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

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.

Keywords: exosomes; focal adhesion pathway; normal mouse serum; radiation pneumonitis.

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.