Zong Lu, Huang Pu, Song Qing, Kang Yan
Department of Obstetrics, The First Affiliated Hospital of Xi'an Jiaotong University Xi'an 710061, Shaanxi, China.
Department of Obstetrics, Shandong Provincial Maternal and Child Health Care Hospital, Cheeloo College of Medicine, Shandong University Ji'nan 250014, Shandong, China.
Am J Transl Res. 2021 Mar 15;13(3):935-951. eCollection 2021.
Neuroinflammation is the most common cause of neurological diseases. Exosomes derived from mesenchymal stem cells (MSCs-exos) have been reported to reduce inflammation and neuronal injury. Its underlying mechanism remains poorly unknown. In this study, identification of bone marrow MSCs-derived exosomes (BMSCs-exos) was conducted by nanosight tracking analysis, transmission electron microscope, and western blot assay. Enzyme-linked immunosorbent (ELISA) was used to analyze microglial M1/M2 polarization and detect levels of inflammatory factors. Cell viability was determined by Cell Counting Kit (CCK)-8 assay. Cell apoptosis was assessed by flow cytometry, caspase-3 activity assay, and DNA fragmentation assay. Quantitative real-time polymerase chain reaction was used to detect gene expression. Luciferase reporter and RNA pull-down assays were exploited to validate the interaction between genes. BMSCs-exos promoted M2 polarization while inhibited M1 polarization in LPS-stimulated BV-2 cells. BMSCs-exos inhibited the secretion of interleukin (IL)-1β, IL-6, and TNF-α, while increased the levels of IL-10. BMSCs-exos resisted the cytotoxicity and apoptosis induced by LPS in HT22 cells. BMSCs-exosomal long noncoding RNA (lncRNA) H19 enhanced the anti-inflammatory ability of BMSCs-exos in BV-2 microglia following LPS stimulation, and strengthened the neuroprotective effect of BMSCs-exos on HT22 cells in the presence of LPS. Moreover, H19 functioned as a sponge for miR-29b-3p. miR-29b-3p mimics abolished the effects of BMSCs-exosomal H19 on M1/M2 polarization and inflammation in LPS-stimulated BV-2 cells. The neuroprotective function of BMSCs-exosomal H19 was attenuated by miR-29b-3p mimics in LPS-stimulated HT22 cells. BMSCs-exosomal H19 modulates LPS-stimulated microglial M1/M2 polarization and alleviates inflammation-mediated neurotoxicity by sponging miR-29b-3p.
神经炎症是神经疾病最常见的病因。据报道,间充质干细胞来源的外泌体(MSCs-exos)可减轻炎症和神经元损伤。但其潜在机制仍知之甚少。在本研究中,通过纳米可视跟踪分析、透射电子显微镜和蛋白质免疫印迹分析对骨髓间充质干细胞来源的外泌体(BMSCs-exos)进行鉴定。采用酶联免疫吸附测定(ELISA)分析小胶质细胞的M1/M2极化并检测炎症因子水平。通过细胞计数试剂盒(CCK)-8法测定细胞活力。通过流式细胞术、半胱天冬酶-3活性测定和DNA片段化测定评估细胞凋亡。采用定量实时聚合酶链反应检测基因表达。利用荧光素酶报告基因和RNA下拉实验验证基因之间的相互作用。BMSCs-exos在脂多糖(LPS)刺激的BV-2细胞中促进M2极化,同时抑制M1极化。BMSCs-exos抑制白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)的分泌,同时提高IL-10水平。BMSCs-exos抵抗LPS诱导的HT22细胞的细胞毒性和凋亡。BMSCs-外泌体长链非编码RNA(lncRNA)H19增强LPS刺激后BMSCs-exos在BV-2小胶质细胞中的抗炎能力,并在存在LPS的情况下增强BMSCs-exos对HT22细胞的神经保护作用。此外,H19作为miR-29b-3p的海绵。miR-29b-3p模拟物消除了BMSCs-外泌体H19对LPS刺激的BV-2细胞中M1/M2极化和炎症的影响。在LPS刺激的HT22细胞中,miR-29b-3p模拟物减弱了BMSCs-外泌体H19的神经保护功能。BMSCs-外泌体H19通过结合miR-29b-3p调节LPS刺激的小胶质细胞M1/M2极化并减轻炎症介导的神经毒性。