破骨细胞刺激性跨膜蛋白诱导铁死亡促进矽肺纤维化  

Mechanism of osteoclast stimulatory transmembrane protein promoting silicosis fibrosis by inducing ferroptosis

作　　者：吴静 左翠云 柯妍妍 汪洁 许雅萍 杜薇 石怡敏 庄云扬 伊雪 WU Jing;ZUO Cuiyun;KE Yanyan;WANG Jie;XU Yaping;DU Wei;SHI Yimin;ZHUANG Yunyang;YI Xue(Public Health School of Fujian Medical University,Fuzhou,Fujian 350122,China;Basic Medicine Department,Xiamen Medical College,Xiamen,Fujian 361023,China;Respiratory Department,Second Affiliated Hospital of Xiamen Medical College,Xiamen,Fujian 361021,China;Department of Cardiothoracic Surgery,Second Affiliated Hospital of Xiamen Medical College,Xiamen,Fujian 361021,China;Insitute of Respiratory Research,Xiamen Medical College,Xiamen,Fujian 361023,China)

机构地区：[1]福建医科大学公共卫生学院,福建福州350122 [2]厦门医学院基础医学部,福建厦门361023 [3]厦门医学院附属第二医院呼吸科,福建厦门361021 [4]厦门医学院附属第二医院胸心外科,福建厦门361021 [5]厦门医学院呼吸疾病研究所,福建厦门361023

出　　处：《环境与职业医学》2023年第11期1257-1263,共7页Journal of Environmental and Occupational Medicine

基　　金：福建省自然科学基金项目(2022J011409,2023J0113);厦门医学院科技计划项目(K2020-03,K2023-07)。

摘　　要：破骨细胞刺激性跨膜蛋白(OC-STAMP)参与二氧化硅(SiO_(2))暴露引起矽肺纤维化,其通过诱导肺泡Ⅱ型上皮细胞铁死亡从而参与矽肺纤维化的作用及相关机制尚不明确。[目的]探讨SiO_(2)暴露下OC-STAMP调控肺泡Ⅱ型上皮细胞铁死亡参与大鼠矽肺纤维化的作用及可能机制。[方法]20只SPF级Wistar雄性大鼠,随机分为2组,每组10只,分别为对照组(Sham组)、SiO_(2)组。SiO_(2)组大鼠通过气管非暴露法一次性给予1 mL 50 mg·L^(-1)的SiO_(2)混悬液建立矽肺动物模型,Sham组大鼠采用同样的方法给予1 mL0.9%氯化钠溶液,8周后处死大鼠。取左下肺固定于戊二醛或多聚甲醛,用透射电镜观察线粒体超微结构;用HE染色、Masson染色、VG染色及普鲁士蓝染色显示肺组织结构变化及铁沉积状况。通过免疫组化和免疫荧光评价OC-STAMP表达水平和肺纤维化程度。采用实时荧光定量PCR检测大鼠肺组织OCSTAMP表达水平及验证OC-STAMP的转染效果。培养MLE-12细胞,通过转染OC-STAMP质粒或OC-STAMP小干扰RNA(siRNA)构建过表达(OCS组)和抑制表达(SI-OC组)模型。采用Western blotting法检测肺组织及MLE-12中谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)等蛋白的相对表达量。[结果]SiO_(2)暴露8周后,HE、Masson和VG染色结果显示造模成功;组织免疫荧光结果显示,OC-STAMP与ATP结合盒亚家族A成员3(ABCA3)共定位于肺泡Ⅱ型上皮中;免疫组化结果显示,SiO_(2)组OC-STAMP、Ⅰ型胶原表达水平与Sham组相比升高(P<0.01);实时荧光定量PCR结果显示,SiO_(2)组肺组织中OC-STAMP的mRNA高于Sham组(P<0.01);肺组织普鲁士蓝染色结果显示在SiO_(2)组可见阳性棕黄色颗粒;经透射电镜观察,Sham组线粒体结构正常,SiO_(2)组线粒体普遍肿胀,线粒体嵴溶解、消失;肺组织免疫印迹结果显示,在SiO_(2)组,SLC7A11、GPX4蛋白表达水平降低(P<0.05,P<0.01),Vimentin蛋白表达水平增加(P<0.01);在转染[Background]Osteoclast stimulatory transmembrane protein(OC-STAMP)is involved in silicosis fibrosis induced by silicon oxide(SiO_(2))exposure.Its role in silicosis fibrosis by inducing ferroptosis of alveolar type II epithelial cells and its related mechanism remain unclear.[Objective]To explore the effect and possible mechanism of OC-STAMP on ferroptosis of alveolar type II epithelial cells and silicosis fibrosis in rats under SiO_(2)exposure.[Methods]Twenty male Wistar rats of SPF grade were randomly divided into two groups:control(Sham)group and SiO_(2)group,15 rats in each group.Rats in the SiO_(2)group were given 1 mL of 50 mg·L^(-1)SiO_(2)suspension at one time through the non-exposed intratracheal instillation method to establish an animal model of silicosis,and rats in the Sham group were give 1 mL of 0.9%sodium chloride solution in the same way.Rats were sacrificed after 8 weeks.Samples of lung tissue were fixed in glutaraldehyde or paraformaldehyde for observing ultrastructure of mitochondria by transmission electron microscopy;HE,Masson,VG,and Prussian blue were used to observe changes in lung tissue structure and iron deposition.The expression level of OC-STAMP and the degree of lung fibrosis were evaluated by immunohistochemistry and immunofluorescence.The expression level of OC-STAMP in rat lung tissue was detected and the transfection effect of OC-STAMP was verified by real-time fluorescence quantitative polymerase chain reaction(RT-PCR).Overexpression(OCS group)and inhibition expression(SI-OC group)models were constructed by OC-STAMP plasmid and OC-STAMP small interfering RNA(siRNA)transfection to cultured MLE-12 cells,respectively.The relative expression levels of glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and other proteins in lung tissue and MLE-12 were detected by Western blotting.[Results]The results of HE,Masson,and VG staining showed that the silicosis modeling was successful after 8 weeks of SiO_(2)exposure.The immunofluorescence results showed that OC-STAMP

关 键 词：矽肺 破骨细胞刺激性跨膜蛋白 铁死亡 纤维化 

分 类 号：R114[医药卫生—卫生毒理学]