关键词: 哮喘, 叉头转录因子1, 骨髓间充质干细胞, 嗜酸性粒细胞, 气道重塑

Abstract:

Objective To discuss the inhibitory effect of bone marrow-derived mesenchymal stem cells （BMSCs） over-expressing forkhead box transcription factor 1 （FOXO1） on the eosinophil infiltration and airway remodeling of the asthmatic mice， and to clarify the possible mechanism. Methods The BMSCs of the mice were isolated， and Oil Red O staining and Alizarin staining were used to identify the BMSCs. Flow cytometry was used to identify the phenotypes of the BMSCs. The BMSCs at logarithmic growth phase were collected and divided into control group （without treatment）， FOXO1-BMSCs group （infected with recombinant lentivirus carrying FOXO1 gene）， and NC-BMSCs group （infected with negative control recombinant lentivirus）. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of FOXO1 mRNA in the BMSCs in various groups； Western blotting method was used to detect the expression levels of FOXO1 protein in the BMSCs in various gorups. Forty mice were randomly divided into control group （given saline）， model group （given saline）， NC-BMSCs group （given NC-BMSCs cell suspension）， and FOXO1-BMSCs group （given FOXO1-BMSCs cell suspension），and there were 10 mice in each group. Except for control group， the mice in the other three groups were sensitized with ovalbumin （OVA） and challenged with aerosol to establish the asthma models. The cell classification counts in bronchoalveolar lavage fluid （BALF） of the mice in various groups were detected；the histopathology of lung tissue of the mice in various groups was observed by HE staining；the expressions of α-smooth muscle actin （α-SMA） and proliferating cell nuclear antigen （PCNA） in lung tissue of the mice in various groups were detected by immunofluorescence method； the bronchial lumen circumference （Pi）， wall area （W）， smooth muscle area （S）， and numbers of smooth muscle cell nuclei （N） of the mice in various groups were detected by using Image-Pro Plus Software，and the ratios of S/Pi， W/Pi， and N/Pi were calculated； the expression levels of matrix metalloproteinase-9 （MMP-9），matrix metalloproteinase-12 （MMP-12）， and tissue inhibitor of metalloproteinase-1 （TIMP-1） proteins in lung tissue of the mice in various groups were detected by Western blotting method. Results The isolated and cultured BMSCs showed a spindle-shaped morphology， and significant red lipid droplets and orange-red precipitates could be observed after Oil Red O and Alizarin staining. The expression amonuts of CD29， CD44， and CD71 on surface of the BMSCs were increased， while the expression amonuts of CD34， CD45， and HLA-DR were decreased. Compared with control group and NC-BMSCs group， the expression levels of FOXO1 mRNA and protein in the BMSCs in FOXO1-BMSCs group were increased （P<0.05）. Compared with model group and NC-BMSCs group， the counts of eosinophils， macrophages， lymphocytes， and neutrophils in bronchoalveolar lavage fluid （BALF） of the mice in FOXO1-BMSCs group were decreased（P<0.05）， and the inflammatory cell infiltration in the airways and alveoli was alleviated； the ratios of S/Pi， W/Pi， and N/Pi were decreased（P<0.05）， and the expression amounts of α-SMA and PCNA in lung tissue were decreased， the expression levels of MMP-9， MMP-12， and TIMP-1 proteins in lung tissue were decreased （P<0.05）. Conclusion The BMSCs over-expressing FOXO1 can attenuate the inflammatory cell infiltration， particularly eosinophil infiltration， and inhibit airway remodeling in the asthmatic mice， thus alleviating the asthma symptoms.

Key words: Asthma, Forkhead box transcription factor 1, Bone marrow mesenchymal stem cells, Eosinophils, Airway remodeling