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Background: Dihydroquercetin (DHQ) is a flavonoid with strong anti-inflammatory and antioxidant effects. However, its protective activity against cigarette smoke-induced ferroptosis in the pathogenesis of chronic obstructive pulmonary disease and its underlying mechanisms remain unclear.Purpose: The present study was conducted to investigate the protective role of DHQ in the pathogenesis of COPD in vivo and in vitro.Methods: A cigarette smoke-induced COPD mouse model was established by cigarette smoke (CS) exposure combined with intraperitoneal injection of cigarette smoke extract (CSE). During the modeling process, the mice were intraperitoneally injected with DHQ daily. HBE cells were cultured with CSE with or without pretreatment with DHQ (40, 80 μM) or ML385 (10 μM). Cell viability was assessed by a cell counting kit 8 (CCK-8). The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by MDA and SOD assay kits, respectively, and reactive oxygen species (ROS) generation was detected by DCFH-DA assays. Protein expression levels of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPx4) and nuclear factor erythroid 2-related factor 2 (Nrf2) were measured by western blot. Lipid peroxidation was determined by C11-BODIPY staining. Transmission electron microscopy was used to observe the morphological features of the mitochondria.Results: Treatment with DHQ significantly elevated ferroptosis-related protein (SLC7A11 and GPx4) expression in vivo and in vitro. The mRNA levels of SLC7A11 and GPx4 were also increased after DHQ treatment. The excessive MDA and ROS production and depleted SOD activity induced by CSE were reversed by DHQ. DHQ notably reduced the increased lipid peroxidation induced by CSE in HBE cells. In addition, treatment with DHQ attenuated the morphological changes in the mitochondria caused by CSE. Moreover, we also found that DHQ increased the levels of Nrf2 in a concentration-dependent manner in the cigarette smoke-induced COPD mouse model and CSE-treated HBE cells. Additionally, after administering an Nrf2-specific inhibitor, ML385, to HBE cells, the elevated SLC7A11 and GPx4 mRNA and protein levels induced by DHQ were reversed. Moreover, ML385 treatment attenuated the protective effect of DHQ on lipid peroxidation.Conclusion: Our results show that treatment with DHQ significantly reverses the ferroptosis induced by cigarette smoke both in vivo and in vitro via a Nrf2-dependent signaling pathway. [ABSTRACT FROM AUTHOR]

• This is the first study to investigate whether pirfenidone could mediate cigarette smoke extract (CSE) induced inflammation and oxidative stress in vitro and in vivo. • Our study reveals the potential role of pirfenidone in regulating inflammatory response and oxidative stress induced by CSE. Antioxidant and anti-inflammatory effects are two main pharmacological mechanisms of pirfenidone (PFD) besides the anti-fibrotic effect. This study aims to investigate whether PFD could mediate cigarette smoke extract (CSE) induced inflammation and oxidative stress in vitro and in vivo. BALB/C mice and alveolar epithelial (A549) cells treated with CSE were established as disease models in vivo and in vitro. Effects of PFD treatment on disease models were further measured. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in lung tissues of mice. CCK-8 assay kit was applied to measure the viability of A549 cells treated by different concentrations of PFD. Inflammation cytokine expression in cell supernatants was measured with ELISA kits. The mRNA and protein levels of inflammation and oxidative stress-related factors were determined by real-time quantitative polymerase chain reaction analysis (RT-qPCR) and Western blotting. Furthermore, myeloperoxidase (MPO), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were measured to detect the antioxidative activity of lung tissues. Moreover, an assay kit with fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used to evaluate the intracellular reactive oxygen species (ROS) generation. In vitro and in vivo, PFD significantly reversed TNF-α, IL-6, CCL2, SOD1, and CAT mRNA level changes led by CSE; in addition, PFD significantly decreased the ratios of p-p65 to p65, p-ikBα to ikBα and increased Nrf-2 protein level compared with CSE group. In mice, high-dose (100 mg/kg/d) PFD significantly reversed MPO and MDA increases induced by CSE. However, PFD didn't significantly reverse T-AOC decrease induced by CSE. In A549 cell supernatant, PFD dramatically reversed the elevated levels of TNF-α and IL-1β induced by CSE. Furthermore, PFD could significantly reverse the increased level of ROS induced by CSE in A549 cells. Our study reveals the potential role of PFD in regulating inflammatory response and oxidative stress induced by CSE. [ABSTRACT FROM AUTHOR]