Background. Lung fibrosis is a severe lung disorder featured by chronic nonspecific inflammation of the interstitial lung and deposition of collagen, leading to lung dysfunction. It has been identified that ferroptosis is involved in the progression of lung injury. Particulate matter (PM2.5) is reported to be correlated with the incidence of pulmonary fibrosis. However, mechanisms underlying ferroptosis in PM2.5-related lung fibrosis is unclear. In this study, we aimed to explore the effect of PM2.5 on ferroptosis in lung fibrosis and the related molecular mechanisms. Methods. PM2.5-treated mouse model and cell model were established. Fibrosis and tissue damage were measured by Masson’s trichrome staining and HE staining. Fibrosis biomarkers, such as α-SMA, collagen I, and collagen III, were examined by histological analysis. The ferroptosis phenotypes, including the levels of iron, Fe²⁺, MDA, and GSH, were measured by commercial kits. ROS generation was checked by DCFH-DA. The oxidative stress indicators, 3-nitro-L-tyrosine (3-NT), 4-HNE, and protein carbonyl, were checked by enzyme linked immunosorbent assay (ELISA). The thiobarbituric acid reactive substances (TBARS) and GSH/GSSG ratio were assessed by TBARS assay kit and GSH/GSSG assay kit, respectively. TGF-β signaling was detected by Western blotting. Results. PM2.5 induced the lung injury and fibrosis in the mice model, along with elevated expression of fibrosis markers. PM2.5 enhanced oxidative stress in the lung of the mice. The SOD2 expression was reduced, and NRF2 expression was enhanced in the mice by the treatment with PM2.5. PM2.5 triggered ferroptosis, manifested as suppressed expression of GPX4 and SLC7A11, decreased levels of iron, Fe²⁺, and MDA, and increased GSH level in mouse model and cell model. The TGF-β and Smad3 signaling was inhibited by PM2.5. ROS inhibitor NAC reversed PM2.5-regulated ROS and ferroptosis in primary mouse lung epithelial cells. Conclusions. Therefore, we concluded that PM2.5 exposure induced lung injury and fibrosis by inducing ferroptosis via TGF-β signaling.

中文翻译：

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PM2.5 暴露通过 TGF-β 信号传导调节铁死亡，诱导肺损伤和纤维化

背景。肺纤维化是一种严重的肺部疾病，其特征是肺间质的慢性非特异性炎症和胶原沉积，导致肺功能障碍。已经确定铁死亡参与肺损伤的进展。据报道，颗粒物（PM2.5）与肺纤维化的发生率相关。然而，PM2.5 相关肺纤维化中铁死亡的机制尚不清楚。本研究旨在探讨PM2.5对肺纤维化铁死亡的影响及其相关分子机制。方法。建立PM2.5处理小鼠模型和细胞模型。通过马森三色染色和HE染色测量纤维化和组织损伤。通过组织学分析检查纤维化生物标志物，例如α -SMA、胶原蛋白 I 和胶原蛋白 III。通过商业试剂盒测量铁死亡表型，包括铁、Fe ²⁺ 、MDA 和 GSH的水平。通过 DCFH-DA 检查 ROS 生成。采用酶联免疫吸附试验（ELISA）检测氧化应激指标3-硝基-L-酪氨酸（3 - NT）、4-HNE、蛋白质羰基。分别采用TBARS测定试剂盒和GSH/GSSG测定试剂盒评估硫代巴比妥酸反应物质(TBARS)和GSH/GSSG比值。通过蛋白质印迹法检测TGF- β信号传导。结果。PM2.5 诱导小鼠模型肺损伤和纤维化，同时纤维化标志物的表达升高。PM2.5 增强了小鼠肺部的氧化应激。PM2.5处理后，小鼠中SOD2表达降低，NRF2表达增强。PM2.5引发铁死亡，表现为小鼠模型和细胞模型中GPX4和SLC7A11的表达受到抑制，铁、Fe ²⁺和MDA水平降低，GSH水平升高。PM2.5 抑制TGF- β和 Smad3 信号传导。ROS 抑制剂 NAC 可逆转原代小鼠肺上皮细胞中 PM2.5 调节的 ROS 和铁死亡。结论。因此，我们得出结论，PM2.5 暴露通过TGF- β信号传导诱导铁死亡，从而导致肺损伤和纤维化。