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
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摘要:
背景 破骨细胞刺激性跨膜蛋白（OC-STAMP）参与二氧化硅（SiO2）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SiO2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）、SiO2组。SiO2组大鼠通过气管非暴露法一次性给予1 mL 50 mg·L−1的SiO2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 mL 0.9%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OC-STAMP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 SiO2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，SiO2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，SiO2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SiO2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，SiO2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SiO2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）；在转染的MLE-12细胞中，与Sham组相比，OCS组SLC7A11、GPX4蛋白表达水平降低（P <0.01，P <0.01）。结论 OC-STAMP可能通过影响铁死亡相关蛋白表达，从而促进SiO2暴露导致的肺纤维化。
Abstract:Background Osteoclast stimulatory transmembrane protein (OC-STAMP) is involved in silicosis fibrosis induced by silicon oxide (SiO2) 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 SiO2 exposure.
Methods Twenty male Wistar rats of SPF grade were randomly divided into two groups: control (Sham) group and SiO2 group, 15 rats in each group. Rats in the SiO2 group were given 1 mL of 50 mg·L−1 SiO2 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 SiO2 exposure. The immunofluorescence results showed that OC-STAMP and ATP binding cassette subfamily A member 3 (ABCA3) co-localized in alveolar type II epithelium. The immunohistochemical results showed that the levels of OC-STAMP and collagen I in the SiO2 group were significantly higher than those in the Sham group (
P <0.01). The RT-PCR results showed that the OC-STAMP mRNA in the lung tissue of the SiO2 group was significantly higher than that of the Sham group (P <0.01). The Prussian blue staining in the lung tissue of the SiO2 group showed positive brownish-yellow particles. Compared with the Sham group which showed normal mitochondrial structure, the mitochondrial structure was generally swollen and the mitochondrial cristae dissolved and disappeared in the SiO2 group by transmission electron microscope observation. The Western blotting results showed that the expression levels of SLC7A11 and GPX4 both decreased in the lung tissue of the SiO2 group (P <0.05,P <0.01), and the expression level of Vimentin increased (P <0.01). In the transfected MLE-12 cells, compared with the Sham group, the expression levels of SLC7A11 and GPX4 in the OCS group were significantly reduced (P <0.05,P <0.01).Conclusion OC-STAMP may affect the expression of proteins related to ferroptosis, and promote lung fibrosis induced by SiO2 exposure.
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