铁死亡在煤尘诱导小鼠煤工尘肺形成中的作用

刘太阳; 孙岳; 宝瑞; 郝玮; 王秋实; 刘耀阳; 王梦; 畅思容; 李媛媛; 刘志宏

doi:10.13213/j.cnki.jeom.2021.21169

铁死亡在煤尘诱导小鼠煤工尘肺形成中的作用

Role of ferroptosis in coal dust induced mouse models of coal workers' pneumoconiosis

摘要

摘要:
背景煤工尘肺（CWP）是一种危害严重的职业病，铁死亡是一种细胞程序性死亡方式，它是否参与了煤尘诱导CWP的形成须进一步探讨。

目的阐明铁死亡在煤尘诱导小鼠CWP形成中的作用。

方法 C57BL/6J小鼠被随机分为生理盐水组和CWP模型组，每组8只。采用气管滴注法造模，分别予以0.1 mL生理盐水或煤尘悬液0.1mL(50 g·L⁻¹) 经气管注入。通过HE染色和Masson染色评价CWP模型组小鼠肺部损伤及纤维化情况；铁离子检测试剂盒检测小鼠肺部铁离子浓度；分别检测丙二醛（MDA）浓度和荧光强度、谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）值评价小鼠肺部脂质过氧化情况；通过Western blotting和实时荧光定量PCR评价谷胱甘肽过氧化物酶4（GPX₄）、铁蛋白的mRNA和蛋白表达水平。

结果 CWP模型组小鼠肺部正常结构受损，肺泡壁厚度增加，出现炎症细胞浸润，胶原蛋白沉积较生理盐水组明显增加。CWP模型组小鼠肺组织铁离子质量浓度（10.75±5.42）mg·L⁻¹，较生理盐水组（1.14±0.37） mg·L⁻¹增加（P<0.01）。CWP模型组小鼠肺组织MDA浓度（37.32±12.18）μmol·L⁻¹较生理盐水组（18.70±8.22）μmol·L⁻¹增加（P<0.01）。CWP模型组鼠肺组织MDA荧光强度高于生理盐水组。CWP模型组小鼠肺组织GSH/GSSG值（1.50±1.70）较生理盐水组（4.95±2.86）降低（P<0.01）。CWP模型组小鼠肺组织GPX₄、铁蛋白的mRNA相对表达水平（14.29±7.21、106.70±36.70）均较生理盐水组（38.84±15.61、225.90±54.34）降低（均P<0.01），同时GPX₄、铁蛋白的蛋白相对表达水平（0.92±0.22、0.97±0.09）也均较生理盐水组（1.47±0.54、1.73±0.34）降低（均P<0.05）。

结论铁死亡可能参与了煤尘诱导小鼠CWP的形成。

Abstract:
Background Coal workers' pneumoconiosis (CWP) is a serious occupational disease. Whether ferroptosis, a form of necrotic regulated cell death, is involved in coal dust induced mouse models of CWP needs further survey.

Objective This experiment is designed to elucidate the role of ferroptosis in the formation of CWP induced by coal dust in mice.

Methods C57BL/6J mice were randomly assigned to a saline group or a CWP group, with eight mice in each group. The mice were treated with 0.1 mL normal saline or 0.1 mL coal dust suspensions (50g·L^-1) via intra-tracheal instillation. HE staining and Masson staining were used to show lung injury and lung fibrosis. Iron concentration in mouse lung tissues was measured using iron assay kit. Lipid peroxidation was estimated in lung tissues by malondialdehyde (MDA) concentration and immunofluorescence intensity, and the ratio of glutathione (GSH) to L-glutathione oxidized (GSSG). Western blotting and real-time fluorescence-based quantitative PCR were used to test protein and mRNA expression levels of glutathione peroxidase 4 (GPX₄) and ferritin in mice.

Results Coal dust injured pulmonary structure, thickened alveolar wall, and caused collagen deposition and infiltration of inflammatory cells in the CWP group. The iron concentration in the CWP group (10.75 ± 5.42) mg·L⁻¹ was higher than that in the saline group (1.14 ± 0.37) mg·L⁻¹ (P < 0.01). The MDA concentration in the CWP group (37.32 ± 12.18) μmol·L ⁻¹ was higher than that in the saline group (18.70 ± 8.22) μmol·L⁻¹ (P <0.01). The immunofluorescence intensity of MDA in the CWP group was stronger than that in the saline group. The GSH/GSSG ratio decreased in CWP treated mice (1.50 ± 1.70) compared with the normal saline treated ones (4.95 ± 2.86) ( P < 0.01). Compared with the saline group (38.84 ± 15.61 for GPX ₄, 225.90 ± 54.34 for ferritin), the relative expression levels of GPX₄ and ferritin mRNA in the CWP group were downregulated (14.29 ± 7.21 for GPX₄, 106.70 ± 36.70 for ferritin) (P < 0.01). Compared with the saline group (1.47 ± 0.54 for GPX ₄, 1.73 ± 0.34 for ferritin), the relative expression levels of GPX₄ and ferritin protein in the CWP group were also downregulated (0.92 ± 0.22 for GPX₄, 0.97 ± 0.09 for ferritin) (P < 0.05).

Conclusion Ferroptosis may be involved in the formation of coal workers' pneumoconiosis induced by coal dust in mice.

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