橘络活性成分小分子果胶调控肺上皮间质转化缓解氡致肺纤维化

The effect and mechanism of low-molecular-weight citrus pectin in alleviating radon-induced pulmonary fibrosis by regulating EMT process

摘要

目的 研究橘络活性成分小分子果胶(low-molecular-weight citrus pectin, LCP)对氡致小鼠肺纤维化的保护作用。方法 将90只雄性C57BL/6J小鼠随机分组,除空白对照组(NC组)外,单纯氡暴露组(Rn组)、LCP干预组(Rn+LCP组)在多功能生态氡室(HD-3)内吸入染氡构建氡致肺损伤小鼠模型,氡剂量设置为30、60、120工作水平月(working level month, WLM),其中Rn+LCP组在氡暴露的同时给予含LCP的饮用水,其余各组给予无菌水喂养。应用小鼠肺功能仪检测小鼠呼吸功能相关指标；通过HE和Masson染色观察各组肺组织病理学及胶原沉积改变,免疫荧光染色检测肺组织中E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)的表达,免疫组化染色检测肺组织中P-Smad3的表达；转录组测序检测各组小鼠肺组织中关键信号分子的表达差异。结果 与NC组相比,Rn组小鼠呼吸功能显著降低；Rn组小鼠肺组织结构紊乱、炎性细胞浸润明显,胶原纤维沉积增多；与Rn组相比,Rn+LCP组小鼠呼吸功能显著改善,肺组织炎性反应和纤维化明显减少。与Rn组相比,Rn+LCP组有89个基因上调,37个基因下调,共126个差异基因,其中大多数基因与上皮间质转化(epithelial-mesenchymal transition, EMT)进程相关。氡暴露可上调小鼠肺组织Smad3磷酸化水平,而LCP能够抑制Smad3磷酸化,调控EMT标志蛋白表达,阻滞EMT进程。结论 LCP可通过调控EMT过程、抑制TGF-β/Smad3信号通路蛋白表达,缓解氡诱导的肺纤维化。这为放射性肺损伤的天然产物干预提供了理论依据,同时为橘络资源高值化开发及氡致肺纤维化靶向药物研发提供新思路。

Abstract

Objective To study the protective effect of low-molecular-weight citrus pectin (LCP) on radon-induced pulmonary fibrosis in mice. Methods Ninety male C57BL/6J mice were randomly divided into control group (NC group), simple radon exposure group (Rn group), and LCP intervention group (Rn+LCP group). A mouse model of radon-induced lung injury was constructed by inhalation of radon in the multifunctional ecological radon chamber (HD-3). The radon dose was set to 30, 60, 120 working level months (WLM). The Rn+LCP group was given drinking water containing LCP while radon exposure, and the other groups were given sterile water. The related indexes of respiratory function of the mice were detected by mouse lung function instrument. The pathological changes of lung tissue in each group were observed by HE and Masson staining. The expression of E-cadherin and N-cadherin in lung tissue was detected by immunofluorescence staining. The expression of P-Smad3 in lung tissue of mice was detected by immunohistochemical staining. Transcriptome sequencing was used to detect the expression differences of key signaling molecules in lung tissues of mice in each group. Results Compared with NC group, the respiratory function of mice in the Rn group was significantly decreased. In the Rn group, the structure of lung tissue was disordered, the infiltration of inflammatory cells was obvious, and the deposition of collagen fibers increased. Compared with the Rn group, the respiratory function of mice in the Rn+LCP group was significantly improved, and the inflammatory response and fibrosis of lung tissue were significantly reduced. Compared with the Rn group, 89 genes were upregulated while 37 genes were downregulated in the Rn+LCP group, with a total of 126 differential genes, most of which were related to the process of epithelial-mesenchymal transition (EMT). Radon exposure dose-dependently upregulated Smad3 phosphorylation level in mouse lung tissue, whereas LCP inhibited Smad3 phosphorylation, regulated the expression of EMT marker proteins, and blocked the EMT process. Conclusion LCP can alleviate radon-induced pulmonary fibrosis by regulating EMT process and inhibiting the expression of TGF-β/Smad3 signaling pathway. It provides theoretical basis for the intervention of natural products in radiation-induced lung injury, and provides new ideas for the high-value development of tangerine collateral resources and the development of targeted drugs for radon-induced pulmonary fibrosis.

关键词

橘络活性成分小分子果胶(LCP) / 氡 / 肺纤维化 / 上皮间质转化(EMT) / TGF-β/Smad3

Key words

low-molecular-weight citrus pectin (LCP) / radon / pulmonary fibrosis / epithelial-mesenchymal transition (EMT) / TGF-β/Smad3

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基金资助

国家自然科学基金资助项目(82273568)