[1]郝海珍,郭铁,余丹.脂肪间充质干细胞来源外泌体对脑缺血再灌注大鼠神经元凋亡及炎症因子影响[J].第三军医大学学报,2019,41(17):1656-1665.
 HAO Haizhen,GUO Tie,YU Dan.Effect of exosomes derived from adipose-derived mesenchymal stem cells on neuron apoptosis and inflammatory cytokines in a rat model of cerebral ischemia reperfusion[J].J Third Mil Med Univ,2019,41(17):1656-1665.
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脂肪间充质干细胞来源外泌体对脑缺血再灌注大鼠神经元凋亡及炎症因子影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第17期
页码:
1656-1665
栏目:
基础医学
出版日期:
2019-09-15

文章信息/Info

Title:
Effect of exosomes derived from adipose-derived mesenchymal stem cells on neuron apoptosis and inflammatory cytokines in a rat model of cerebral ischemia reperfusion
作者:
郝海珍郭铁余丹
570208 海口,中南大学湘雅医学院附属海口医院神经内科
Author(s):
HAO Haizhen GUO Tie YU Dan

Department of Neurology, the Affiliated Haikou Hospital of Xiangya Medical School, Central South University, Haikou, Hainan Province, 570208, China

关键词:
脂肪间充质干细胞外泌体神经元凋亡细胞色素C白细胞介素-1&beta脑缺血再灌注损伤
Keywords:
adipose-derived mesenchymal stem cells exosomes neuronal apoptosis cytochrome C interleukin-1&beta cerebral ischemia reperfusion injury
分类号:
R322.8; R364.4; R392.3
文献标志码:
A
摘要:

目的探讨脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADMSCs)来源外泌体对脑缺血再灌注大鼠神经元凋亡及炎症因子影响。方法取10只健康雄性8周龄SD大鼠附睾旁脂肪组织,提取ADMSCs进行细胞培养,取第3代ADMSCs分别在常氧(21%)和低氧(1%)条件培养,24 h后取培养皿上清液,试剂盒提取法分离外泌体。60只健康雄性8周龄SD大鼠按随机数字表法等分为5组:假手术组、缺血再灌注组、PBS组、低氧外泌体组、常氧外泌体组(n=12),后4组采用Longa改良线栓法建立脑缺血再灌注模型,其中缺血再灌注组仅进行造模,不进行干预,PBS组经尾静脉注射PBS 2 mL ,低氧外泌体组和常氧外泌体组分别同时给予等量低氧外泌体和常氧外泌体100 μg。在大鼠脑缺血再灌注损伤后24 h,采用Longa法测定各组大鼠神经功能缺损评分,TTC染色测量脑梗死体积,TUNEL法检测神经元凋亡情况,HE染色观察组织病理学变化,免疫组化检测细胞色素C(CytC)和白细胞介素-1β(IL-1β)的表达。结果与PBS组相比,低氧外泌体组和常氧外泌体组病理学改变减轻,神经功能缺损评分、脑梗死体积百分比、细胞色素C和IL-1β的表达及神经元凋亡指数均明显降低(P<0.05),低氧外泌体组较常氧外泌体组降低更为明显(P<0.05)。结论ADMSCs来源外泌体能减少脑缺血再灌注大鼠神经元凋亡和炎症因子IL-1β的表达,有神经保护作用,其机制可能是外泌体抑制了线粒体介导的细胞凋亡和IL-1β参与的炎症级联反应。

Abstract:

ObjectiveTo investigate the effect of exosomes derived from adipose-derived mesenchymal stem cells (ADMSCs) on neuron apoptosis and inflammatory cytokines in cerebral ischemia reperfusion (I/R) mice. MethodsADMSCs were extracted from adipose tissue adjacent to the epididymis in 10 8-week-old healthy male SD rats, and the cells of the third generation were cultured under normal oxygen (21%) and hypoxia (1%) conditions, respectively. After 24 h, the culture supernatant was collected and exosomes were separated by kit extraction method. Sixty 8-week-old healthy male SD rats were randomly divided into 5 groups, that is, sham-operation group, I/R group, PBS group, hypoxic exosomes group, and normoxic exosomes group (n=12). Rat model of cerebral I/R was established by insertion of a thread through the internal carotid artery in the latter 4 groups. The rats of PBS group were injected intraperitoneally with 2 mL PBS, while those of the hypoxic and normoxic exosomes groups were given 100 μg of hypoxic and normoxic exosomes, respectively. In 24 h after cerebral I/R injury, neurological function was evaluated by Longa score, infarct volumes were measured by TTC staining, neuronal apoptosis was detected by TUNEL assay, the expression of cytochrome C (CytC) and interleukin-1β (IL-1β) was detected by immunohistochemical staining. HE staining was employed to observe the morphological changes of the injury. ResultsCompared with the PBS group, the hypoxic and normoxic exosomes groups demonstrated significant decreases in pathological changes, neurological functional score, percentage of infarct volumes, expression levels of Cyt C and IL-1β, and neuronal apoptosis index (P<0.05). While, the decreases were more significant in the hypoxic than the normoxic exosomes group (P<0.05). ConclusionADMSCs-derived exosomes can reduce the apoptosis of neurons and the expression of IL-1β after cerebral I/R injury and show neuroprotective effects, which may be due to exosomes’ inhibition of mitochondrial-mediated apoptosis and IL-1β-involved inflammatory cascade reactions.

参考文献/References:

[1]ELTZSCHIG H K, ECKLE T. Ischemia and reperfusion: from mechanism to translation[J]. Nat Med, 2011, 17(11): 1391-1401. DOI:10.1038/nm.2507.
[2]丁新生, 冯美江. 缺血性卒中的病理生理学机制与细胞凋亡[J]. 国际脑血管病杂志, 2006, 14(1): 5-10. DOI:10.3760/cma.j.issn.1673-4165.2006.01.004.
DING X S, FENG M J. Pathophysiology and apoptosis in ischemic stroke[J]. Int J Cerebrovasc Dis, 2006, 14(1): 5-10. DOI:10.3760/cma.j.issn.1673-4165.2006.01.004.
[3]SADAT S, GEHMERT S, SONG Y H, et al. The cardioprotective effect of mesenchymal stem cells is mediated by IGF-Ⅰ and VEGF[J]. Biochem Biophys Res Commun, 2007, 363(3): 674-679. DOI:10.1016/j.bbrc.2007.09.058.
[4]CHUNG T N, KIM J H, CHOI B Y, et al. Adipose-derived mesenchymal stem cells reduce neuronal death after transient global cerebral ischemia through prevention of blood-brain barrier disruption and endothelial damage[J]. Stem Cells Transl Med, 2015, 4(2): 178-185. DOI:10.5966/sctm.2014-0103.
[5]LI D H, FANG Y, WANG P, et al. Autologous transplantation of adipose-derived mesenchymal stem cells attenuates cerebral ischemia and reperfusion injury through suppressing apoptosis and inducible nitric oxide synthase[J]. Int J Mol Med, 2012, 29(5): 848-854. DOI:10.3892/ijmm.2012.909.
[6]THRY C, ZITVOGEL L, AMIGORENA S. Exosomes: composition, biogenesis and function[J]. Nat Rev Immunol, 2002, 2(8): 569-579. DOI:10.1038/nri855.
[7]LUO Q C, GUO D F, LIU G R, et al. Exosomes from MiR-126-overexpressing adscs are therapeutic in relieving acute myocardial ischaemic injury[J]. Cell Physiol Biochem, 2017, 44(6): 2105-2116. DOI:10.1159/000485949.
[8]QU Y, ZHANG Q D, CAI X B, et al. Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation[J]. J Cell Mol Med, 2017, 21(10): 2491-2502. DOI:10.1111/jcmm.13170.
[9]BAI Y, HAN Y D, YAN X L, et al. Adipose mesenchymal stem cell-derived exosomes stimulated by hydrogen peroxide enhanced skin flap recovery in ischemia-reperfusion injury[J]. Biochem Biophys Res Commun, 2018, 500(2): 310-317. DOI:10.1016/j.bbrc.2018.04.065.
[10]LEE M, BAN J J, YANG S, et al. The exosome of adipose-derived stem cells reduces β-amyloid pathology and apoptosis of neuronal cells derived from the transgenic mouse model of Alzheimer’s disease[J]. Brain Res, 2018, 1691: 87-93. DOI:10.1016/j.brainres.2018.03.034.
[11]LEE M, BAN J J, KIM K Y, et al. Adipose-derived stem cell exosomes alleviate pathology of amyotrophic lateral sclerosis in vitro[J]. Biochem Biophys Res Commun, 2016, 479(3): 434-439. DOI:10.1016/j.bbrc.2016.09.069.
[12]HUANG X, DING J, LI Y F, et al. Exosomes derived from PEDF modified adipose-derived mesenchymal stem cells ameliorate cerebral ischemia-reperfusion injury by regulation of autophagy and apoptosis[J]. Exp Cell Res, 2018, 371(1): 269-277. DOI:10.1016/j.yexcr.2018.08.021.
[13]LONGA E Z, WEINSTEIN P R, CARLSON S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
[14]WEXLER E J, PETERS E E, GONZALES A, et al. An objective procedure for ischemic area evaluation of the stroke intraluminal thread model in the mouse and rat[J]. J Neurosci Methods, 2002, 113(1): 51-58.
[15]SKOG J, WRDINGER T, VAN RIJN S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers[J]. Nat Cell Biol, 2008, 10(12): 1470-1476. DOI:10.1038/ncb1800.
[16]CHEN K H, CHEN C H, WALLACE C G, et al. Intravenous administration of xenogenic adipose-derived mesenchymal stem cells(ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke[J]. Oncotarget, 2016, 7(46): 74537-74556. DOI:10.18632/oncotarget.12902.
[17]EL ANDALOUSSI S, MGER I, BREAKEFIELD X O, et al. Extracellular vesicles: biology and emerging therapeutic opportunities[J]. Nat Rev Drug Discov, 2013, 12(5): 347-357. DOI:10.1038/nrd3978.
[18]UENO Y, CHOPP M, ZHANG L, et al. Axonal outgrowth and dendritic plasticity in the cortical peri-infarct area after experimental stroke[J]. Stroke, 2012, 43(8): 2221-2228. DOI:10.1161/STROKEAHA.111.646224.
[19]YIN K J, HAMBLIN M, CHEN Y E. Angiogenesis-regulating microRNAs and ischemic stroke[J]. Curr Vasc Pharmacol, 2015, 13(3): 352-365.
[20]CLARKSON A N, OVERMAN J J, ZHONG S, et al. AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke[J]. J Neurosci, 2011, 31(10): 3766-3775. DOI:10.1523/JNEUROSCI.5780-10.2011.
[21]FRHBEIS C, FRHLICH D, KUO W P, et al. Extracellular vesicles as mediators of neuron-Glia communication[J]. Front Cell Neurosci, 2013, 7: 182. DOI:10.3389/fncel.2013.00182.
[22]ZHANG Y, YU M, TIAN W D. Physiological and pathological impact of exosomes of adipose tissue[J]. Cell Prolif, 2016, 49(1): 3-13. DOI:10.1111/cpr.12233.
[23]CHAN S Y, ZHANG Y Y, HEMANN C, et al. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2[J]. Cell Metab, 2009, 10(4): 273-284. DOI:10.1016/j.cmet.2009.08.015.
[24]BROUGHTON B R S, REUTENS D C, SOBEY C G. Apoptotic mechanisms after cerebral ischemia[J]. Stroke, 2009, 40(5): e331-e339. DOI:10.1161/strokeaha.108.531632.
[25]MARTINOU J C, YOULE R J. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics[J]. Dev Cell, 2011, 21(1): 92-101. DOI:10.1016/j.devcel.2011.06.017.
[26]SMITH D J, NG H, KLUCK R M, et al. The mitochondrial gateway to cell death[J]. IUBMB Life, 2008, 60(6): 383-389. DOI:10.1002/iub.44.
[27]HUANG X P, TAN H, CHEN B Y, et al. Astragalus extract alleviates nerve injury after cerebral ischemia by improving energy metabolism and inhibiting apoptosis[J]. Biol Pharm Bull, 2012, 35(4): 449-454.
[28]秦华平, 杨常春, 张一, 等. 深低温对全脑缺血大鼠海马线粒体细胞色素C释放及细胞凋亡影响[J]. 中华神经医学杂志, 2012, 11(2): 138-140. DOI:10.3760/cma.j.issn.1671-8925.2012.02.007.
QIN H P, YANG C C, ZHANG Y, et al. Effect of profound hypothermia on cytochrome C release and cell apoptosis in the Hippocampus after global ischemia in rats[J]. Chin J Neuromed, 2012, 11(2): 138-140. DOI:10.3760/cma.j.issn.1671-8925.2012.02.007.
[29]HOEHN B, YENARI M A, SAPOLSKY R M, et al. Glutathione peroxidase overexpression inhibits cytochrome C release and proapoptotic mediators to protect neurons from experimental stroke[J]. Stroke, 2003, 34(10): 2489-2494. DOI:10.1161/01.STR.0000091268.25816.19.
[30]CASO J R, MORO M A, LORENZO P, et al. Involvement of IL-1beta in acute stress-induced worsening of cerebral ischaemia in rats[J]. Eur Neuropsychopharmacol, 2007, 17(9): 600-607. DOI:10.1016/j.euroneuro.2007.02.009.
[31]CLARK W M, RINKER L G, LESSOV N S, et al. Lack of interleukin-6 expression is not protective against focal central nervous system ischemia[J]. Stroke, 2000, 31(7): 1715-1720.
[32]SHEN J L, XU S X, ZHOU H, et al. IL-1β induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells[J]. Sci Rep, 2017, 7: 41067. DOI:10.1038/srep41067.
[33]SRIVASTAVA K, ROM W, JAGIRDAR J, et al. Crucial role of interleukin-1 β and nitric oxide synthase in silica-induced inflammation and apoptosis in mice[J]. Am J Respir Crit Care Med, 2002, 165(4): 527-533. DOI:10.1164/ajrccm.165.4.2106009.
[34]HARA H, FRIEDLANDER R M, GAGLIARDINI V, et al. Inhibition of interleukin 1 converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage[J]. Proc Natl Acad Sci U S A, 1997, 94(5): 2007-2012. DOI:10.1073/pnas.94.5.2007.
[35]AL DERA H. Neuroprotective effect of resveratrol against late cerebral ischemia reperfusion induced oxidative stress damage involves upregulation of osteopontin and inhibition of interleukin-1beta[J]. J Physiol Pharmacol, 2017, 68(1): 47-56.

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更新日期/Last Update: 2019-09-09