Young Sca-1+ bone marrow stem cell-derived exosomes preserve visual function via the miR-150-5p/MEKK3/JNK/c-Jun pathway to reduce M1 microglial polarization
Yuan Wang, Weipei Qin, Qi Wang, Xin-na Liu, Xianghui Li, X. Ye, Ying Bai, Yan Zhang, Pan Liu, Xinlin Wang, Yuhang Zhou, Huiping Yuan, Zhengbo Shao
Abstract
Abstract 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.