Ma Jia, Wang Jing
Clinical Laboratory Diagnosis Center, People's Hospital of Ningxia Hui Autonomous Region, Yinchuan 750001; Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western, College of Life Science, Ningxia University, Yinchuan 750021, China.
Clinical Laboratory Diagnosis Center, People's Hospital of Ningxia Hui Autonomous Region, Yinchuan 750001, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Aug;38(8):699-706.
Objective To investigate the potential role of Wnt5a signaling in SiO-induced ferroptosis in mouse alveolar macrophages. Methods C57BL/6 mice were treated by intratracheal instillation of crystalline silica (SiO) or saline, and plasma and bronchial alveolar lavage fluid (BALF) were harvested at 24 hours post injury. Immunofluorescence cytochemical staining was used to detect the expression of Wnt5a, p65 (NF-κB p65) and Toll-like receptor 4 (TLR4) in alveolar macrophages. ELISA was employed to detect the levels of inflammatory cytokines IL-6 and TNF-α in plasma and BALF. In vitro, the RAW264.7 cells were treated with 0, 50, 100, 150, 200 and 300 μg/mL SiO, while the levels expression of p65(p-NF-κB, p65), Wnt5a, cleaved caspase-1(c-caspase-1), gasdermin D, NLR family pyrin domain containing 3(NLRP3), glutathione peroxidase 4(GPX4), NADPH oxidase 1(NOX1) and transferrin were then determined by Western blot; CellROX fluorescent probe loading assay was used to detect the release of reactive oxygen species (ROS); real time quantitative PCR tested the mRNA levels of Wnt5a, IL-6, IL-10 and TNF-α. Wnt5a recombinant protein; small molecule antagonist BOX5 were used to activate and inhibit the Wnt5a signaling to investigate the role of Wnt5a in ferroptosis, respectively. Results The stimulation of SiO could significantly activate Wnt5a and other inflammatory signaling pathways, meanwhile, release inflammatory factors such as IL-6 and TNF-α, and inhibit the expression of GPX4 protein. The Wnt5a recombinant protein and SiO could synergistically inhibit the expression of GPX4, whereas BOX5 reduced SiO-induced GPX4 and Ferroptosis. Conclusion Wnt5a signaling plays a positive feedback role in SiO-induced ferroptosis in mouse alveolar macrophages.
目的 探讨Wnt5a信号通路在二氧化硅（SiO）诱导的小鼠肺泡巨噬细胞铁死亡中的潜在作用。方法 通过气管内滴注结晶二氧化硅（SiO）或生理盐水处理C57BL/6小鼠，在损伤后24小时收集血浆和支气管肺泡灌洗液（BALF）。采用免疫荧光细胞化学染色检测肺泡巨噬细胞中Wnt5a、p65（NF-κB p65）和Toll样受体4（TLR4）的表达。采用酶联免疫吸附测定法（ELISA）检测血浆和BALF中炎性细胞因子白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平。体外实验中，将RAW264.7细胞分别用0、50、100、150、200和300μg/mL的SiO处理，然后通过蛋白质免疫印迹法检测p65（磷酸化核因子κB，p65）、Wnt5a、裂解的半胱天冬酶-1（c-半胱天冬酶-1）、gasdermin D、含NLR家族pyrin结构域蛋白3（NLRP3）、谷胱甘肽过氧化物酶4（GPX4）、烟酰胺腺嘌呤二核苷酸磷酸氧化酶1（NOX1）和转铁蛋白的表达水平；采用CellROX荧光探针负载测定法检测活性氧（ROS）的释放；实时定量聚合酶链反应检测Wnt5a、IL-6、IL-10和TNF-α的信使核糖核酸（mRNA）水平。分别使用Wnt5a重组蛋白和小分子拮抗剂BOX5激活和抑制Wnt5a信号通路，以研究Wnt5a在铁死亡中的作用。结果 SiO刺激可显著激活Wnt5a和其他炎性信号通路，同时释放IL-6和TNF-α等炎性因子，并抑制GPX4蛋白的表达。Wnt5a重组蛋白与SiO可协同抑制GPX4的表达，而BOX5可减轻SiO诱导的GPX4表达及铁死亡。结论 Wnt5a信号通路在SiO诱导的小鼠肺泡巨噬细胞铁死亡中起正反馈作用。
