Wnt5a正反馈调控二氧化硅诱导的小鼠肺泡巨噬细胞铁死亡

Wnt5a regulates SiO2-induced ferroptosis in mouse alveolar macrophages by positive feedback

目的探讨Wnt5a信号在二氧化硅诱导的小鼠巨噬细胞铁死亡过程中的调控作用。方法设置C57BL/6小鼠生理盐水组和二氧化硅处理组,收集支气管肺泡灌洗液(BALF)。免疫荧光细胞化学染色法检测小鼠BALF细胞Wnt5a、核因子κB p65(NF-κB p65)和Toll样受体4(TLR4)蛋白的表达;ELISA检测小鼠血浆和BALF上清白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)的水平。体外培养小鼠RAW264.7巨噬细胞,Western blot法检测(50、100、150、200、300)μg/mL二氧化硅刺激下RAW264.7细胞磷酸化的NF-κB p65(p-NF-κB p65)、Wnt5a、含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)、裂解型胱天蛋白酶1(c-caspase-1)、消皮素D(gasdermin D)以及铁死亡蛋白谷胱甘肽过氧化物酶4(GPX4)、尼克酰胺腺嘌呤二核苷酸磷酸氧化酶1(NOX1)和转铁蛋白(transferrin)的表达;CellROX~?荧光探针负载法检测活性氧(ROS)的水平;实时荧光定量PCR检测Wnt5a、IL-6、IL-10以及TNF-α的mRNA表达水平;采用Wnt5a重组蛋白和小分子拮抗剂BOX5分别激活和抑制Wnt5a信号,观察Wnt5a信号在铁死亡发生过程中的调控作用。结果二氧化硅刺激激活小鼠肺泡巨噬细胞和RAW264.7小鼠巨噬细胞Wnt5a信号及相关炎性信号,释放炎性因子IL-6及TNF-α,抑制铁死亡调控因子GPX4的表达;激活Wnt5a信号协同二氧化硅作用进一步下调GPX4的表达,促进二氧化硅诱导巨噬细胞铁死亡,而Wnt5a抑制剂BOX5减缓二氧化硅的抑制GPX4作用。结论Wnt5a信号在二氧化硅诱导的小鼠巨噬细胞铁死亡发生过程中具有正反馈调控作用。

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-αin plasma and BALF.In vitro,the RAW264.7 cells were treated with 0,50,100,150,200 and 300μg/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;CellROXfluorescent 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 SiOcould significantly activate Wnt5a and other inflammatory signaling pathways,meanwhile,release inflammatory factorssuch as IL-6 and TNF-α,and inhibit the expression of GPX4 protein.The Wnt5a recombinant protein and SiOcould synergistically inhibit the expression of GPX4,whereas BOX5 reduced SiO-induced GPX4 and Ferroptosis.ConclusionWnt5a signaling plays a positive feedback role in SiO-induced ferroptosis in mouse alveolar macrophages.