ReN细胞来源纳米囊泡中的let-7i-5p在帕金森病模型中通过抗凋亡作用发挥保护效应

The let-7i-5p in ReN cell-derived nanovesicles exerts neuroprotective effects via anti-apoptotic mechanisms in Parkinson's disease models

摘要

目的: 探究人类神经干细胞系ReN细胞来源的纳米囊泡（nanovesicle，NV）对帕金森病（Parkinson's disease，PD）的神经保护作用及其潜在分子机制。方法: 使用1-甲基-4-苯基-1，2，3，6-四氢吡啶（1-methyl-4-phenyl-1，2，3，6-tetrahydro-pyridine，MPTP）20 mg/kg腹腔注射C57BL/6小鼠，建立PD小鼠模型，并通过尾静脉注射挤压ReN细胞获得的NV（ReN-NV）及经狂犬病病毒糖蛋白肽（rabies virus glycoprotein，RVG）表面修饰的ReN-NV（RVG-ReN-NV）进行干预。采用转棒实验和爬杆实验评估小鼠运动功能变化，并通过免疫荧光染色检测小鼠黑质区域的酪氨酸羟化酶（tyrosine hydroxylase，TH）、小胶质细胞标志物Iba1的表达，以及纹状体区域TH的表达；利用蛋白免疫印迹法检测纹状体区域的TH蛋白水平。使用1-甲基-4-苯基吡啶阳离子（1-methyl-4-phenylpyridinium ion，MPP⁺）1 mmol/L处理人神经母细胞瘤细胞SH-SY5Y，构建PD细胞模型，分别通过CCK-8法检测细胞活力，乳酸脱氢酶（lactate dehydrogenase，LDH）法评估细胞毒性，蛋白免疫印迹法检测Bcl-2和Bax的蛋白表达。结果: 行为学实验结果显示，MPTP处理后小鼠爬杆掉落潜伏期缩短及总下杆时间延长，提示帕金森样运动功能障碍形成。免疫荧光染色结果显示，ReN-NV能够改善MPTP诱导的小鼠黑质及纹状体TH阳性神经元减少，并降低黑质区域Iba1表达；其中RVG-ReN-NV干预组改善效果更为显著。蛋白免疫印迹结果表明，ReN-NV可上调纹状体TH蛋白表达水平。体外实验结果显示，ReN-NV能够提高MPP⁺诱导的SH-SY5Y细胞活力，降低LDH释放水平，并上调抗凋亡蛋白Bcl-2表达、下调促凋亡蛋白Bax表达。结论: ReN-NV对MPTP/MPP⁺诱导的PD模型具有显著神经保护作用，且经RVG修饰可进一步增强其脑靶向保护效果，其机制可能与let-7i-5p介导的抑制细胞凋亡途径有关。

Abstract

Objective: To investigate the neuroprotective effects of nanovesicles（NVs）derived from the human neural stem cell line ReN cell and to explore the underlying molecular mechanisms in Parkinson's disease（PD）. Methods: PD mouse models were established in C57BL/6 mice by intraperitoneal injection of 20 mg/kg 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine（MPTP）. Mice were treated via tail vein injection with nanovesicles obtained from extruded ReN cells（ReN-NV）or RVG peptide-modified ReN-NV（RVG-ReN-NV）. Motor function was evaluated by the rotarod test and the pole test，and immunofluorescence staining was performed to assess the expression of tyrosine hydroxylase（TH）and the microglial marker Iba1 in the substantia nigra，as well as TH expression in the striatum. Western blot analysis was used to determine TH protein levels in the striatum. In vitro，a PD cell model was established by treating human neuroblastoma SH-SY5Y cells with 1 mmol/L 1-methyl-4-phenylpyridinium ion（MPP⁺）. Cell viability was assessed using the CCK-8 assay，cytotoxicity was evaluated by lactate dehydrogenase（LDH）release assay，and the protein expression levels of Bcl-2 and Bax were measured by Western blotting. Results: The behavioral results showed that MPTP-treated mice displayed a significantly reduced latency to fall during the pole test，accompanied by an increased total descent time，suggesting the establishment of Parkinsonian-like motor impairment. Immunofluorescence analysis showed that ReN-NV alleviated the loss of TH-positive neurons in the substantia nigra and striatum induced by MPTP and reduced Iba1 expression in the substantia nigra，with RVG-ReN-NV exhibiting a more pronounced protective effect. Western blot analysis demonstrated that ReN-NV upregulated TH protein expression in the striatum. In vitro experiments showed that ReN-NV increased the viability of MPP⁺-treated SH-SY5Y cells，reduced LDH release，upregulated the expression of the anti-apoptotic protein Bcl-2，and downregulated the expression of the pro-apoptotic protein Bax. Conclusion: ReN cell-derived nanovesicles exert significant neuroprotective effects in MPTP/MPP⁺-induced PD models. RVG modification further enhances their brain-targeting protective efficacy，and the underlying mechanism may be associated with let-7i-5p-mediated inhibition of apoptotic pathways.

关键词

外泌体 / 帕金森病 / 纳米囊泡 / 狂犬病毒糖蛋白肽

Key words

exosomes / Parkinson's disease / nanovesicles / rabies virus glycoprotein peptide

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基金资助

国家自然科学基金(32371053)

国家自然科学基金(81973308)

江苏省高校自然科学基金(18KJB180017)