School of Medicine, Hunan Normal University, Changsha, Hunan Province 410013, PR China; Key Laboratory of Molecular Epidemiology of Hunan Province, Hunan Normal University Changsha, Hunan Province 410013, PR China.
Department of Pneumoconiosis, Beidaihe Sanitarium for China Coal Miners, Qinhuangdao, Hebei Province 066104, PR China.
Life Sci. 2020 Sep 15;257:118043. doi: 10.1016/j.lfs.2020.118043. Epub 2020 Jul 2.
Alveolar macrophages (AMs) are the primary targets of silicosis. Blockade of autophagy may aggravate the apoptosis of AMs. Trehalose (Tre), a transcription factor EB (TFEB) activator, may impact the autophagy-lysosomal system in AMs during silicosis. However, the mechanism by which Tre acts upon AMs in silicosis is unknown.
We collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups. AMs from the two groups were then exposed to Tre. Western blot was used to measure the expression of autophagy-associated proteins. Lysosomal-associated membrane protein 1 (LAMP1) expression was observed using immunofluorescence and western blot. Apoptosis of the AMs was detected by TUNEL assay and western blot.
Tre induced localization of TFEB to the nucleus in the AMs of both groups. After Tre exposure, LAMP1 levels increased and LC3 levels decreased in the AMs of both groups, suggesting that Tre may increase the function of the autophagy-lysosomal system. The LC3-II/I ratio in the Tre-exposed AMs was lower than in the AMs not exposed to Tre. The LC3-II/I ratio in AMs subjected to Tre plus Bafilomycin (Baf) was higher than the ratio in cells exposed to Tre or Baf individually. Additionally, p62 levels decreased after Tre stimulation in the AMs of both groups. This indicates that Tre may accelerate the process of autophagic degradation. We also found decreased levels of cleaved caspase-3 after Tre treatment in the AMs of both groups. However, p-mTOR (Ser2448) and p-mTOR (Ser2481) levels did not change significantly after Tre treatment, suggesting that the mTOR signaling pathway was not affected by Tre treatment.
Our findings suggest that the restoration of autophagy-lysosomal function by Tre may be a potential protective strategy against silicosis.
肺泡巨噬细胞（AMs）是矽肺的主要靶细胞。自噬阻断可能会加重 AMs 的细胞凋亡。海藻糖（Tre）是转录因子 EB（TFEB）的激活剂，可能会影响矽肺时 AMs 的自噬溶酶体系统。然而，Tre 在矽肺 AMs 中的作用机制尚不清楚。
我们从 20 名接触二氧化硅的男性工人中收集 AMs，并将其分为观察者和矽肺患者组。然后将两组 AMs 暴露于 Tre 中。使用 Western blot 测定自噬相关蛋白的表达。通过免疫荧光和 Western blot 观察溶酶体相关膜蛋白 1（LAMP1）的表达。通过 TUNEL 测定和 Western blot 检测 AMs 的凋亡。
Tre 诱导两组 AMs 中 TFEB 向核内定位。Tre 暴露后，两组 AMs 中的 LAMP1 水平升高，LC3 水平降低，提示 Tre 可能增加自噬溶酶体系统的功能。与未暴露于 Tre 的 AMs 相比，暴露于 Tre 的 AMs 中的 LC3-II/I 比值降低。Tre 加 Bafilomycin（Baf）处理的 AMs 中的 LC3-II/I 比值高于单独暴露于 Tre 或 Baf 的细胞。此外，Tre 刺激后两组 AMs 中的 p62 水平降低。这表明 Tre 可能加速自噬降解过程。我们还发现两组 AMs 中 Tre 处理后 cleaved caspase-3 水平降低。然而，Tre 处理后 p-mTOR（Ser2448）和 p-mTOR（Ser2481）水平没有明显变化，提示 mTOR 信号通路不受 Tre 处理的影响。
我们的研究结果表明，Tre 恢复自噬溶酶体功能可能是一种潜在的矽肺保护策略。
