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

Effect of inhalation of human umbilical cord mesenchymal stem cells-derived extracellular vesicles on airway inflammation in neutrophilic asthmatic mice

摘要

目的: 探究雾化人脐带间充质干细胞源胞外囊泡(human umbilical cord mesenchymal stem cells-derived extracellular vesicles,hUCMSC-EVs)对屋尘螨(house dust mite,HDM)脂多糖(lipopolysaccharides,LPS)诱导的中性粒细胞性哮喘小鼠气道炎症的影响及其潜在机制。方法: 采用超高速离心法提取hUCMSC-EVs;透射电镜观察hUCMSC-EVs形态;蛋白免疫印迹法检测细胞外囊泡中分化簇81(cluster of differentiation 81,CD81)、热休克蛋白70(heat shock protein 70,HSP70)、肿瘤易感基因101(tumor susceptibility gene 101,TSG101)和钙联蛋白(Calnexin)表达。构建HDM-LPS诱导的中性粒细胞性哮喘小鼠模型并给予hUCMSC-EVs雾化干预,将15只6~8周龄、雌性BALB/c小鼠随机分为对照组、HDM-LPS组和HDM-LPS+EVs组,每组5只;HE和过碘酸雪夫(periodic acid-Schiff,PAS)染色法分别观察肺脏气道周围炎性细胞浸润和杯状细胞化生情况;血细胞计数板和瑞氏吉姆萨染色分别计数肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中炎症细胞和中性粒细胞总数;qRT-PCR检测小鼠肺脏组织C-X-C基序趋化因子配体15(C-X-C motif chemokineligand 15,Cxcl15)和Cxcl5 mRNA表达;采用相关试剂盒检测各组小鼠肺组织中亚铁离子和丙二醛含量,蛋白免疫印迹法检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)表达。激光共聚焦显微镜观察人支气管上皮细胞株HBE135-E6E7对CM-Dil标记的hUCMSC-EVs的摄取情况;体外应用HDM-LPS、HDM-LPS+EVs处理HBE135-E6E7细胞,qRT-PCR检测各组细胞中IL-8 mRNA和Cxcl5 mRNA表达,采用相关试剂盒和蛋白免疫印迹法分别检测各组亚铁离子、丙二醛和GPX4表达。结果: HUCMSC-EVs呈杯托样膜性结构且表达CD81、HSP70和TSG101。与对照组相比,HDM-LPS组小鼠气道周围炎性细胞浸润和杯状细胞化生程度明显,炎症和PAS染色评分显著升高(P<0.01),BALF中总细胞和中性粒细胞数明显增多(P<0.01),肺组织中Cxcl15 mRNA、Cxcl5 mRNA、亚铁离子和丙二醛含量显著增加而GPX4表达明显降低(P<0.01或P<0.05);与HDM-LPS组相比,HDM-LPS+EVs组小鼠气道炎症和PAS评分显著降低,BALF中总细胞和中性粒细胞数均明显减少(P<0.01),肺脏Cxcl15 mRNA、Cxcl5 mRNA、亚铁离子和丙二醛含量明显降低而GPX4蛋白表达明显升高(P<0.01或P<0.05)。体外实验结果显示,hUCMSC-EVs可经HBE135-E6E7细胞内吞;与HDM-LPS组相比,HDM-LPS+EVs组细胞中亚铁离子、丙二醛含量、IL-8 mRNA和Cxcl5 mRNA表达均显著降低而GPX4表达明显增加(P<0.01或P<0.05)。结论: 雾化hUCMSC-EVs可显著改善HDM-LPS诱导的中性粒细胞性哮喘小鼠气道炎症,其可能是通过抑制支气管上皮细胞铁死亡实现抗炎作用。

Abstract

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.

关键词

人脐带间充质干细胞源胞外囊泡 / 雾化吸入 / 中性粒细胞性哮喘 / 气道炎症 / 铁死亡

Key words

human umbilical cord mesenchymal stem cells-derived extracellular vesicles / nebulized inhalation / neutrophilic asthma / airway inflammation / ferroptosis

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基金资助

国家自然科学基金资助项目(82300035)

国家自然科学基金资助项目(81900562)

江苏省自然科学基金资助项目(BK20251848)

江苏省卫健委医学科研项目(H2023021)

江苏省卫健委“血地寄防”项目(x202306)

镇江市临床医学重点实验室项目(SS2023012)