缺氧的hUCMSC产生的细胞外囊泡减轻了慢性哮喘小鼠的过敏性气道炎症和气道重塑
2021/03/22
摘要
背景：作为间充质干细胞（MSCs）的主要功能形式之一，源自MSC的细胞外囊泡（MSC-EVs）在过敏性哮喘的实验模型中已显示出替代的治疗选择。氧浓度在MSCs的自我更新，增殖和EV释放中起着重要作用，最近的一项研究发现，低氧条件下的培养可增强MSCs的抗哮喘作用。然而，尚不清楚低氧MSC产生的细胞外囊泡（Hypo-EV）在哮喘中的潜力。
方法：用卵清蛋白（OVA）对BALB / c雌性小鼠进行致敏和激发，每组每周接受PBS，正常氧人脐带MSC-EV（Nor-EV）或Hypo-EV。治疗后，对动物实施安乐死，并收集其肺和支气管肺泡灌洗液（BALF）。通过使用苏木精和曙红（HE），高碘酸席夫（PAS）和Masson的三色染色，酶联免疫吸附测定（ELISA），Western印迹分析和实时PCR，观察气道和肺实质炎症反应及胶原纤维含量变化。
结果：低氧环境可以促进人脐带间充质干细胞（hUCMSC）释放更多的细胞外囊泡。在OVA动物中，Nor-EV或Hypo-EV的使用可显着改善哮喘小鼠的BALF总细胞，嗜酸性粒细胞和促炎性介质（IL-4和IL-13）。此外，Hypo-EV在抑制哮喘小鼠气道炎症方面通常比Nor-EV更有效。与Nor-EV相比，Hypo-EV进一步阻止了小鼠慢性过敏性气道重塑，同时伴随着促纤维化标志物α-平滑肌肌动蛋白（α-SMA），胶原蛋白1和TGF-β1-p-smad2/3信号通路的表达降低。在体外，Hypo-EV降低了TGF-β1刺激的HLF-1细胞（人肺成纤维细胞）中p-smad2 / 3，α-SMA和胶原蛋白1的表达。此外，我们显示，与Nor-EV相比，Hypo-EV富含miR-146a-5p，Hypo-EV的给药未调控哮喘小鼠肺组织和TGF-β1处理过的HLF-1中的miR-146a-5p的表达。更重要的是，Hypo-EVs中miR-146a-5p表达的降低损害了OVA小鼠中Hypo-EV介导的肺保护。
结论：我们的发现提供了第一个证据，即缺氧的hUCMSC衍生的EV减轻了慢性哮喘小鼠的过敏性气道炎症和气道重塑，可能为治疗哮喘创造新的途径。
Hypoxic hUCMSC-derived extracellular vesicles attenuate allergic airway inflammation and airway remodeling in chronic asthma mice
Dong L, Wang Y, Zheng T, et al.
Abstract
Background: As one of the main functional forms of mesenchymal stem cells (MSCs), MSC-derived extracellular vesicles (MSC-EVs) have shown an alternative therapeutic option in experimental models of allergic asthma. Oxygen concentration plays an important role in the self-renewal, proliferation, and EV release of MSCs and a recent study found that the anti-asthma effect of MSCs was enhanced by culture in hypoxic conditions. However, the potential of hypoxic MSC-derived EVs (Hypo-EVs) in asthma is still unknown.
Methods: BALB/c female mice were sensitized and challenged with ovalbumin (OVA), and each group received PBS, normoxic human umbilical cord MSC-EVs (Nor-EVs), or Hypo-EVs weekly. After treatment, the animals were euthanized, and their lungs and bronchoalveolar lavage fluid (BALF) were collected. With the use of hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome staining, enzyme-linked immune sorbent assay (ELISA), Western blot analysis, and real-time PCR, the inflammation and collagen fiber content of airways and lung parenchyma were investigated.
Results: Hypoxic environment can promote human umbilical cord MSCs (hUCMSCs) to release more EVs. In OVA animals, the administration of Nor-EVs or Hypo-EVs significantly ameliorated the BALF total cells, eosinophils, and pro-inflammatory mediators (IL-4 and IL-13) in asthmatic mice. Moreover, Hypo-EVs were generally more potent than Nor-EVs in suppressing airway inflammation in asthmatic mice. Compared with Nor-EVs, Hypo-EVs further prevented mouse chronic allergic airway remodeling, concomitant with the decreased expression of pro-fibrogenic markers α-smooth muscle actin (α-SMA), collagen-1, and TGF-β1-p-smad2/3 signaling pathway. In vitro, Hypo-EVs decreased the expression of p-smad2/3, α-SMA, and collagen-1 in HLF-1 cells (human lung fibroblasts) stimulated by TGF-β1. In addition, we showed that miR-146a-5p was enriched in Hypo-EVs compared with that in Nor-EVs, and Hypo-EV administration unregulated the miR-146a-5p expression both in asthma mice lung tissues and in TGF-β1- treated HLF-1. More importantly, decreased miR-146a-5p expression in Hypo-EVs impaired Hypo-EV-mediated lung protection in OVA mice.
Conclusion: Our findings provided the first evidence that hypoxic hUCMSC-derived EVs attenuated allergic airway inflammation and airway remodeling in chronic asthma mice, potentially creating new avenues for the treatment of asthma.
背景：作为间充质干细胞（MSCs）的主要功能形式之一，源自MSC的细胞外囊泡（MSC-EVs）在过敏性哮喘的实验模型中已显示出替代的治疗选择。氧浓度在MSCs的自我更新，增殖和EV释放中起着重要作用，最近的一项研究发现，低氧条件下的培养可增强MSCs的抗哮喘作用。然而，尚不清楚低氧MSC产生的细胞外囊泡（Hypo-EV）在哮喘中的潜力。
方法：用卵清蛋白（OVA）对BALB / c雌性小鼠进行致敏和激发，每组每周接受PBS，正常氧人脐带MSC-EV（Nor-EV）或Hypo-EV。治疗后，对动物实施安乐死，并收集其肺和支气管肺泡灌洗液（BALF）。通过使用苏木精和曙红（HE），高碘酸席夫（PAS）和Masson的三色染色，酶联免疫吸附测定（ELISA），Western印迹分析和实时PCR，观察气道和肺实质炎症反应及胶原纤维含量变化。
结果：低氧环境可以促进人脐带间充质干细胞（hUCMSC）释放更多的细胞外囊泡。在OVA动物中，Nor-EV或Hypo-EV的使用可显着改善哮喘小鼠的BALF总细胞，嗜酸性粒细胞和促炎性介质（IL-4和IL-13）。此外，Hypo-EV在抑制哮喘小鼠气道炎症方面通常比Nor-EV更有效。与Nor-EV相比，Hypo-EV进一步阻止了小鼠慢性过敏性气道重塑，同时伴随着促纤维化标志物α-平滑肌肌动蛋白（α-SMA），胶原蛋白1和TGF-β1-p-smad2/3信号通路的表达降低。在体外，Hypo-EV降低了TGF-β1刺激的HLF-1细胞（人肺成纤维细胞）中p-smad2 / 3，α-SMA和胶原蛋白1的表达。此外，我们显示，与Nor-EV相比，Hypo-EV富含miR-146a-5p，Hypo-EV的给药未调控哮喘小鼠肺组织和TGF-β1处理过的HLF-1中的miR-146a-5p的表达。更重要的是，Hypo-EVs中miR-146a-5p表达的降低损害了OVA小鼠中Hypo-EV介导的肺保护。
结论：我们的发现提供了第一个证据，即缺氧的hUCMSC衍生的EV减轻了慢性哮喘小鼠的过敏性气道炎症和气道重塑，可能为治疗哮喘创造新的途径。
（中日友好医院呼吸与危重症医学科 李春晓 摘译 林江涛 审校）
（Stem Cell Res Ther, 2021, 12(1): 4.）
（Stem Cell Res Ther, 2021, 12(1): 4.）
Hypoxic hUCMSC-derived extracellular vesicles attenuate allergic airway inflammation and airway remodeling in chronic asthma mice
Dong L, Wang Y, Zheng T, et al.
Abstract
Background: As one of the main functional forms of mesenchymal stem cells (MSCs), MSC-derived extracellular vesicles (MSC-EVs) have shown an alternative therapeutic option in experimental models of allergic asthma. Oxygen concentration plays an important role in the self-renewal, proliferation, and EV release of MSCs and a recent study found that the anti-asthma effect of MSCs was enhanced by culture in hypoxic conditions. However, the potential of hypoxic MSC-derived EVs (Hypo-EVs) in asthma is still unknown.
Methods: BALB/c female mice were sensitized and challenged with ovalbumin (OVA), and each group received PBS, normoxic human umbilical cord MSC-EVs (Nor-EVs), or Hypo-EVs weekly. After treatment, the animals were euthanized, and their lungs and bronchoalveolar lavage fluid (BALF) were collected. With the use of hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome staining, enzyme-linked immune sorbent assay (ELISA), Western blot analysis, and real-time PCR, the inflammation and collagen fiber content of airways and lung parenchyma were investigated.
Results: Hypoxic environment can promote human umbilical cord MSCs (hUCMSCs) to release more EVs. In OVA animals, the administration of Nor-EVs or Hypo-EVs significantly ameliorated the BALF total cells, eosinophils, and pro-inflammatory mediators (IL-4 and IL-13) in asthmatic mice. Moreover, Hypo-EVs were generally more potent than Nor-EVs in suppressing airway inflammation in asthmatic mice. Compared with Nor-EVs, Hypo-EVs further prevented mouse chronic allergic airway remodeling, concomitant with the decreased expression of pro-fibrogenic markers α-smooth muscle actin (α-SMA), collagen-1, and TGF-β1-p-smad2/3 signaling pathway. In vitro, Hypo-EVs decreased the expression of p-smad2/3, α-SMA, and collagen-1 in HLF-1 cells (human lung fibroblasts) stimulated by TGF-β1. In addition, we showed that miR-146a-5p was enriched in Hypo-EVs compared with that in Nor-EVs, and Hypo-EV administration unregulated the miR-146a-5p expression both in asthma mice lung tissues and in TGF-β1- treated HLF-1. More importantly, decreased miR-146a-5p expression in Hypo-EVs impaired Hypo-EV-mediated lung protection in OVA mice.
Conclusion: Our findings provided the first evidence that hypoxic hUCMSC-derived EVs attenuated allergic airway inflammation and airway remodeling in chronic asthma mice, potentially creating new avenues for the treatment of asthma.
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MSC产生的外泌体可通鼻内注射使小鼠产生分泌IL-10的肺间质巨噬细胞来减轻过敏性哮喘
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