Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China.
Future Medical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin, China.
J Nanobiotechnology. 2023 Jun 15;21(1):194. doi: 10.1186/s12951-023-01944-w.
BACKGROUND: Polarization of microglia, the resident retinal immune cells, plays important roles in mediating both injury and repair responses post-retinal ischemia-reperfusion (I/R) injury, which is one of the main pathological mechanisms behind ganglion cell apoptosis. Aging could perturb microglial balances, resulting in lowered post-I/R retinal repair. Young bone marrow (BM) stem cell antigen 1-positive (Sca-1) cells have been demonstrated to have higher reparative capabilities post-I/R retinal injury when transplanted into old mice, where they were able to home and differentiate into retinal microglia. METHODS: Exosomes were enriched from young Sca-1 or Sca-1 cells, and injected into the vitreous humor of old mice post-retinal I/R. Bioinformatics analyses, including miRNA sequencing, was used to analyze exosome contents, which was confirmed by RT-qPCR. Western blot was then performed to examine expression levels of inflammatory factors and underlying signaling pathway proteins, while immunofluorescence staining was used to examine the extent of pro-inflammatory M1 microglial polarization. Fluoro-Gold labelling was then utilized to identify viable ganglion cells, while H&E staining was used to examine retinal morphology post-I/R and exosome treatment. RESULTS: Sca-1 exosome-injected mice yielded better visual functional preservation and lowered inflammatory factors, compared to Sca-1, at days 1, 3, and 7 days post-I/R. miRNA sequencing found that Sca-1 exosomes had higher miR-150-5p levels, compared to Sca-1 exosomes, which was confirmed by RT-qPCR. Mechanistic analysis found that miR-150-5p from Sca-1 exosomes repressed the mitogen-activated protein kinase kinase kinase 3 (MEKK3)/JNK/c-Jun axis, leading to IL-6 and TNF-α downregulation, and subsequently reduced microglial polarization, all of which contributes to reduced ganglion cell apoptosis and preservation of proper retinal morphology. CONCLUSION: This study elucidates a potential new therapeutic approach for neuroprotection against I/R injury, via delivering miR-150-5p-enriched Sca-1 exosomes, which targets the miR-150-5p/MEKK3/JNK/c-Jun axis, thereby serving as a cell-free remedy for treating retinal I/R injury and preserving visual functioning.
背景：小胶质细胞极化是视网膜固有免疫细胞的重要作用之一，在介导视网膜缺血再灌注（I/R）损伤后的损伤和修复反应中发挥重要作用，这是节细胞凋亡的主要病理机制之一。衰老可能会扰乱小胶质细胞的平衡，导致 I/R 后视网膜修复能力下降。已经证明，年轻的骨髓（BM）干细胞抗原 1 阳性（Sca-1）细胞在移植到老年小鼠后，在 I/R 视网膜损伤后具有更高的修复能力，它们能够归巢并分化为视网膜小胶质细胞。 方法：从小鼠的 Sca-1 或 Sca-1 细胞中分离出外泌体，并在视网膜 I/R 后注入玻璃体。采用生物信息学分析，包括 miRNA 测序，分析外泌体的内容物，并通过 RT-qPCR 进行验证。然后进行 Western blot 以检查炎症因子和潜在信号通路蛋白的表达水平，同时进行免疫荧光染色以检查促炎 M1 小胶质细胞极化的程度。然后使用氟金标识别存活的节细胞，并用 H&E 染色检查 I/R 后和外泌体处理后的视网膜形态。 结果：与 Sca-1 相比，在 I/R 后第 1、3 和 7 天，Sca-1 外泌体注射小鼠的视觉功能保存更好，炎症因子水平更低。miRNA 测序发现，与 Sca-1 外泌体相比，Sca-1 外泌体具有更高水平的 miR-150-5p，通过 RT-qPCR 进行验证。机制分析发现，Sca-1 外泌体中的 miR-150-5p 抑制丝裂原活化蛋白激酶激酶激酶 3（MEKK3）/JNK/c-Jun 轴，导致 IL-6 和 TNF-α下调，进而减少小胶质细胞极化，所有这些都有助于减少节细胞凋亡并保持适当的视网膜形态。 结论：这项研究通过递送富含 miR-150-5p 的 Sca-1 外泌体，阐明了一种针对 I/R 损伤的神经保护的潜在新治疗方法，该方法靶向 miR-150-5p/MEKK3/JNK/c-Jun 轴，从而作为一种无细胞疗法来治疗视网膜 I/R 损伤并维持视觉功能。
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