黑色素纳米颗粒促线粒体转移增强干细胞神经保护

Melanin nanoparticles enhance stem cell-mediated neuroprotection by promoting mitochondrial transfer

摘要

目的 探讨黑色素纳米颗粒（MNPs）预处理对骨髓间充质干细胞（BMSCs）线粒体转移的调控作用，及其增强 BMSCs 对缺血缺氧损伤神经元的保护效应与潜在机制。方法 采用氧-葡萄糖剥夺（OGD）处理原代皮层神经元，构建体外缺血性损伤模型。将神经元随机分为 7 组：Normal control 组、OGD 组、OGD+MNPs 组、OGD+BMSCs 组、 OGD+MNPs-BMSCs 组、 Transwell+BMSCs 组、 Transwell+MNPs-BMSCs 组。通过 MitoTracker Green/Red 双标记示踪结合激光共聚焦显微镜、流式细胞术定量分析线粒体跨细胞转移效率；检测各组神经元细胞存活率、活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）及磷酸戊糖途径关键代谢物还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、还原型谷胱甘肽（GSH）水平。采用蛋白免疫印迹法检测线粒体外膜转位酶 20（TOMM20）、线粒体融合素 2（MFN2）、动力蛋白相关蛋白 1（DRP1）的表达变化。结果 与 OGD+BMSCs 组相比，OGD+MNPs-BMSCs 组神经元存活率升高（ P ＜ 0.000 1）。MNPs 预处理可使 BMSCs 向神经元的线粒体转移效率提升 1.6 倍（ P ＜ 0.05），且该过程依赖细胞间直接接触（Transwell 物理隔离后转移效率下降， P ＜ 0.05）。机制层面，与 OGD 组比较，OGD+MNPs-BMSCs 组神经元 ROS、MDA 水平降低，SOD 活性升高（均 P ＜ 0.05）；磷酸戊糖途径代谢活性增强，NADPH、GSH 水平增加（均 P ＜ 0.05）；线粒体动力学相关蛋白 TOMM20、MFN2 表达上调，DRP1 表达下调（均 P ＜ 0.05）。结论 MNPs 可通过促进 BMSCs 向缺血缺氧损伤神经元的功能性线粒体转移，增强神经元内源性抗氧化能力，激活磷酸戊糖途径代谢稳态，并改善线粒体融合-裂变失衡与生物发生障碍，发挥协同神经保护作用。

Abstract

Objective To investigate the regulatory effect of melanin nanoparticle (MNP) pretreatment on mitochondrial transfer from bone marrow mesenchymal stem cells (BMSCs), as well as its protective effect and potential mechanisms in enhancing BMSC-mediated protection of neurons injured by ischemia and hypoxia. Methods Primary cortical neurons were treated with oxygen-glucose deprivation (OGD) to establish an in vitro ischemic injury model. The neurons were randomly divided into seven groups: the Normal control group, OGD group, OGD + MNPs group, OGD + BMSCs group, OGD + MNPs-BMSCs group, Transwell + BMSCs group, and Transwell + MNPs-BMSCs group. MitoTracker Green/Red dual-label tracing combined with laser confocal microscopy and flow cytometry was used to quantitatively analyze the efficiency of intercellular mitochondrial transfer. Neuronal cell viability, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and levels of the key metabolites of the pentose phosphate pathway, reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH), were measured in each group. Western blotting was used to detect changes in the expression of translocase of outer mitochondrial membrane 20 (TOMM20), mitofusin 2 (MFN2), and dynamin-related protein 1 (DRP1). Results Compared with the OGD + BMSCs group, the OGD + MNPs-BMSCs group showed increased neuronal viability ( P ＜ 0.000 1). MNP pretreatment increased the efficiency of mitochondrial transfer from BMSCs to neurons by 1.6-fold ( P ＜ 0.05), and this process depended on direct intercellular contact, as the transfer efficiency decreased after physical separation by Transwell ( P ＜ 0.05). Mechanistically, compared with the OGD group, neurons in the OGD + MNPs-BMSCs group showed decreased ROS and MDA levels and increased SOD activity (all P ＜ 0.05). Metabolic activity of the pentose phosphate pathway was enhanced, and NADPH and GSH levels were increased (both P ＜ 0.05). The expression levels of the mitochondrial dynamics-related proteins TOMM20 and MFN2 were upregulated, while DRP1 expression was downregulated (all P ＜ 0.05). Conclusions MNPs can promote functional mitochondrial transfer from BMSCs to neurons injured by ischemia and hypoxia, thereby enhancing endogenous antioxidant capacity in neurons, activating metabolic homeostasis of the pentose phosphate pathway, and improving mitochondrial fusion-fission imbalance and biogenesis impairment, ultimately exerting a synergistic neuroprotective effect.

关键词

缺血性卒中 / 黑色素纳米颗粒 / 骨髓间充质干细胞 / 线粒体转移 / 线粒体动力学 / 能量代谢

Key words

Ischemic stroke / Melanin nanoparticles / Bone marrow mesenchymal stem cells / Mitochondrial transfer / Mitochondrial dynamics / Energy metabolism

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基金资助

广西自然科学基金(2025GXNSFAA069341)

广西自然科学基金区域高发疾病研究联合专项(2023GXNSFAA026197)