电针结合脑内注射VEGF修复大鼠脑缺血后再灌注损伤的机制研究
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摘要
目的观察电针及电针结合脑内注射血管内皮生长因子(VEGF)对脑缺血后再灌注损伤大鼠caspase12、caspasse3、葡萄糖调节蛋白-78(GRP78)的影响,探讨电针结合脑内VEGF对脑缺血后再灌注损伤的修复机制。方法将60只雄性SD大鼠随机分为假手术组、模型组、电针组、电针+VEGF组,每组15只。后3组采用线栓法进行大脑中动脉缺血(MCAO)法制作脑缺血后再灌注损伤模型。电针组及电针+VEGF组造模后1d开始电针干预(穴位:百会、曲池、足三里),1次/d,每次30min,共14d。电针+VEGF组大鼠于造模成功后24h行第一次电针干预后,向侧脑室内一次性注入10μL VEGF165(0.025μg/μL)。每组于0d(造模后1d,开始电针干预前)、7d、14d时,各取5只大鼠进行神经功能评分(mNSS)后处死取材,尼氏染色观察脑梗死区组织形态,免疫组化法检测大鼠缺血脑组织GRP78活性,real-time PCR法检测大鼠缺血脑组织caspase12、caspase3、GRP78mRNA表达量。结果 0d、7d、14d时,与假手术组比较,模型组大鼠mNSS评分升高(P<0.05),镜下可见脑梗死征象(尼氏小体数量明显减少且排列混乱,结构不清晰),GRP78免疫阳性细胞数量增多,GRP78mRNA表达升高(P<0.05),caspase12及caspase3mRNA表达升高(P<0.05)。7d和14d时,与模型组比较,电针组以及电针+VEGF组大鼠的mNSS评分降低(P<0.05),镜下脑梗死征象减轻,GRP78免疫阳性细胞数量增多,GRP78mRNA表达增多(P<0.05),caspase12、caspase3mRNA表达降低(P<0.05),电针+VEGF组上述指标变化均较电针组明显(P<0.05)。假手术组各时点间上述指标差异无统计学意义(P>0.05),其余3组mNSS评分(P<0.05)、脑梗死征象、caspase12、caspase3mRNA表达随治疗时间降低(P<0.05),GRP78免疫阳性细胞数量随治疗时间增多,GRP78mRNA表达随治疗时间升高(P<0.05)。结论电针结合脑内注射VEGF可促进脑缺血损伤组织修复,其机制可能与下调caspase12、caspase3基因,上调GRP78基因的表达有关,且其效果比单纯使用电针法更具优势。
Objective To determine the effects of electroacupuncture(EA)and intracerebral injection of vascular endothelial growth factor(VEGF)on caspase12,caspase3,and glucose regulated protein 78kD(GRP78)genes of rats with cerebral ischemia reperfusion injury.Methods 60 SD rats were randomly divided into shamoperation group,model group,EA group and EA+VEGF group with 15 rats in each group.Middle cerebral artery occlusion(MCAO)method was used to establish the model of cerebral ischemia reperfusion injury.Electroacupuncture intervention was introduced 1day after the injury in the EA group and EA+VEGF group:30minutes each session and once a day for a total of 14 d [acupoint selection:Baihui(GV 20),Quchi(Li 11),Zusanli(ST36)].The rats in the EA+VEGF group were also injected with 10μL of VEGF165(0.025μg/μL)into the lateral ventricle after the first session of EA.Five rats in each group were sacrificed after obtaining a neurological function score(mNSS)at day 0(1dafter modeling,before EA intervention),day 7and day 14,respectively.Nissl staining was used to observe the histomorphology of cerebral infarction areas.Immunohistochemistry was used to detect GRP78 activity in the ischemic brain tissues.Real-time fluorescence quantitative PCR(real-time PCR)was used to detect the expressions of caspase12,caspase3 and GRP78mRNA in the ischemic brain tissues.ResultsCompared with the sham-operation group,rats in the model group had higher mNSS scores(P<0.05),showed signs of cerebral infarction(with reduced numbers of and disordered Nissl bodies and unclear structure),increased GRP78 immunopositive cells,increased expression of GRP78 mRNA(P<0.05),and increased expressions of caspase12 and caspase3mRNA(P<0.05).Compared with the model group,EA and EA+VEGF decreased mNSS scores at day 7and 14(P<0.05),showing alleviated signs of cerebral infarction,increased GRP78 immunopositive cells(P<0.05),increased GRP78 mRNA expression(P<0.05),and decreased caspase12 and caspase3mRNA expressions(P<0.05).The most obvious changes were found in the EA+VEGF group(P<0.05).No significant changes were observed in the sham-operation group over time(P<0.05).In comparison,mNSS scores,the signs of cerebral infarction,and the expressions of caspase12 and caspase3decreased over time in the other groups(P<0.05),accompanied with increased GRP78 immunopositive cells and the expression of GRP78 gene(P<0.05).Conclusion Electroacupuncture and intracerebral injection of VEGF promote tissue repair of rats with cerebral ischemic injury,possibly through down-regulating the expressions of caspase12 and caspase3genes and upregulating the expression of GRP78 gene.The effect of electroacupuncture in combination with intracerebral injection of VEGF is superior to that of the single use of electroacupuncture.
Objective To determine the effects of electroacupuncture(EA)and intracerebral injection of vascular endothelial growth factor(VEGF)on caspase12,caspase3,and glucose regulated protein 78kD(GRP78)genes of rats with cerebral ischemia reperfusion injury.Methods 60 SD rats were randomly divided into shamoperation group,model group,EA group and EA+VEGF group with 15 rats in each group.Middle cerebral artery occlusion(MCAO)method was used to establish the model of cerebral ischemia reperfusion injury.Electroacupuncture intervention was introduced 1day after the injury in the EA group and EA+VEGF group:30minutes each session and once a day for a total of 14 d [acupoint selection:Baihui(GV 20),Quchi(Li 11),Zusanli(ST36)].The rats in the EA+VEGF group were also injected with 10μL of VEGF165(0.025μg/μL)into the lateral ventricle after the first session of EA.Five rats in each group were sacrificed after obtaining a neurological function score(mNSS)at day 0(1dafter modeling,before EA intervention),day 7and day 14,respectively.Nissl staining was used to observe the histomorphology of cerebral infarction areas.Immunohistochemistry was used to detect GRP78 activity in the ischemic brain tissues.Real-time fluorescence quantitative PCR(real-time PCR)was used to detect the expressions of caspase12,caspase3 and GRP78mRNA in the ischemic brain tissues.ResultsCompared with the sham-operation group,rats in the model group had higher mNSS scores(P<0.05),showed signs of cerebral infarction(with reduced numbers of and disordered Nissl bodies and unclear structure),increased GRP78 immunopositive cells,increased expression of GRP78 mRNA(P<0.05),and increased expressions of caspase12 and caspase3mRNA(P<0.05).Compared with the model group,EA and EA+VEGF decreased mNSS scores at day 7and 14(P<0.05),showing alleviated signs of cerebral infarction,increased GRP78 immunopositive cells(P<0.05),increased GRP78 mRNA expression(P<0.05),and decreased caspase12 and caspase3mRNA expressions(P<0.05).The most obvious changes were found in the EA+VEGF group(P<0.05).No significant changes were observed in the sham-operation group over time(P<0.05).In comparison,mNSS scores,the signs of cerebral infarction,and the expressions of caspase12 and caspase3decreased over time in the other groups(P<0.05),accompanied with increased GRP78 immunopositive cells and the expression of GRP78 gene(P<0.05).Conclusion Electroacupuncture and intracerebral injection of VEGF promote tissue repair of rats with cerebral ischemic injury,possibly through down-regulating the expressions of caspase12 and caspase3genes and upregulating the expression of GRP78 gene.The effect of electroacupuncture in combination with intracerebral injection of VEGF is superior to that of the single use of electroacupuncture.
引文
[1]孟文婷,李东翔,佟玲.缺血性脑卒中的治疗研究进展.中国新药杂志,2016(10):1114-1120.
[2]彭智远,刘旺华,曹雯.脑缺血再灌注损伤细胞凋亡机制的研究进展.中华中医药学刊,2017(8):1957-1961.
[3]吉康祥,李捷,邱彩霞,等.脑缺血后细胞凋亡机制的研究现状.中国卒中杂志,2010,5(1):66-72.
[4]ZHANG H,LI H,LIU X,et al.Effect of caspase-9inhibition on endoplasmic reticulum stress induced cortical neuronal injury in rats.Int J Clin Exp Med,2013,6(7):546-551.
[5]MARTI HJ,BENANDIN M,BELLAIL A,et al.Hypoxiainduced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia,Am JPathol,2000,156(3):965-976.
[6]KOVCA Z,IKEZAKI K,SAMOTO K,et al.VEGF and flt:expression time kinetics in rat brain infarct.Stroke,1996,27(10):1865-1873.
[7]KAZEMI M,CARRER A,MOIMAS S,et al.VEGF121and VEGF165differentially promote vessel maturation and tumor growth in mice and humans.Cancer Gene Therapy,2016,23(5):125-132.
[8]WANG P,MU YY,CHEN J,et al.Electroacupuncture on serum interleukin level in rat models of cerebral ischemiareperfusion injury.J Acupunct Tuina Sci,2015,13(1):9-14.
[9]李潇潇,卢圣锋,朱冰梅,等.兴奋性氨基酸毒性与缺血性脑中风及针刺的调整作用.针刺研究,2016,41(2):180-185.
[10]LONGA EZ,WEINSTEIN PR,CARlSON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke,1989,20(1):84-91.
[11]郭义,方剑乔主编.实验针灸学.北京:中国中医药出版社,2012:45-59.
[12]孟云,陈吉相,孙圣刚.脑室注射VEGF165对实验性脑出血大鼠血脑屏障的影响.卒中与神经疾病,2006,13(2):49-57.
[13]刘红玉,房伟.缺血性脑卒中发病机制的研究进展.中国现代医生,2010,48(25):11-12.
[14]陈志强.缺血性脑血管病的研究进展.亚太传统医药,2010,6(7):159-162.
[15]杨扬,韩正学.血管内皮细胞在组织缺血再灌注损伤中作用机制的研究进展.北京口腔医学,2017(5):294-296.
[16]黄萍,郭俐宏,陈少秀.电针联合穴位注射单唾液酸四己糖神经节苷脂钠对大鼠脑缺血/再灌注损伤的保护作用.中国中医急症,2016,25(7):1335-1338.
[17]杨杨,孙华.近10年针刺治疗脑缺血再灌注损伤的研究进展.中国针灸,2015,35(7):749-752.
[18]王林林,陈岚榕,许文威,等.古代针灸治疗中风后肢体偏瘫取穴规律探究.陕西中医药大学学报,2018,12(3):150-156.
[19]HONG JL,KIM KS,PARK IH,et al.Human neural stem cells over-expressing VEGF provide neuroprotection,angiogenesis and functional recovery in mouse stroke model.PLoS One,2007,2(1):e156[2018-03-17].http://doi.org/10.1371/jounal.pone.0000156.
[20]宋学萍,孙盛同,郭阳,等.不同注射VEGF途径对局灶性脑缺血再灌注大鼠侧支循环形成的影响.中华神经医学杂志,2013,12(7):661-664.
[21]吕哲,张颖,宋永周,等.内质网应激预处理对大鼠局灶性脑缺血再灌注后听皮层损伤的保护作用.实用医学杂志,2017,33(16):2646-2649.
[22]LI WH,YU J,LIN YP,et al.Effect of electroacupuncture at Neiguan(PC 6)and Baihui(GV 20)on CHOP and caspase-12gene expressions in rats after ischemia-reperfusion injury.J Acupunct Tuina Sci,2017,15(1):8-13.
[23]申龙健,殷香宇,孔迪,等.内质网应激与细胞死亡.神经损伤与功能重建,2015(3):230-232.
[2]彭智远,刘旺华,曹雯.脑缺血再灌注损伤细胞凋亡机制的研究进展.中华中医药学刊,2017(8):1957-1961.
[3]吉康祥,李捷,邱彩霞,等.脑缺血后细胞凋亡机制的研究现状.中国卒中杂志,2010,5(1):66-72.
[4]ZHANG H,LI H,LIU X,et al.Effect of caspase-9inhibition on endoplasmic reticulum stress induced cortical neuronal injury in rats.Int J Clin Exp Med,2013,6(7):546-551.
[5]MARTI HJ,BENANDIN M,BELLAIL A,et al.Hypoxiainduced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia,Am JPathol,2000,156(3):965-976.
[6]KOVCA Z,IKEZAKI K,SAMOTO K,et al.VEGF and flt:expression time kinetics in rat brain infarct.Stroke,1996,27(10):1865-1873.
[7]KAZEMI M,CARRER A,MOIMAS S,et al.VEGF121and VEGF165differentially promote vessel maturation and tumor growth in mice and humans.Cancer Gene Therapy,2016,23(5):125-132.
[8]WANG P,MU YY,CHEN J,et al.Electroacupuncture on serum interleukin level in rat models of cerebral ischemiareperfusion injury.J Acupunct Tuina Sci,2015,13(1):9-14.
[9]李潇潇,卢圣锋,朱冰梅,等.兴奋性氨基酸毒性与缺血性脑中风及针刺的调整作用.针刺研究,2016,41(2):180-185.
[10]LONGA EZ,WEINSTEIN PR,CARlSON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke,1989,20(1):84-91.
[11]郭义,方剑乔主编.实验针灸学.北京:中国中医药出版社,2012:45-59.
[12]孟云,陈吉相,孙圣刚.脑室注射VEGF165对实验性脑出血大鼠血脑屏障的影响.卒中与神经疾病,2006,13(2):49-57.
[13]刘红玉,房伟.缺血性脑卒中发病机制的研究进展.中国现代医生,2010,48(25):11-12.
[14]陈志强.缺血性脑血管病的研究进展.亚太传统医药,2010,6(7):159-162.
[15]杨扬,韩正学.血管内皮细胞在组织缺血再灌注损伤中作用机制的研究进展.北京口腔医学,2017(5):294-296.
[16]黄萍,郭俐宏,陈少秀.电针联合穴位注射单唾液酸四己糖神经节苷脂钠对大鼠脑缺血/再灌注损伤的保护作用.中国中医急症,2016,25(7):1335-1338.
[17]杨杨,孙华.近10年针刺治疗脑缺血再灌注损伤的研究进展.中国针灸,2015,35(7):749-752.
[18]王林林,陈岚榕,许文威,等.古代针灸治疗中风后肢体偏瘫取穴规律探究.陕西中医药大学学报,2018,12(3):150-156.
[19]HONG JL,KIM KS,PARK IH,et al.Human neural stem cells over-expressing VEGF provide neuroprotection,angiogenesis and functional recovery in mouse stroke model.PLoS One,2007,2(1):e156[2018-03-17].http://doi.org/10.1371/jounal.pone.0000156.
[20]宋学萍,孙盛同,郭阳,等.不同注射VEGF途径对局灶性脑缺血再灌注大鼠侧支循环形成的影响.中华神经医学杂志,2013,12(7):661-664.
[21]吕哲,张颖,宋永周,等.内质网应激预处理对大鼠局灶性脑缺血再灌注后听皮层损伤的保护作用.实用医学杂志,2017,33(16):2646-2649.
[22]LI WH,YU J,LIN YP,et al.Effect of electroacupuncture at Neiguan(PC 6)and Baihui(GV 20)on CHOP and caspase-12gene expressions in rats after ischemia-reperfusion injury.J Acupunct Tuina Sci,2017,15(1):8-13.
[23]申龙健,殷香宇,孔迪,等.内质网应激与细胞死亡.神经损伤与功能重建,2015(3):230-232.