正常小鼠血清通过抑制focal adhesion信号通路减轻小鼠放射性肺炎

doi:10.12122/j.issn.1673-4254.2024.05.01

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (5): 801-809.doi: 10.12122/j.issn.1673-4254.2024.05.01

• 基础研究 • 下一篇

正常小鼠血清通过抑制focal adhesion信号通路减轻小鼠放射性肺炎

苑通(), 郭玉莹, 张俊伶(), 樊赛军()

中国医学科学院//北京协和医学院放射医学研究所，天津市放射医学与分子核医学重点实验室，天津 300192

收稿日期:2023-11-03 接受日期:2024-01-05 出版日期:2024-05-20 发布日期:2024-06-06
通讯作者: 张俊伶,樊赛军 E-mail:yuantong1233@163.com;zhangjunling@irm-cams.ac.cn;fansaijun@irm-cams.ac.cn
作者简介:苑通，在读博士研究生，E-mail: yuantong1233@163.com
基金资助:
中国医学科学院医学与健康科技创新工程重大协同创新项目(2021-I2M-1-042);中国医学科学院国家医学健康科技战略平台与体系建设项目(2022-I2M-2-003);国家自然科学基金(82273577)

Normal mouse serum alleviates radiation pneumonitis in mice by inhibiting the focal adhesion signaling pathway

Tong YUAN(), Yuying GUO, Junling ZHANG(), Saijun FAN()

Institute of Radiation Medicine, Chinese Academy of Medical Science//Peking Union Medical College; Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Tianjin 300192, China

Received:2023-11-03 Accepted:2024-01-05 Online:2024-05-20 Published:2024-06-06
Contact: Junling ZHANG, Saijun FAN E-mail:yuantong1233@163.com;zhangjunling@irm-cams.ac.cn;fansaijun@irm-cams.ac.cn
Supported by:
the CAMS Innovation Fund for Medical Sciences(2021-I2M-1-042);National Natural Science Foundation of China(82273577)

摘要/Abstract

摘要：

目的探讨正常小鼠血清（NMS）对放射性肺炎的治疗作用及可能机制。方法建立胸腔照射诱导的放射性肺炎模型，将小鼠分为对照组、静脉注射血清组、照射组和照射后静脉注射血清组。注射血清组小鼠在照射后立即静脉注射正常小鼠血清100 μL，对照组小鼠注射100 μL 生理盐水，隔天注射1次，共注射8次。照射后15 d取材，HE染色检测肺组织形态学变化，ELISA检测小鼠肺组织和血清中炎症因子肿瘤坏死因子-α（TNF-α）、转化生长因子-β（TGF-β）、白细胞介素-1α（IL-1α）、白细胞介素-6（IL-6）水平；流式细胞术检测肺组织内淋巴细胞比例变化。外泌体miRNA高通量测序探索处理后小鼠的信号通路变化，qRT-PCR检测免疫相关基因的表达水平，使用Western blotting检测黏着斑通路talin-1、tensin 2、FAK、vinculin、α-actinin和paxillin蛋白的表达。结果与照射组相比，照射后注射血清组小鼠肺脏器系数、血清及肺组织上清液中炎症因子TNF-α、TGF-β、IL-1α、IL-6水平显著降低（P<0.05），CD45⁺、CD4⁺、T_reg淋巴细胞在小鼠肺组织中的浸润程度显著下降（P<0.05）；肺中Egfr和Pik3cd的mRNA和蛋白表达水平显著下调，talin-1、tensin 2、FAK、vinculin、α-actinin和paxillin蛋白的表达水平也显著降低（P<0.05）。结论正常小鼠血清通过抑制focal adhesion信号通路关键蛋白的表达减轻电离辐射诱发的小鼠放射性肺炎。

关键词: 正常小鼠血清, 放射性肺炎, 外泌体, 粘着斑途径

Abstract:

Objective To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism. Methods Mouse models of radiation pneumonitis induced by thoracic radiation exposure were given intravenous injections of 100 μL normal mouse serum or normal saline immediately after the exposure followed by injections once every other day for a total of 8 injections. On the 15th day after irradiation, histopathological changes of the lungs of the mice were examined using HE staining, the levels of TNF-α, TGF-β, IL-1α and IL-6 in the lung tissue and serum were detected using ELISA, and the percentages of lymphocytes in the lung tissue were analyzed with flow cytometry. High-throughput sequencing of exosome miRNA was carried out to explore the changes in the signaling pathways. The mRNA expression levels of the immune-related genes were detected by qRT-PCR, and the protein expressions of talin-1, tensin2, FAK, vinculin, α-actinin and paxillin in the focal adhesion signaling pathway were detected with Western blotting. Results In the mouse models of radiation pneumonitis, injections of normal mouse serum significantly decreased the lung organ coefficient, lowered the levels of TNF-α, TGF-β, IL-1α and IL-6 in the serum and lung tissues, and ameliorated infiltration of CD45⁺, CD4⁺ and T_reg lymphocytes in the lung tissue (all P<0.05). The expression levels of Egfr and Pik3cd genes at both the mRNA and protein levels and the protein expressions of talin-1, tensin2, FAK, vinculin, α‑actinin and paxillin were all significantly down-regulated in the mouse models after normal mouse serum treatment. Conclusion Normal mouse serum ameliorates radiation pneumonitis in mice by inhibiting the expressions of key proteins in the Focal adhesion signaling pathway.

Key words: normal mouse serum, radiation pneumonitis, exosomes, focal adhesion pathway

苑通, 郭玉莹, 张俊伶, 樊赛军. 正常小鼠血清通过抑制focal adhesion信号通路减轻小鼠放射性肺炎[J]. 南方医科大学学报, 2024, 44(5): 801-809.

Tong YUAN, Yuying GUO, Junling ZHANG, Saijun FAN. Normal mouse serum alleviates radiation pneumonitis in mice by inhibiting the focal adhesion signaling pathway[J]. Journal of Southern Medical University, 2024, 44(5): 801-809.

图/表 7

Fig.1 Normal mouse serum effectively protects normal histological structure of the lung tissue of irradiated mice. A: Gross observation of mouse lungs. B: Comparison of lung organ coefficients among the 4 groups. C: HE staining of the lung tissues in the 4 groups. Data are presented as Mean±SD (n=5). *P<0.05, ***P<0.001.

Fig.2 Intravenous injections of normal mouse serum down-regulate the levels of inflammatory factors in the lung tissue and serum of irradiated mice. A-D: Levels of TGF-β, TNF-α, IL-1α, and IL-6 in the lung tissue, respectively. E-H: Serum levels of TGF-β, TNF-α, IL-1α, and IL-6 in the serum, respectively. Data are presented as Mean±SD (n=8 in panel A and panel E, and n=5 in the rest panels). *P<0.05, **P<0.01, ***P<0.001.

Fig.3 NMS reduces lymphocyte infiltration in the lung tissue of irradiated mice A-D: Results of fluorescence-activated cell sorting showing gating for CD45+, CD4+ and Treg cells and their percentages in total cells. Data are presented as Mean±SD (n=5). *P<0.05, **P<0.01.

Fig.4 Statistics of the differential miRNAs in irradiated and NMS-treated mice (A) and Venn diagram of the differential miRNAs (B).

Fig.5 Number of immune-related differential genes and their expression at mRNA and protein levels in irradiated and NMS-treated mice. A: Classification of KEGG pathways. B, C: Relative mRNA expressions of Gsk3b, Cul3, Tiam1, Prkci, Egfr and Pik3cd in the lung tissue. D, E: Western blots of EGFR and P110δ proteins and their relative expression levels. Data are presented as Mean±SD (n=5 in panel B and panel C, and n=3 in panel D and panel E). *P<0.05, **P<0.01, ***P<0.001.

Fig.6 Normal mouse serum significantly down-regulates the expression of Focal adhesion pathway-related proteins in irradiated mice. A: Enrichment bubble chart of KEGG pathway. B-D: Western blotting of the key proteins in the focal adhesion signaling pathway and their relative expression levels in the lung tissues. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001.

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正常小鼠血清通过抑制focal adhesion信号通路减轻小鼠放射性肺炎

Normal mouse serum alleviates radiation pneumonitis in mice by inhibiting the focal adhesion signaling pathway

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