H2S调控BMSCs外泌体相关miR-9/TN-C轴对HIBD后的神经保护作用及机制研究

Neonatal hypoxic-ischemic brain damage (HIBD) is a leading cause of mortality and neurological disabilities in infants and young children. Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes have been reported as a promising target in HIBD therapy. Our previous work has demonstrated that H2S-preconditioned BMSCs could enhance the therapeutic effects of BMSCs on neuronal injury and neurological recovery. Moreover, H2S also exerted neuroprotective roles in HIBD. However, the role of H2S-preconditioned BMSCs on neonatal HIBD remains unknown. Recently, we found that exosomes derived from BMSCs mainly targeted to the neurons in lesioned hemisphere in HIBD mice. Exosomes derived from H2S-preconditioned BMSCs achieved more powerful effects in alleviating brain injury following HIBD in mice. Based on the next-generation sequencing analysis, we hypothesize that these effects are more likely to rely on microRNA(miRNA) system delivered by the H2S-triggered exosome from BMSCs, which suppresses neuronal apoptosis and neuroinflammation, promotes synaptic remodeling. This research gives a priority to miR-9, one that was significantly upregulated in BMSCs-derived exosome after H2S stimuli. We are to demonstrate miR-9 and its promising target tenascin-c (TN-C) neuroprotective unit in HIBD in vivo and vitro and clarify the mechanism via neuronal conditional TN-C deficient mice. This project is to present new stubborn evidence to enrich clinical treatment to HIBD.

缺氧缺血性脑损伤（HIBD）是引起新生儿死亡及幸存者长期神经功能缺陷的重要原因。骨髓间充质干细胞(BMSCs)来源的外泌体具有的神经保护作用为改善HIBD神经功能提供了新思路。我们曾报道H2S预处理的BMSCs移植于缺血再灌注模型后可显著减轻脑损伤，而H2S在HIBD新生小鼠中也有显著的神经保护作用，但其作用是否依赖BMSCs尚不清楚。我们最新研究发现BMSCs外泌体主要靶向至HIBD新生小鼠受损脑区神经元，而H2S预处理的BMSCs外泌体更能显著抑制神经元凋亡和炎症反应，促进突触重塑，从而发挥更强的神经保护作用。进一步对H2S预处理的BMSCs外泌体进行miRNA测序后发现miR-9/TN-C轴可能起关键作用。本课题拟通过体外实验联合神经元条件敲除TN-C小鼠模型，全面阐明H2S诱导的BMSCs外泌体对HIBD后的神经保护作用及机制，为研发H2S改良外泌体治疗HIBD的策略提供重要支持。

我们关注间充质干细胞( mesenchymal stem cells, MSCs)来源的细胞外囊泡（extracellular vesicles, EVs）中携带的miRNAs作为一个潜在的与这种神经保护作用相关的因素的作用。基于现有的研究数据，我们本研究旨在探讨H2S预处理MSCs后分泌的EVs（H2S-EVs）与普通EVs相比是否具有更优越的神经保护作用及其发挥更优越神经保护作用的可能机制。利用流式、免疫组化、蛋白印迹等分子生物学方法，结合行为学等方法，探讨H2S-EVs 和MSCs-EVs在新生儿缺氧缺血性脑损伤（hypoxia-ischemia, HI）中的神经保护作用及机制，结果如下：（1）H2S-EVs的分布：免疫荧光结果显示H2S-EVs同普通EVs结果一致，大量聚集于右侧（损伤侧）半球的Iba-1+小胶质细胞内。同时在右侧半球损伤区NeuN+神经元以及GFAP+星形胶质细胞中也发现了一定数量的H2S-EVs；（2）H2S-EVs改善新生小鼠HI后72 h脑损伤；（3） H2S-EVs抑制新生小鼠HI后72 h的细胞凋亡；（4）H2S-EVs对新生小鼠HI后72 h右侧（损伤侧）皮层的发挥免疫调控作用；（5）H2S-EVs促进新生小鼠HI后远期神经功能恢复；（6）H2S-EVs通过上调HI小鼠脑内的miR-7b-5p，减轻脑损伤及神经炎症发挥神经保护作用；（7）H2S-EVs通过调控miR-7b-5p/FOS轴抑制HI诱导的脑损伤及神经炎症，发挥神经保护作用。本研究表明，H2S-EVs比普通EVs具有更优越的神经保护作用，同时也阐明了H2S-EVs具有更优越神经保护作用的可能机制。H2S预处理MSCs后上调了EVs中miR-7b-5p的表达，H2S-EVs作为高丰度miR-7b-5p的天然递送载体将miR-7b-5p递送至HI小鼠右侧（损伤侧）原驻小胶质细胞/浸润单核-巨噬细胞，抑制原驻小胶质细胞/浸润单核-巨噬细胞活化的同时促进其向M2型极化，减轻神经炎症，从而改善HI小鼠远期学习和记忆功能。此外，对EVs进行改造也可成为围生期脑损伤的潜在治疗策略。