOX4, and autophagy impairment, as well as improving lung fibrosis induced by smoking stimulation in vivo. In vitro, CSE treatment elevated NOX4 protein expression and ROS production, resulting in the accumulation of impaired autophagosomes in fibroblasts. LC3B depletion enhanced CSE-induced collagen synthesis. Treatment with antioxidants or NOX4 siRNA inhibited CSE-induced insufficient autophagic flux and collagen production. In contrast, the action of Ang-(1-7) opposed the effects of CSE. In conclusion, Ang-(1-7) improves smoking-induced pulmonary fibrosis via attenuating the impaired autophagy caused by NOX4-dependent ROS in vivo and in vitro.
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