低氧hUCMSC-EVs通过转移CAV-1减轻慢性哮喘小鼠的气道上皮屏障缺陷
2024/11/29
摘要
背景：低氧人脐带间充质干细胞衍生的细胞外囊泡（Hypo-EVs）可抑制慢性哮喘小鼠的气道炎症和重塑，但其确切机制仍不清楚。最近，气道上皮屏障缺陷被认为是哮喘的关键治疗靶点。本研究的目的是探讨在哮喘条件下，Hypo-EVs 是否以及如何保护气道上皮屏障免受破坏。
方法：在卵清蛋白（OVA）诱导的哮喘小鼠以及 IL-4 和 IL-13 诱导的 HBE135-E6E7 细胞模型中，通过检测细胞单层渗漏和连接蛋白表达，评估了 Hypo-EVs 对气道上皮屏障缺陷的治疗效果。通过 Western 印迹法测定了Hypo-EVs蛋白水平，并采用基因敲除法研究了Hypo-EVs中的生物因子。
结果：雾化吸入 Hypo-EVs 直接缓解了哮喘小鼠的气道上皮屏障缺陷，表现为与支气管上皮细胞共定位、白蛋白浓度降低以及 ZO-1 和 E-cadherin 表达增加。在体外，Hypo-EVs 处理可显著缓解气道细胞通透性的增加，并上调 ZO-1 和 E-cadherin 蛋白的表达。基于 WB 分析，我们发现CAV-1在 Hypo-EVs 中强烈富集。在体外和体内，CAV-1蛋白水平被敲除会明显削弱Hypo-EVs介导的屏障保护作用。此外，CAV-1的敲除还明显削弱了Hypo-EVs对哮喘小鼠气道炎症和重塑的有益作用。此外，我们还发现，IL-4/IL-13 诱导的气道上皮屏障缺陷主要与 STAT6 磷酸化（p-STAT6）的激活有关，而过表达 CAV-1 或 Hypo-EVs 可抑制 IL-4/IL-13 诱导的 HBE135-E6E7 细胞中 p-STAT6 的水平。
结论：雾化Hypo-EVs可通过输送CAV-1抑制p-STAT6的表达来减轻哮喘的气道上皮屏障缺陷，并可用于治疗其他屏障缺陷疾病。
Nebulization of Hypoxic hUCMSC-EVs Attenuates Airway Epithelial Barrier Defects in Chronic Asthma Mice by Transferring CAV-1
Luo X, Wang Y, Mao Y, Xu X, Gu W, Li W, Mao C, Zheng T, Dong L.
Abstract
Background:Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions.
Methods:The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs.
Results:Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells.
Conclusion:Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases
背景：低氧人脐带间充质干细胞衍生的细胞外囊泡（Hypo-EVs）可抑制慢性哮喘小鼠的气道炎症和重塑，但其确切机制仍不清楚。最近，气道上皮屏障缺陷被认为是哮喘的关键治疗靶点。本研究的目的是探讨在哮喘条件下，Hypo-EVs 是否以及如何保护气道上皮屏障免受破坏。
方法：在卵清蛋白（OVA）诱导的哮喘小鼠以及 IL-4 和 IL-13 诱导的 HBE135-E6E7 细胞模型中，通过检测细胞单层渗漏和连接蛋白表达，评估了 Hypo-EVs 对气道上皮屏障缺陷的治疗效果。通过 Western 印迹法测定了Hypo-EVs蛋白水平，并采用基因敲除法研究了Hypo-EVs中的生物因子。
结果：雾化吸入 Hypo-EVs 直接缓解了哮喘小鼠的气道上皮屏障缺陷，表现为与支气管上皮细胞共定位、白蛋白浓度降低以及 ZO-1 和 E-cadherin 表达增加。在体外，Hypo-EVs 处理可显著缓解气道细胞通透性的增加，并上调 ZO-1 和 E-cadherin 蛋白的表达。基于 WB 分析，我们发现CAV-1在 Hypo-EVs 中强烈富集。在体外和体内，CAV-1蛋白水平被敲除会明显削弱Hypo-EVs介导的屏障保护作用。此外，CAV-1的敲除还明显削弱了Hypo-EVs对哮喘小鼠气道炎症和重塑的有益作用。此外，我们还发现，IL-4/IL-13 诱导的气道上皮屏障缺陷主要与 STAT6 磷酸化（p-STAT6）的激活有关，而过表达 CAV-1 或 Hypo-EVs 可抑制 IL-4/IL-13 诱导的 HBE135-E6E7 细胞中 p-STAT6 的水平。
结论：雾化Hypo-EVs可通过输送CAV-1抑制p-STAT6的表达来减轻哮喘的气道上皮屏障缺陷，并可用于治疗其他屏障缺陷疾病。
(中日友好医院呼吸与危重症医学科 沈焜路 摘译 林江涛 审校)
(Int J Nanomedicine. 2024 Oct 29; DOI: 10.2147/IJN.S476151)
(Int J Nanomedicine. 2024 Oct 29; DOI: 10.2147/IJN.S476151)
Nebulization of Hypoxic hUCMSC-EVs Attenuates Airway Epithelial Barrier Defects in Chronic Asthma Mice by Transferring CAV-1
Luo X, Wang Y, Mao Y, Xu X, Gu W, Li W, Mao C, Zheng T, Dong L.
Abstract
Background:Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions.
Methods:The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs.
Results:Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells.
Conclusion:Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases
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