| Objective:The purpose of this study was to investigate the therapeutic effect and mechanism of human umbilical cord mesenchymal stem cells(h UC-MSCs)on acute lung injury(ALI)mouse model,and provide a basis for the clinical research of MSCs in the treatment of ALI.Method:C57BL/6 male mice aged 7-8 weeks were injected with LPS(1.5 mg/kg)through tail vein to establish the ALI mouse model.24 h after LPS injection,the histopathological changes of lung tissue were detected by HE staining to determine the model preparation.To investigate the therapeutic effect of h UC-MSCs on ALI,30 min after LPS injection,mice injected with h UC-MSCs(1×10~6/mice)through tail vein were used as treatment group(LPS+MSCs group),and mice injected with normal saline(NS,200μl/mice)through tail vein were used as untreated group(LPS+NS group);The mice were killed and the lung tissues and blood samples were taken for subsequent studies at 4 h or 24 h after LPS injection:1.Histopathological changes in mouse lung were assessed by HE staining;2.Blood routine and coagulation function test in mice;3.The expression of apoptosis-related protein and inflammatory factors in lung tissue was detected by Western blot;4.Activation of neutrophils in peripheral blood and lung tissue of mice was assessed by flow cytometry and Western blot;5.Phenotypic changes of macrophages in mouse lung tissue were detected by immunohistochemistry and flow cytometry;6.The protein levels of Hypoxia-inducible factor 1α(HIF-1α)in mouse lung tissue were detected by Western blot.To further investigate the effects of h UC-MSCs on the biological properties and functions of macrophages,RAW264.7 cells(a mouse macrophage cell line)were stimulated with LPS for 24 h in vitro while co-cultured with or without h UC-MSCs:1.Differential expression of genes in RAW264.7 cells were analyzed by transcriptomics;2.The changes of M1/M2 phenotype,phagocytosis,inflammatory factors and ROS in RAW264.7 cells were determined by flow cytometry and q PCR;3.The protein levels of HIF-1αin RAW264.7 cells were detected by Western blot.Results:The results of animal experiments showed that h UC-MSCs significantly alleviated the pathological damage of lung tissue in ALI mice,including improved alveolar structure and inhibited the thickening of alveolar walls.h UC-MSCs significantly improved peripheral immune imbalance in ALI mice,increased the proportion of lymphocytes and decreased the proportion of neutrophils in peripheral blood.h UC-MSCs improved lung cell apoptosis,decreased IL-1βexpression,and promoted TGF-βexpression.h UC-MSCs inhibited the activation of neutrophils in lung tissue and blood of ALI mice,including inhibited the expression of myeloperoxidase in neutrophils in lung tissue,inhibited the shedding of CD62L and the expression of CD11b in neutrophils in peripheral blood.h UC-MSCs also promoted the increase of M2-type macrophages in the lung tissue of ALI mice.h UC-MSCs also promoted the degradation of HIF-1αin lung tissue of ALI mice.After co-culture of macrophages and h UC-MSCs in vitro,the macrophages transcriptomic results showed that the differential genes in the LPS group and the LPS+MSCs group were mainly enriched in the TNF and AGE-RAGE signaling pathways,and h UC-MSCs were significantly down-regulated the expressions of Edn1,IL-1β,IL-6,Ptgs2,and Nox1 in macrophages.q RT-PCR results further confirmed that h UC-MSCs reduced the levels of IL-1βand IL-6 in macrophages,and at the same time reduced the expression levels of pro-inflammatory factor TNF-αand oxidative stress molecule ROS.Co-culture with h UC-MSCs promoted the polarization of macrophages to M2 type,increased the phagocytic ability of macrophages,and promote the degradation of HIF-1αin macrophages.Conclusion:h UC-MSCs could significantly ameliorate the pathological damage of lung tissue in LPS-induced ALI mice,inhibit neutrophil hyperactivation,and promote the polarization of lung macrophages to anti-inflammatory M2 type.Further in vitro experiments showed that h UC-MSCs could promote the polarization to M2 type and phagocytic capacity of macrophages under inflammatory conditions,and inhibit the production of pro-inflammatory factors and ROS in macrophages.Our results suggested that h UC-MSCs alleviate LPS induced ALI may though inhibiting inflammation and oxidative stress.This study provides theoretical and experimental basis for the clinical study of h UC-MSCs in the treatment of ALI. |
