let-7c参与肺泡巨噬细胞M2极化在煤矿粉尘致大鼠肺组织上皮-间质转化中的作用
Involvement of let-7c in alveolar macrophage M2 polarization in coal mine dust-induced epithelial-mesenchymal transition in rat lung tissue
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摘要:
背景 煤工尘肺（CWP）是一种危害严重的职业性肺病，是我国法定职业病之一。上皮-间质转化（EMT）参与尘肺病肺组织弥漫性纤维化，其机制可能与
let-7c 调控巨噬细胞极化有关。目的 通过动物体内实验，探讨
let-7c 参与巨噬细胞极化在不同含量游离SiO2的煤矿粉尘致大鼠肺组织EMT中的作用。方法 将SD大鼠随机分为对照组、5% SiO2组、30% SiO2组和99.9% SiO2组，每组16只。各组大鼠分别气管滴注100 μL 20 mg·mL-1混悬液（5% SiO2、30% SiO2、99.9% SiO2）或生理盐水，并在1、3个月时，收集支气管肺泡灌洗液。采用免疫荧光检测肺泡巨噬细胞（AMs）中M1标志物（CD86）或M2标志物（CD206）相对表达水平。采用苏木素-伊红（HE）染色和Masson染色观察肺组织炎症和纤维化。采用蛋白质免疫印迹法检测转化生长因子-β1（TGF-β1）、E-钙黏连蛋白及波形蛋白表达水平；采用荧光定量PCR检测
let-7c 、c/EBP-δ 基因表达水平。结果 HE及Masson染色结果显示：与对照组相比，随着染尘时间的增加，5% SiO2组、30% SiO2组和99.9% SiO2组大鼠肺组织纤维化程度逐渐加重。与对照组相比，5% SiO2组、30% SiO2组和99.9% SiO2组大鼠CD86、CD206的表达在1个月时均逐渐增加（
F =330.904、146.801，P <0.05），3个月时各组CD86的表达逐渐减少（F =331.781，P <0.05），而CD206的表达增加（F =1164.190，P <0.05）。1个月时，5% SiO2组、30% SiO2组和99.9% SiO2组TGF-β1（F =8.847，P <0.05）及波形蛋白（F =13.275，P <0.05）表达逐渐增加，E-钙黏连蛋白（F =6.253，P <0.05）表达逐渐减少；3个月时，各组TGF-β1（F =16.833，P <0.05）及波形蛋白（F =55.021，P <0.05）表达增加，E-钙黏连蛋白减少（F =12.790，P <0.05）。PCR结果显示：与对照组相比，5% SiO2组、30% SiO2组和99.9% SiO2组let-7c mRNA的表达在1个月和3个月时均增加（F =11.251、28.136，P <0.05）；各组c/EBP-δ mRNA的表达在1个月和3个月时均减少（F =49.204、177.090，P <0.05）。结论 在粉尘刺激下，
let-7c 通过调节巨噬细胞极化促进EMT的发生，从而参与肺纤维化的形成，进而影响CWP的进展。Abstract:Background Coal workers' pneumoconiosis (CWP) is a serious occupational lung disease and one of the prescript occupational diseases in China. Epithelial-mesenchymal transition (EMT) is involved in the diffuse fibrosis of lung tissue of pneumoconiosis patients, and its mechanism may be related to the polarization of macrophages regulated by
let-7c .Objective To investigate the effect of
let-7c on the regulation of macrophage polarization in EMT in rats induced by coal dust exposure with different content of free SiO2.Methods SD rats were randomly divided into a control group, a 5% SiO2 group, a 30% SiO2 group, and a 99.9% SiO2 group, with 16 rats in each group. The rats in each group were tracheally titrated with 100 μL of 20 mg·mL−1 suspension (5% SiO2, 30% SiO2, and 99.9% SiO2) or normal saline, respectively. Alveolar lavage fluid was collected at the ends of the 1st month and the 3rd month. The relative expression levels of M1 or M2 markers, CD86 or CD206, in alveolar macrophages (AMs) were detected by immunofluorescence. The inflammation and fibrosis of lung tissue were observed by hematoxylin-eosin (HE) staining and Masson staining. The expression levels of transforming growth factor-β1 (TGF-β1), E-cadherin, and vimentin were detected by Western blotting. The expression levels of
let-7c andc/EBP-δ genes were detected by real-time fluorescence quantitative PCR.Results The HE and Masson staining results showed that compared with the control group, the degree of pulmonary fibrosis in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group gradually increased with the increase of dust exposure time. Compared with the control group, the expressions of CD86 and CD206 in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group gradually increased at the end of the 1st month (
F =330.904, 146.801,P <0.05), and the expression of CD86 in each group decreased gradually at the end of the 3rd month (F =331.781,P <0.05), but the expression of CD206 increased (F =1164.190,P <0.05). At the end of the 1st month, the expressions of TGF-β1 (F =8.847,P <0.05) and vimentin (F =13.275,P <0.05) gradually increased, and the expression of E-cadherin (F =6.253,P <0.05) gradually reduced in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group. At the end of the 3rd month, the expressions of TGF-β1 (F =16.833,P <0.05) and vimentin (F =55.021,P <0.05) increased, and the expression of E-cadherin (F =12.790,P <0.05) gradually decreased in all groups. The PCR results showed that compared with the control group, the expression oflet-7c mRNA in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group increased at the ends of the 1st month and the 3rd month (F =11.251, 28.136,P <0.05). The expression ofc/EBP-δ mRNA decreased in all groups at the ends of the 1st month and the 3rd month (F =49.204, 177.090,P <0.05).Conclusion In response to dust stimulation,
let-7c promotes EMT by modulating macrophage polarization, which is involved in the formation of pulmonary fibrosis and thus influences the progression of CWP .
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