葛根素对局灶性脑缺血大鼠半暗带微血管内皮细胞的保护作用及其机制
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摘要
目的:研究葛根素对局灶性脑缺血大鼠半暗带微血管内皮细胞的保护作用及其机制。方法:线栓法制作永久局灶性脑缺血大鼠模型,用神经行为学缺损评分法、组织形态学方法、免疫学技术方法,评价葛根素对模型大鼠神经功能改善作用、对半暗带脑组织保护作用和抗半暗带微血管内皮细胞凋亡机制。结果:在模型大鼠缺血半暗带区域,葛根素100,50和25 mg·kg-1剂量均能显著促进微血管Bcl-2蛋白表达,抑制Bax,Caspase-3和Cleaved caspase-3蛋白表达;显著降低微血管内皮细胞凋亡率;减轻半暗带脑组织病理损害程度。同时减少脑梗死面积百分比,改善大鼠的神经功能。结论:葛根素可抗半暗带微血管内皮细胞凋亡,减轻局灶性脑缺血对大鼠的神经损害。
Objective: To study the protective effect and mechanism of puerarin on microvascular endothelial cells( MECs) in ischemic penumbra of focal cerebral ischemia rats. Methods: The permanent focal cerebral ischemia rat model was established by suture embolization. With modified neurological severity scores,histomorphometry and immunological techniques,the improvement of neurological function in rats and the protection on ischemic penumbra of puerarin were evaluated,and the anti-apoptotic effects on MECs in ischemic penumbra was evaluated too.Results: Within the ischemic penumbra region of model rats,puerarin of all the doses( 100,50 and 25 mg·kg-1)significantly enhanced the Bcl-2 protein expression,decreased the Bax,Caspase-3 and Cleaved caspase-3 protein expression,and reduced the apoptosis rate of MECs,the degree of pathological damage and the percentage of infarct volume. Puerarin notably ameliorated the neurological function of focal cerebral ischemia rats. Conclusion:Puerarin can reduce the neurological damage induced by focal cerebral ischemia in rats by preventing microvascular endothelial cell apoptosis in the ischemic penumbra.
Objective: To study the protective effect and mechanism of puerarin on microvascular endothelial cells( MECs) in ischemic penumbra of focal cerebral ischemia rats. Methods: The permanent focal cerebral ischemia rat model was established by suture embolization. With modified neurological severity scores,histomorphometry and immunological techniques,the improvement of neurological function in rats and the protection on ischemic penumbra of puerarin were evaluated,and the anti-apoptotic effects on MECs in ischemic penumbra was evaluated too.Results: Within the ischemic penumbra region of model rats,puerarin of all the doses( 100,50 and 25 mg·kg-1)significantly enhanced the Bcl-2 protein expression,decreased the Bax,Caspase-3 and Cleaved caspase-3 protein expression,and reduced the apoptosis rate of MECs,the degree of pathological damage and the percentage of infarct volume. Puerarin notably ameliorated the neurological function of focal cerebral ischemia rats. Conclusion:Puerarin can reduce the neurological damage induced by focal cerebral ischemia in rats by preventing microvascular endothelial cell apoptosis in the ischemic penumbra.
引文
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[17] XU X,ZHENG X,ZHOU Q,et al. Inhibition of excitatory amino acid efflux contributes to protective effects of puerarin against cerebral ischemia in rats[J]. Biomed Environ Sci,2007,20(4):336-342.
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[21] LIU X,MEI Z,QIAN J,et al. Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway[J]. NRR,2013,8(34):3203-3215.
[22] ZHANG H,LIU Y,LAO M,et al. Puerarin protects Alzheimer's disease neuronal cybrids from oxidant-stress induced apoptosis by inhibiting pro-death signaling pathways[J]. Exp Gerontol,2011,46(1):30-37.
[23] GAO L,JI X,SONG J,et al. Puerarin protects against ischemic brain injury in a rat model of transient focal ischemia[J]. Neurol Res,2009,31(4):402-406.
[24] WANG N,ZHANG Y,WU L,et al. Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J]. Neuropharmacol,2014,79:282-289.
[25] PAN HP,LI G. Protecting mechanism of puerarin on the brain neurocyte of rat in acute local ischemia brain injury and local cerebral ischemia-reperfusion injury[J]. Yakugaku Zasshi,2008,128(11):1689-1698.
[26] YANG Y,SHUAIB A,LI Q. Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method[J]. J Neurosci Meth,1998,84(84):9-16.
[27] YU Q,TAO H,WANG X,et al. Targeting brain microvascular endothelial cells:a therapeutic approach to neuroprotection against stroke[J]. NRR,2015,10(11):1882-1891.
[28] BLEAU C,FILLIOL A,SAMSON M,et al. Brain invasion by mouse hepatitis virus depends on impairment of tight junctions and beta interferon production in brain microvascular endothelial cells[J]. J Virol,2015,89(19):9896-9908.
[29] WANG X,YU X,XIE C,et al. Rescue of brain function using tunneling nanotubes between neural stem cells and brain microvascular endothelial cells[J]. Mol Neurobiol,2015,53(4):2480-2488.
[30] SHIMIZU F,SANO Y,SAITO K,et al. Pericyte-derived glial cell line-derived neurotrophic factor increase the expression of claudin-5 in the blood-brain barrier and the blood-nerve barrier[J]. Neurochem Res,2012,37(2):401-409.
[31] REUTER B,RODEMER C,GRUDZENSKI S,et al. Temporal profile of matrix metalloproteinases and their inhibitors in a human endothelial cell culture model of cerebral ischemia[J]. Cerebrovasc Dis,2013,35(6):514-520.
[32] O'DONNELL MJ,XAVIER D,LIU L,et al. Risk factors for ischaemic and intracerebral haemorrhagic stroke in 22 countries(the INTERSTROKE study):a case-control study[J]. Lancet,2010,376(9735):112-123.
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[34] ZHANG Y,ZHANG X,PARK TS,et al. Cerebral endothelial cell apoptosis after ischemia|[ndash]|reperfusion:role of PARP activation and AIF translocation[J]. J Cerebr Blood F Met,2005,25(7):868-877.
[35] KRUEGER M,BECHMANN I,IMMIG K,et al. Blood-brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia[J]. J Cerebr Blood F Met,2015,35(2):292-303.
[36] LIU Y,PAN Q,ZHAO Y,et al. MicroRNA-155 regulates ROS production,NO generation,apoptosis and multiple functions of human brain microvessel endothelial cells under physiological and pathological conditions[J]. J Cell Biochem,2015,116(12):2870-2881.
[37] WU KW,KOU ZW,MO JL,et al. Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures[J]. Neuroscience,2016,334:275-282.
[38] LAPI D,COLANTUONI A. Remodeling of cerebral microcirculation after ischemia-reperfusion[J]. J Vasc Res,2015,52(1):22-31.
[39] ROCHFORT KD,COLLINS LE,MCLOUGHLIN A,et al. Sheardependent attenuation of cellular ROS levels can suppress proinflammatory cytokine injury to human brain microvascular endothelial barrier properties[J]. J Cereb Blood Flow Metab,2015,35(10):1648-1656.
[40] RADAK D,KATSIKI N,RESANOVIC I,et al. Apoptosis and acute brain ischemia in ischemic stroke[J]. Curr Vasc Pharmacol,2016,15(2):115-122.
[41] ZHANG P,YANG L,HE H,et al. Differential variations of autophagy and apoptosis in permanent focal cerebral ischaemia rat model[J]. Brain Injury,2017,31(1):1151-1158.
[42] TIAN S,WU JF,LI C,et al. Apoptotic cell characteristics of rat brain microvascular endothelia induced by different degrees of hypoperfusion[J]. Int J Clin Exp Med,2017,10(11):11360-11368.
[43]王琴,徐晓玉.葛根素注射剂致溶血的研究进展[J].中国中药杂志,2011,36(10):1402-1405.
[44]孙露,丁选胜,张东蕾.丙二醇对葛根素溶血率影响的研究[J].南京中医药大学学报,2012,28(6):570-573.
[45]原欢欢,张继芬,王琴,等.梓葛冻干粉针的局部刺激性、过敏性、溶血性和急性毒性研究[J].中国药理学通报,2013,29(6):880-881.
[2] FURIE KL,KASNER SE,ADAMS RJ,et al. Guidelines for the prevention of stroke in patients with stroke or transient ischemic attack:a guideline for healthcare professionals from the american heart association/American stroke association[J]. Stroke,2012,42(1):227-276.
[3]刘庆焕,王宏,张岩,等.复方红景天提取物对大、小鼠脑缺血模型的保护作用[J].中国现代应用药学,2018,35(4):524-528.
[4] MOZAFFARIAN D,BENJAMIN EJ,GO AS,et al. Heart disease and stroke statistics-2016 update:a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee[J]. Circulation,2016,133(4):e38-e360.
[5] RABINSTEIN AA. Treatment of acute ischemic stroke[J]. Continuum,2017,23(1):62-81.
[6] PAN Q,HE C,LIU H,et al. Microvascular endothelial cellsderived microvesicles imply in ischemic stroke by modulating astrocyte and blood brain barrier function and cerebral blood flow[J]. Molecular Brain,2016,9(1):63-76.
[7]俞燕,刘强,杨吕洪.清心饮对柯萨奇B3病毒感染心脏微血管内皮细胞的保护作用[J].中国现代应用药学,2017,34(1):20-24.
[8] AKANUMA S,OHTSUKI S,DOI Y,et al. ATP-binding cassette transporter A1(ABCA1)deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-beta peptide(1-40)at the blood-brain barrier[J]. Neurochem Int,2008,52(6):956-961.
[9] MA SC,LI Q,PENG JY,et al. Claudin-5 regulates blood-brain barrier permeability by modifying brain microvascular endothelial cell proliferation,migration,and adhesion to prevent lung cancer metastasis[J]. Cns Neurosci Ther,2017,23(12):947-960.
[10] LI H,GAO A,FENG D,et al. Evaluation of the protective potential of brain microvascular endothelial cell autophagy on bloodbrain barrier integrity during experimental cerebral ischemiareperfusion injury[J]. Transl Stroke Res,2014,5(5):618-626.
[11] DECLVES X,STRAZIELLE N,SCHERRMANN JM,et al.Drug metabolism at the blood-brain and blood-CSF barriers[M].Springer New York,Drug Delivery to the Brain,2014:101-124.
[12] NAKAMURA K,AGO T. Roles of brain microvascular pericyte in physiological and ischemic conditions[J]. J Cerebr Blood F Met,2014,25(2):109-115.
[13] SHIMIZU F,KANDA T. The blood-brain barrier in neuroinflammation[M]. Springer New York,Neuron-Glia Interaction in Neuroinflammation,2013:157-179.
[14] DANEMAN R. The blood-brain barrier in health and disease[J]. Ann Neurol,2012,72(5):648-672.
[15] ZLOKOVIC BV. The blood-brain barrier in health and chronic neurodegenerative disorders[J]. Neuron,2008,57(2):178-201.
[16] JIANG M,YUN Q,NIU G,et al. Puerarin prevents inflammation and apoptosis in the neurocytes of a murine Parkinson's disease model[J]. Genet Mol Res,2016,15(4). doi:10. 4238/gmr. 15047501.
[17] XU X,ZHENG X,ZHOU Q,et al. Inhibition of excitatory amino acid efflux contributes to protective effects of puerarin against cerebral ischemia in rats[J]. Biomed Environ Sci,2007,20(4):336-342.
[18]黄雄峰,汪建民.葛根素的神经保护作用机制研究进展[J].中国实验方剂学杂志,2015,21(4):224-230.
[19] LIU B,TAN Y,WANG D,et al. Puerarin for ischaemic stroke[J]. Cochrane DB Syst Rev,2016,2(2):CD004955.
[20] MAHDY HM,MOHAMED MR,EMAM MA,et al. The anti-apoptotic and anti-inflammatory properties of puerarin attenuate 3-nitropropionic-acid induced neurotoxicity in rats[J]. Can J Physiol Pharmacol,2014,92(3):252-258.
[21] LIU X,MEI Z,QIAN J,et al. Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway[J]. NRR,2013,8(34):3203-3215.
[22] ZHANG H,LIU Y,LAO M,et al. Puerarin protects Alzheimer's disease neuronal cybrids from oxidant-stress induced apoptosis by inhibiting pro-death signaling pathways[J]. Exp Gerontol,2011,46(1):30-37.
[23] GAO L,JI X,SONG J,et al. Puerarin protects against ischemic brain injury in a rat model of transient focal ischemia[J]. Neurol Res,2009,31(4):402-406.
[24] WANG N,ZHANG Y,WU L,et al. Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J]. Neuropharmacol,2014,79:282-289.
[25] PAN HP,LI G. Protecting mechanism of puerarin on the brain neurocyte of rat in acute local ischemia brain injury and local cerebral ischemia-reperfusion injury[J]. Yakugaku Zasshi,2008,128(11):1689-1698.
[26] YANG Y,SHUAIB A,LI Q. Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method[J]. J Neurosci Meth,1998,84(84):9-16.
[27] YU Q,TAO H,WANG X,et al. Targeting brain microvascular endothelial cells:a therapeutic approach to neuroprotection against stroke[J]. NRR,2015,10(11):1882-1891.
[28] BLEAU C,FILLIOL A,SAMSON M,et al. Brain invasion by mouse hepatitis virus depends on impairment of tight junctions and beta interferon production in brain microvascular endothelial cells[J]. J Virol,2015,89(19):9896-9908.
[29] WANG X,YU X,XIE C,et al. Rescue of brain function using tunneling nanotubes between neural stem cells and brain microvascular endothelial cells[J]. Mol Neurobiol,2015,53(4):2480-2488.
[30] SHIMIZU F,SANO Y,SAITO K,et al. Pericyte-derived glial cell line-derived neurotrophic factor increase the expression of claudin-5 in the blood-brain barrier and the blood-nerve barrier[J]. Neurochem Res,2012,37(2):401-409.
[31] REUTER B,RODEMER C,GRUDZENSKI S,et al. Temporal profile of matrix metalloproteinases and their inhibitors in a human endothelial cell culture model of cerebral ischemia[J]. Cerebrovasc Dis,2013,35(6):514-520.
[32] O'DONNELL MJ,XAVIER D,LIU L,et al. Risk factors for ischaemic and intracerebral haemorrhagic stroke in 22 countries(the INTERSTROKE study):a case-control study[J]. Lancet,2010,376(9735):112-123.
[33] GARBUZOVA-DAVIS S,RODRIGUES MC,HERNANDEZONTIVEROS DG,et al. Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model[J]. Plos One,2013,8(5):e63553.
[34] ZHANG Y,ZHANG X,PARK TS,et al. Cerebral endothelial cell apoptosis after ischemia|[ndash]|reperfusion:role of PARP activation and AIF translocation[J]. J Cerebr Blood F Met,2005,25(7):868-877.
[35] KRUEGER M,BECHMANN I,IMMIG K,et al. Blood-brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia[J]. J Cerebr Blood F Met,2015,35(2):292-303.
[36] LIU Y,PAN Q,ZHAO Y,et al. MicroRNA-155 regulates ROS production,NO generation,apoptosis and multiple functions of human brain microvessel endothelial cells under physiological and pathological conditions[J]. J Cell Biochem,2015,116(12):2870-2881.
[37] WU KW,KOU ZW,MO JL,et al. Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures[J]. Neuroscience,2016,334:275-282.
[38] LAPI D,COLANTUONI A. Remodeling of cerebral microcirculation after ischemia-reperfusion[J]. J Vasc Res,2015,52(1):22-31.
[39] ROCHFORT KD,COLLINS LE,MCLOUGHLIN A,et al. Sheardependent attenuation of cellular ROS levels can suppress proinflammatory cytokine injury to human brain microvascular endothelial barrier properties[J]. J Cereb Blood Flow Metab,2015,35(10):1648-1656.
[40] RADAK D,KATSIKI N,RESANOVIC I,et al. Apoptosis and acute brain ischemia in ischemic stroke[J]. Curr Vasc Pharmacol,2016,15(2):115-122.
[41] ZHANG P,YANG L,HE H,et al. Differential variations of autophagy and apoptosis in permanent focal cerebral ischaemia rat model[J]. Brain Injury,2017,31(1):1151-1158.
[42] TIAN S,WU JF,LI C,et al. Apoptotic cell characteristics of rat brain microvascular endothelia induced by different degrees of hypoperfusion[J]. Int J Clin Exp Med,2017,10(11):11360-11368.
[43]王琴,徐晓玉.葛根素注射剂致溶血的研究进展[J].中国中药杂志,2011,36(10):1402-1405.
[44]孙露,丁选胜,张东蕾.丙二醇对葛根素溶血率影响的研究[J].南京中医药大学学报,2012,28(6):570-573.
[45]原欢欢,张继芬,王琴,等.梓葛冻干粉针的局部刺激性、过敏性、溶血性和急性毒性研究[J].中国药理学通报,2013,29(6):880-881.