雾化吸入人脐带间充质干细胞源胞外囊泡对中性粒细胞性哮喘小鼠气道炎症的影响OA

Objective:To investigate the effect of nebulized human umbilical cord mesenchymal stem cells-derived extracellular vesicles(hUCMSC-EVs)on house dust mite(HDM)-and lipopolysaccharide(LPS)-induced neutrophilic inflammation in asthmatic mice and its potential mechanism.Methods:HUCMSC-EVs were isolated using ultra-high-speed centrifugation.The morphology of hUCMSC-EVs was observed via transmission electron microscopy.Western blotting was performed to detect the expression of cluster of differentiation 81(CD81),heat shock protein 70(HSP70),tumor susceptibility gene 101(TSG101)and Calnexin in the EVs.A mouse model of neutrophilic asthma induced by HDM-LPS was established and treated with nebulized hUCMSC-EVs.Fifteen 6-8-week-old female BALB/c mice were randomly divided into three groups:control group,HDM-LPS group,and HDM-LPS+EVs group(5 mice per group).Hematoxylin-eosin(HE)staining was used to observe inflammatory cell infiltration around the pulmonary airways,while periodic acid-Schiff(PAS)staining was applied to detect goblet cell metaplasia in the lungs.A hemocytometer was used to count the total number of inflammatory cells in bronchoalveolar lavage fluid(BALF),and Wright-Giemsa staining was employed to count the total number of neutrophils in BALF.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was conducted to measure the mRNA expression levels of C-X-C motif chemokine ligand 15(Cxcl15)and Cxcl5 in mouse lung tissues.Relevant kits were used to detect the contents of ferrous irons and malondialdehyde(MDA)in lung tissues,and Western blotting was used to determine the expression of glutathione peroxidase 4(GPX4).In vitro,HBE135-E6E7 cells were treated with HDM-LPS or HDM-LPS+EVs;laser confocal microscopy was used to observe the uptake of CM-Dil-labeled hUCMSC-EVs by the huaman bronchial epithelial cell line HBE135-E6E7 cells.In vitro,HBE135-E6E7 cells were treated with HDM-LPS or HDM-LPS+EVs.IL-8 and Cxcl5 mRNA expression was analyzed by qRT-PCR,while ferrous ion,MDA,and GPX4 levels were determined using kits and Western blotting,respectively.Results:HUCMSC-EVs exhibited cuplike membranous structure and expressed CD81,HSP70 and TSG101.Compared with the control group,the HDM-LPS group exhibited obvious inflammatory cell infiltration around airways and airway epithelial goblet cell metaplasia,with significantly increased inflammation score and PAS staining scores(all P＜0.01).The total number of cells and neutrophils in the BALF was significantly increased(all P＜0.01).In lung tissues,the levels of Cxcl15 mRNA,Cxcl5 mRNA,ferrous ions,and MDA were significantly upregulated,while GPX4 expression was significantly downregulated(P＜0.01 or P＜0.05).Compared with the HDM-LPS group,the HDM-LPS+EVs group showed significantly reduced airway inflammation and PAS scores in mice,along with a marked decrease in total cell and neutrophil counts in BALF(P＜0.01).In lung tissues,the levels of Cxcl15 mRNA,Cxcl5 mRNA,ferrous ions,and MDA were reduced,whereas GPX4 expression was increased(P＜0.01 or P＜0.05).In vitro experiments revealed that hUCMSC-EVs could be endocytosed by HBE135-E6E7 cells;and compared with the HDM-LPS group,the HDM-LPS+EVs group showed significantly reduced levels of ferrous ions,MDA,and the expression of IL-8 mRNA and Cxcl5 mRNA,while GPX4 expression was markedly upregulated(P＜0.01 or P＜0.05).Conclusion:Nebulized hUCMSC-EVs could significantly alleviate airway inflammation in mice with neutrophilic asthma induced by HDM-LPS,likely through inhibition of ferroptosis in bronchial epithelial cells,thereby exerting anti-inflammatory effects.