Bone marrow mesenchymal stem cell exosomes alleviate oxygen-glucose deprivation/reperfusion injury in hippocampal neurons

doi:10.3969/j.issn.2095-4344.1749

Abstract

Abstract:

BACKGROUND: Increasing attentions have been focused on postoperative neurological complications after deep hypothermic circulatory arrest. The core mechanism of neurological complications is global ischemia-reperfusion injury.
OBJECTIVE: To investigate the protective effect and mechanism of bone mesenchymal stem cell exosomes on oxygen-glucose deprivation/reperfusion injury in hippocampal neurons.
METHODS: Primary cultured fetal rat hippocampal neurons (7 days) were divided into control group, oxygen-glucose deprivation/reperfusion group (OGD/R group) and oxygen-glucose deprivation/reperfusion with adding mesenchymal stem cell exosomes (exosomes group). After 2 hours of oxygen-glucose deprivation/reperfusion, the hippocampal neurons in the latter two groups were cultured under normal conditions. After modeling, hippocampal neurons in all the three groups were cultured in nerve cell culture medium, followed by addition of bone marrow mesenchymal stem cell exosomes in the exosomes group, and addition of PBS in the other two groups. After 24 hours of continuous culture, western blot assay was used; after 48 hours of culture, the survival rate and the growth of neurites in hippocampal neurons were counted and observed by immunofluorescence staining.
RESULTS AND CONCLUSION: Compared with the control group, the survival rate of hippocampal neurons in the OGD/R group was significantly reduced, the length of neurites and the number of branches were significantly impaired, and the expression level of pro-inflammatory factors was significantly increased. Compared with the OGD/R group, the survival rate of hippocampal neurons, the length of neuritis, and the number of branches in the exosomes group were significantly increased in the exosomes group, the expression levels of hepatocyte growth factor and brain-derived neurotrophic factor significantly increased, and the expression of pro-inflammatory factors obviously decreased. The results suggest that exosomes from bone marrow mesenchymal stem cells can alleviate oxygen-glucose deprivation/reperfusion injury in hippocampal neurons. The mechanism may be related to inhibiting secretion of pro-inflammatory factors secretion and promoting expression of hepatocyte growth factor and brain-derived neurotrophic factor.

Key words: bone marrow mesenchymal stem cells, exosomes, hippocampal neurons, oxygen-glucose deprivation/reperfusion injury, deep hypothermic circulatory arrest, hepatocyte growth factor, brain-derived neurotrophic factor, cyclooxygenase 2, inducible nitric oxide synthase, tumor necrosis factor alpha, pro-inflammatory factor, National Natural Science Foundation of China

CLC Number:

Shi Jiang, Gao Shilun, Liu Jinduo, Gu Tianxiang, Shi Enyi. Bone marrow mesenchymal stem cell exosomes alleviate oxygen-glucose deprivation/reperfusion injury in hippocampal neurons[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3316-3322.

Figures/Tables 2

2.1 骨髓间充质干细胞外泌体形态及特点倒置显微镜下可见SD大鼠骨髓间充质干细胞贴壁生长，长梭形，见图2A。通过超高速离心法从骨髓间充质干细胞条件培养基中提取外泌体。在透射电镜下观察，其外泌体为圆形或茶托样膜性囊泡，直径为30-100 nm，见图2B。Western blot显示骨髓间充质干细胞外泌体蛋白标志物Alix、TSG101和CD63表达阳性，见图2C。ZateView检测外泌体的粒径峰值为75 nm，粒子浓度为6.7×1012 L-1，见图2D。 2.2 骨髓间充质干细胞外泌体增加氧糖剥夺再灌注损伤后海马神经细胞的存活率，促进海马神经细胞突起再生免疫荧光染色用于观察海马神经细胞突起生长情况，与正常对照组海马神经细胞突起长度(275.27±19.87) μm和分支数(17.89±2.67)比较，氧糖剥夺再灌注损伤组明显降低海马神经细胞突起长度(65.14±18.14) μm和分支数(5.00±1.87) (P < 0.001)；与氧糖剥夺再灌注损伤组比较，骨髓间充质干细胞外泌体组增加突起长度(162.22±13.43) μm及分支数(15.22±1.39)(P < 0.001)，见图3。海马神经细胞存活率通过神经细胞计数评估，氧糖剥夺再灌注损伤组显著降低神经细胞存活率(46.07±3.78)%(相对于正常对照组，P < 0.001)，见图3。与氧糖剥夺再灌注损伤组比较，骨髓间充质干细胞外泌体组显著提高神经细胞生存率(74.14±4.20)%(P < 0.001)。 2.3 骨髓间充质干细胞外泌体增加氧糖剥夺再灌注损伤后海马神经细胞内的神经营养因子表达水平为了进一步探究骨髓间充质干细胞外泌体对海马神经细胞氧糖剥夺再灌注损伤后的保护作用机制，通过Western blot检测脑源性神经营养因子、肝细胞生长因子表达水平，见图4A。与正常对照组比较，氧糖剥夺再灌注损伤组轻微提升脑源性神经营养因子表达量及下调肝细胞生长因子的表达量，但差异无显著性意义(P > 0.05)。与氧糖剥夺再灌注损伤组比较，骨髓间充质干细胞外泌体组显著增加海马神经细胞内的脑源性神经营养因子和肝细胞生长因子表达水平(P < 0.01)，见图4B，C。 2.4 骨髓间充质干细胞外泌体抑制氧糖剥夺再灌注损伤后海马神经细胞的促炎因子表达炎症反应在海马神经细胞损伤中发挥着重要作用。实验通过Western blot检测促炎因子环氧化酶2、诱导型一氧化氮合酶和肿瘤坏死因子α表达水平，见图5A。与正常对照组比较，氧糖剥夺再灌注损伤组显著提高环氧化酶2、诱导型一氧化氮合酶和肿瘤坏死因子α的表达水平(P < 0.001)。与氧糖剥夺再灌注损伤组比较，骨髓间充质干细胞外泌体组显著抑制环氧化酶2、诱导型一氧化氮合酶和肿瘤坏死因子α的表达(P < 0.001)，见图5B-D。"

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