养脑方对永久性局灶性脑缺血大鼠恢复早期神经功能的影响
|
摘要
目的:观察养脑方对永久性局灶性脑缺血大鼠早期行为学的影响,探讨其促进神经功能恢复的作用机制。方法:建立永久性大脑中动脉栓塞模型,随机分为假手术组、模型组、尼莫地平组和养脑方组。术后3 d至14 d定时灌胃给药,术后3、7、10和14 d行Longa评分及横木行走试验评分,并观察脑组织形态结构,检测皮质缺血区HIF-1α和VEGF基因转录水平以及观察皮质缺血区HIF-1α、VEGF蛋白表达。结果:养脑方能改善大鼠左侧姿势反射及运动协调整合的能力(P<0.01);养脑方组和尼莫地平组皮质缺血区脑组织中HIF-1α、VEGF蛋白表达明显高于假手术组和模型组(P<0.01);养脑方组皮质缺血区脑组织VEGF蛋白表达高于尼莫地平组(P<0.01);养脑方组皮质缺血区脑组织HIF-1α、VEGF基因转录水平与尼莫地平组比较,差异无统计学意义(P>0.05)。结论:养脑方能改善pMCAO脑缺血大鼠恢复早期的神经功能,其机制可能与上调HIF-1α、VEGF的表达有关。
Objective: To observe the effect of Yangnao Prescription(养脑方) on behavior improvement for the rat model with permanent cerebral ischemia in early recovery stage, and to discuss the neural functional recovery mechanism. Methods: The rat models with permanent middle cerebral artery occlusion(pMCAO) were established. All experimental animals were divided into sham operation group, model group, Nimodipine group and Yangnao Prescription group. Then, each group was given gavage once a day after 3～14 days of operation. At the3 thday, 7 thday, 10 thday and 14 thday, neurological deficits and bar walking test were evaluated again in every group. The morphological structure in ischemic cerebral cortex were observed by hematoxylin eosin staining. The transcriptional level of HIF-1α and VEGF was detected in the right ischemic cerebral cortex by employing RTPCR. The expression of HIF-1α and VEGF were examined in the right ischemic cerebral cortex by employing the immunohistochemistry assay. Results: Yangnao prescription improved the ability of left postural reflex and motor coordination and integration in rats(P<0.01); the expression of HIF-1α and VEGF in cortical ischemic brain tissue of Yangnao prescription and Nimodipine group was significantly higher than that of sham operation group and model group(P<0.01); the expression of VEGF in cortical ischemic brain tissue of Yangnao prescription group was higher than that of Nimodipine group(P<0.01); Compared with Nimodipine group, the transcription levels of HIF-1α and vascular endothelial growth factor in cerebral ischemic area of Yangnao prescription had no significant difference(P>0.05). Conclusion: Yangnao prescription can extremely improve neurological function for the rat model with permanent focal cerebral ischemia in early recovery stage. The potential mechanism was related to activate HIF-1α and promote the expression of VEGF.
Objective: To observe the effect of Yangnao Prescription(养脑方) on behavior improvement for the rat model with permanent cerebral ischemia in early recovery stage, and to discuss the neural functional recovery mechanism. Methods: The rat models with permanent middle cerebral artery occlusion(pMCAO) were established. All experimental animals were divided into sham operation group, model group, Nimodipine group and Yangnao Prescription group. Then, each group was given gavage once a day after 3～14 days of operation. At the3 thday, 7 thday, 10 thday and 14 thday, neurological deficits and bar walking test were evaluated again in every group. The morphological structure in ischemic cerebral cortex were observed by hematoxylin eosin staining. The transcriptional level of HIF-1α and VEGF was detected in the right ischemic cerebral cortex by employing RTPCR. The expression of HIF-1α and VEGF were examined in the right ischemic cerebral cortex by employing the immunohistochemistry assay. Results: Yangnao prescription improved the ability of left postural reflex and motor coordination and integration in rats(P<0.01); the expression of HIF-1α and VEGF in cortical ischemic brain tissue of Yangnao prescription and Nimodipine group was significantly higher than that of sham operation group and model group(P<0.01); the expression of VEGF in cortical ischemic brain tissue of Yangnao prescription group was higher than that of Nimodipine group(P<0.01); Compared with Nimodipine group, the transcription levels of HIF-1α and vascular endothelial growth factor in cerebral ischemic area of Yangnao prescription had no significant difference(P>0.05). Conclusion: Yangnao prescription can extremely improve neurological function for the rat model with permanent focal cerebral ischemia in early recovery stage. The potential mechanism was related to activate HIF-1α and promote the expression of VEGF.
引文
[1]夏苏英,杨励,朱志明,等.养脑方治疗脑梗塞恢复期临床观察[J].中华中医药学刊,2014,32(7):1735-1739.
[2] Liu X,Wang Z,Wang P,et al.Green tea polyphenols alleviate early BBB damage during experimental focal cerebral ischemia through regulating tight junctions and PKCalpha signaling[J].BMC Complement Altern Med,2013,21(13):187.
[3] Nagasawa H, Kogure K.Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion[J].Stroke,1989,20(8):1037-1043.
[4]王旋,顾振纶,秦振红,等.银杏内酯A和B混合物对大鼠永久性局灶性脑缺血的保护作用[J].中草药,2007,38(2):241-244.
[5] Longa E Z,Weinstein P R,Carlson S,et al.Reversible middle cerebral artery occlusion[J].Stroke,1989,20(1):84-91.
[6]温仲民,包仕尧.大鼠急性局灶性脑缺血动物模型实验研究[J].中国临床神经科学,2004,12(4):409-411.
[7] Mani N, Khaibullina A, Krum J M, et al.Astrocyte growth effects of vascular endothelial growth factor(VEGF)app lication to perinatal neocortical explants:receptor mediation and signal transduction pathways[J].Exp Neurol,2005,192(2):394-406.
[8] Marti H J,Bernaudin M,Bellail A,et al.Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia[J].Am J Pathol,2000,156(3):965-976.
[9] Noiri E,Lee E,Testa J,et al.Podokinesis in endothelial cell migration:role of nitric oxide[J].Am J Physiol,1998,274(1):236-244.
[10] Storkebaum E, Lambrechts D, Canllelict P.VEGF:once regarded as a specific angiogenic factor,now implicated in neuroprotection[J].Bjoessays,2004,26(9):943-954.
[11] Kirby E D, Kuwahara A A, Messer R L,et al.Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF[J].Proc Natl Acad Sci U S A,2015,31(13):4128-4133.
[12] Lian Jin H,Pennant W A,Hyung Lee M,et al.Neural Stem Cells Modified by a Hypoxia-Inducible VEGF Gene Expression System Improve Cell Viability under Hypoxic Conditions and Spinal Cord Injury[J].Spine(Phila Pa 1976),2011,36(11):857-864.
[13] Jin K, Zhu Y, Sun Y, et al.Vascular endothelial growth factor(VEGF)stimulates neurogenesis in vitro and in vivo[J].Proc Natl Acad Sci USA,2002,99(18):11946-11950.
[14] Krum J M, Khaibullina A.Inhibition of endogenous VEGF impedes revascularization and astroglial proliferation:roles for VEGF in brain repair[J].Exp Neurol,2003,181(2):241-258.
[15] Jin K, Mao X O, Batteur S P, et al.Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor[J].Neuroscience,2001,108(2):351-358.
[16] Pettersson A, Nagy J A, Brown L F, et al.Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor[J].Lab In-vest,2000,80(1):99-115.
[17] Lee J W, Bae S H, Jeong J W, et al.Hypoxia-inducible factor(HIF-1)alpha:its protein stability and biological functions[J].Exp Mol Med,2004,36(1):1-12.
[18] Wiener C M,Booth G,Semenza G L.In vivo expression of m RNAs encoding hypoxia-inducible factor 1[J].Biochem Biophys Res Commun,1996,225(2):485-488.
[19] Ruiz de Almodovar,Coulon,Salin,Chounlamountri,et al.Matrix-binding vascular endothelial growth factor(VEGF)isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1[J].Neurosci,2010,30(45):15052-15066.
[20] Lee J W, Bae S H, Jeong J W, et al.Hypoxia-inducible factor alpha(HIF-1):its protein stability and biological functions[J].Exp Mol Med,2004,36(1):1-12.
[21] Wiener C M,Booth G,Semenza G L.In vivo expression of m RNAs encoding hypoxia2-inducible factor 1[J].Biochem Biophys Res Commun,1996,225(2):485-488.
[22]王永刚,李彩红,于远望,等.双参通冠方对心肌梗死大鼠HIF-1α和VEGF表达的影响[J].中医药导报,2017,23(8):25-27,34.
[23]杨涛,胡亚辉,李天喜,等.补肾祛痰活血汤对脑缺血再灌注损伤大鼠脑组织BBB及VEGF的影响[J].中医药导报,2016,22(15):28-31.
[24]张琳,徐曦,马勇.脑出血小鼠行为学改变和低氧诱导因子-1a表达[J].陕西医学杂志,2011,11(40):1466-1468.
[25]韩岚,梁杰,张艳艳,等.桃红四物汤对产后血瘀大鼠血清NO、子宫组织VEGF的表达及对PI3K/Akt信号通路的影响[J].中华中医药杂志,2016,31(5):1625-1629.
[26]张涓娟,蒲宇,李勇,等.PI3K/AKT和MAPK/ERK1/2信号通路对胰腺癌PANC-1细胞VEGF表达的影响[J].川北医学院学报,2014,29(1):44-48.
[27]李燕,朱君,张煜.PI3K/AKT信号转导通路和VEGF在子宫内膜异位症中的表达[J].中国性科学,2017,26(4):45-47.
[28]余雪梅,吴啸天,李文明,等.SHH信号通路对肝癌细胞Hep3B中血管内皮生长因子表达的影响[J].检验医学与临床,2017,14(3):336-338.
[29]国伟.SHH信号通路在新生血管形成中的作用[D].青岛:青岛大学,2017.
[2] Liu X,Wang Z,Wang P,et al.Green tea polyphenols alleviate early BBB damage during experimental focal cerebral ischemia through regulating tight junctions and PKCalpha signaling[J].BMC Complement Altern Med,2013,21(13):187.
[3] Nagasawa H, Kogure K.Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion[J].Stroke,1989,20(8):1037-1043.
[4]王旋,顾振纶,秦振红,等.银杏内酯A和B混合物对大鼠永久性局灶性脑缺血的保护作用[J].中草药,2007,38(2):241-244.
[5] Longa E Z,Weinstein P R,Carlson S,et al.Reversible middle cerebral artery occlusion[J].Stroke,1989,20(1):84-91.
[6]温仲民,包仕尧.大鼠急性局灶性脑缺血动物模型实验研究[J].中国临床神经科学,2004,12(4):409-411.
[7] Mani N, Khaibullina A, Krum J M, et al.Astrocyte growth effects of vascular endothelial growth factor(VEGF)app lication to perinatal neocortical explants:receptor mediation and signal transduction pathways[J].Exp Neurol,2005,192(2):394-406.
[8] Marti H J,Bernaudin M,Bellail A,et al.Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia[J].Am J Pathol,2000,156(3):965-976.
[9] Noiri E,Lee E,Testa J,et al.Podokinesis in endothelial cell migration:role of nitric oxide[J].Am J Physiol,1998,274(1):236-244.
[10] Storkebaum E, Lambrechts D, Canllelict P.VEGF:once regarded as a specific angiogenic factor,now implicated in neuroprotection[J].Bjoessays,2004,26(9):943-954.
[11] Kirby E D, Kuwahara A A, Messer R L,et al.Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF[J].Proc Natl Acad Sci U S A,2015,31(13):4128-4133.
[12] Lian Jin H,Pennant W A,Hyung Lee M,et al.Neural Stem Cells Modified by a Hypoxia-Inducible VEGF Gene Expression System Improve Cell Viability under Hypoxic Conditions and Spinal Cord Injury[J].Spine(Phila Pa 1976),2011,36(11):857-864.
[13] Jin K, Zhu Y, Sun Y, et al.Vascular endothelial growth factor(VEGF)stimulates neurogenesis in vitro and in vivo[J].Proc Natl Acad Sci USA,2002,99(18):11946-11950.
[14] Krum J M, Khaibullina A.Inhibition of endogenous VEGF impedes revascularization and astroglial proliferation:roles for VEGF in brain repair[J].Exp Neurol,2003,181(2):241-258.
[15] Jin K, Mao X O, Batteur S P, et al.Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor[J].Neuroscience,2001,108(2):351-358.
[16] Pettersson A, Nagy J A, Brown L F, et al.Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor[J].Lab In-vest,2000,80(1):99-115.
[17] Lee J W, Bae S H, Jeong J W, et al.Hypoxia-inducible factor(HIF-1)alpha:its protein stability and biological functions[J].Exp Mol Med,2004,36(1):1-12.
[18] Wiener C M,Booth G,Semenza G L.In vivo expression of m RNAs encoding hypoxia-inducible factor 1[J].Biochem Biophys Res Commun,1996,225(2):485-488.
[19] Ruiz de Almodovar,Coulon,Salin,Chounlamountri,et al.Matrix-binding vascular endothelial growth factor(VEGF)isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1[J].Neurosci,2010,30(45):15052-15066.
[20] Lee J W, Bae S H, Jeong J W, et al.Hypoxia-inducible factor alpha(HIF-1):its protein stability and biological functions[J].Exp Mol Med,2004,36(1):1-12.
[21] Wiener C M,Booth G,Semenza G L.In vivo expression of m RNAs encoding hypoxia2-inducible factor 1[J].Biochem Biophys Res Commun,1996,225(2):485-488.
[22]王永刚,李彩红,于远望,等.双参通冠方对心肌梗死大鼠HIF-1α和VEGF表达的影响[J].中医药导报,2017,23(8):25-27,34.
[23]杨涛,胡亚辉,李天喜,等.补肾祛痰活血汤对脑缺血再灌注损伤大鼠脑组织BBB及VEGF的影响[J].中医药导报,2016,22(15):28-31.
[24]张琳,徐曦,马勇.脑出血小鼠行为学改变和低氧诱导因子-1a表达[J].陕西医学杂志,2011,11(40):1466-1468.
[25]韩岚,梁杰,张艳艳,等.桃红四物汤对产后血瘀大鼠血清NO、子宫组织VEGF的表达及对PI3K/Akt信号通路的影响[J].中华中医药杂志,2016,31(5):1625-1629.
[26]张涓娟,蒲宇,李勇,等.PI3K/AKT和MAPK/ERK1/2信号通路对胰腺癌PANC-1细胞VEGF表达的影响[J].川北医学院学报,2014,29(1):44-48.
[27]李燕,朱君,张煜.PI3K/AKT信号转导通路和VEGF在子宫内膜异位症中的表达[J].中国性科学,2017,26(4):45-47.
[28]余雪梅,吴啸天,李文明,等.SHH信号通路对肝癌细胞Hep3B中血管内皮生长因子表达的影响[J].检验医学与临床,2017,14(3):336-338.
[29]国伟.SHH信号通路在新生血管形成中的作用[D].青岛:青岛大学,2017.