补阳还五汤调节脑缺血星形胶质细胞GLT1和GS的机制研究
发布时间：2018-09-13 11:27
【摘要】：目的： 脑血管疾病(cerebrovascular disease)是一类严重影响人类身体健康的疾病,在全球,脑血管疾病是致死和致残的主要疾病之一,尤其是临床上较常见的缺血性脑血管病(ischemic cerebrovascular disease, ICVD)。ICVD的主要发病机制主要包括能量耗竭、酸中毒,氧自由基产生、损害,一氧化氮和一氧化氮合成酶,钙超载,炎症因子,细胞凋亡调控因子,兴奋性氨基酸(excitatory amino acid, EAA)。其中EAA是众多发病机制中最为关键的一个,它能引起其他机制的产生。谷氨酸(glutamate, Glu)是中枢神经系统中含量最高的兴奋性氨基酸,而胞外过多的Glu是会加重脑缺血损伤。据报告,补阳还五汤(Buyang Huanwu Decoction, BYHWD)能减少神经细胞外的谷氨酸浓度,而Glu的清除主要依赖于星形胶质细胞上谷氨酸转运体-1(glutamate transporter-1, GLT-1或GLT1)和谷氨酰胺合成酶(glutamine synthetase, GS)。所以,本实验主要运用免疫组织荧光化学和蛋白印迹western-blot等技术,探索补阳还五汤能否增加脑缺血后星形胶质细胞GLT1和GS蛋白的表达,从而减少细胞外谷氨酸的浓度；除此,本实验还对其作用机制进行初步探索,希望能为补阳还五汤治疗脑缺血提供更多的实验依据。 方法： 1.补阳还五汤对局灶性脑缺血星形胶质细胞GLT1和GS表达的影响 健康成年的SD大鼠,采用大脑中动脉栓塞(middle cerebral artery occlusion, MCAO)法制备模型,手术结束大鼠清醒后,进行神经功能评分,评分合格的大鼠随机分为模型组(1d、3d、5d、7d)和药物组(1d、3d、5d、7d),各组大鼠再缺血2h再灌24h后给药,其中模型组每次给予2ml的生理盐水灌胃,2次/d,药物组给予每次补阳还五汤(16g/kg)灌胃,2次/d。各组实验结束后,将动物进行全身灌注,取出大脑制备冰冻切片,采用免疫组织化学染色法和免疫组织荧光化学染色法分别检测各组GLT1和GS的表达,并用免疫荧光双标法对GLT1和胶质细胞原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)、GS和GFAP的表达进行检测,显微镜下观察并获取图片,用ImageJ进行图片分析。 2.补阳还五汤影响局灶性脑缺血星形胶质细胞GLT1和GS表达的机制研究 健康成年的SD大鼠,随机分为假手术组、模型组、补阳还五汤组(BYHWD)、补阳还五汤联合垂体腺苷酸环化酶激活多肽拮抗剂(pituitary adenylate cyclase activating polypeptide (6-38), PACAP(6-38))组(BYHWD+PACAP(6-38)),每组18只。各组大鼠在缺血2h后尾静脉注射2次药物,分别为再灌前的15min和再灌后48h,其中假手术组、模型组和BYHWD组给予1ml生理盐水,BYHWD+PACAP(6-38)给予PACAP(6-38)(1.49mmol/ml).此外,各组大鼠在缺血2h再灌24h后灌胃给药,假手术组和模型组每次给予2ml的生理盐水灌胃,2次/d, BYHWD组和BYHWD+PACAP(6-38)组给予补阳还五汤(16g/kg/次,2次/d),各组连续给药3d。动物在最后一次灌胃给药后12h内取材,其中每组抽取3只制备冰冻切片,采用免疫组织荧光化学法检钡PACAP38. GLT1、GS和GFAP的表达；剩下的动物麻醉脱臼处死,取出大脑,冰上分离缺血侧大脑的海马组织,采用western-blot蛋白印迹法检钡GLT1、GS和GFAP的表达,紫外分光光度法检测谷氨酸浓度。 结果： 脑缺血后海马CA1区GLT1和GS的表达都是先升高后下降,药物组的两种蛋白表达均比同时间点的模型组高,且具有差异(P0.01)；而给药3d后的GLT1和GS表达明显比其他给药组高(P0.01)。 脑缺血后,BYHWD组大鼠海马CA1区的PACAP表达明显比模型组高(P0.01),而BYHWD+PACAP(6-38)组大鼠缺血侧大脑海马GLT1和GS蛋白表达比BYHWD组的弱；并且BYHWD+PACAP(6-38)组的谷氨酸浓度与BYHWD组的相比有所升高；此外,GFAP在BYHWD+PACAP(6-38)组的表达比BYHWD组强。 结论： 补阳还五汤能通过PACAP介导星形胶质细胞GLTl和GS表达,减少缺血后谷氨酸浓度,从而保护神经元。
[Abstract]:Objective:
Cerebrovascular disease (cvd) is a kind of disease that seriously affects human health. globally, CVD is one of the main diseases causing death and disability, especially ischemic cerebrovascular disease (ICVD). the main pathogenesis of icVD mainly includes energy depletion, acid depletion. Poisoning, oxygen free radical production, damage, nitric oxide and nitric oxide synthase, calcium overload, inflammatory factors, apoptosis regulators, excitatory amino acids (EAA). EAA is one of the most important pathogenesis, which can cause other mechanisms. Glutamate (Glu) is the central nervous system. Buyang Huanwu Decoction (BYHWD) has been reported to reduce the concentration of extracellular glutamate, and glutamate transporter-1 (GLT-1) or GLT-1 (glutamate transporter-1, GLT-1) is the most abundant excitatory amino acid in astrocytes. In this study, immunohistochemistry and Western-blot were used to explore whether Buyang Huanwu decoction could increase the expression of GLT1 and GS protein in astrocytes after cerebral ischemia, thereby reducing the concentration of extracellular glutamate. We hope to provide more experimental evidence for the treatment of cerebral ischemia by buyanghuan Five Decoction.
Method:
1. the effect of buyanghuanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were divided into model group (1d, 3d, 5d, 7d) and drug group (1d, 3d, 5d, 7d) by middle cerebral artery occlusion (MCAO). The rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day, and the rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day. The expression of GLT1 and glial fibrillary acidic protein (GFAP), GS and GFAP were detected by fluorescence double labeling. The pictures were observed under microscope and analyzed by ImageJ.
2. the mechanism of Buyang huanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were randomly divided into sham operation group, model group, BYHWD group, pituitary adenylate cyclase activating polypeptide (6-38), PACAP (6-38) group (BYHWD + PACAP (6-38), 18 rats in each group were injected into tail vein 2 hours after ischemia. The rats in sham operation group, model group and BYHWD group were given 1 ml normal saline, BYHWD + PACAP (6-38) were given PACAP (6-38) (1.49 mmol/ml). In addition, the rats in each group were given 2 ml normal saline after 2 h of ischemia and 24 h of reperfusion respectively. HWD+PACAP(6-38) group was given Buyang Huanwu Decoction(16g/kg/time,twice/day) for 3 days. Animals were taken within 12 hours after the last intragastric administration. Three of them were taken from each group to prepare frozen sections and the expression of barium PACAP38.GLT1,GS and GFAP were detected by immunohistochemistry. The expression of barium GLT1, GS and GFAP in hippocampus of ischemic brain was detected by Western blot and the concentration of glutamate was detected by ultraviolet spectrophotometry.
Result:
After cerebral ischemia, the expression of GLT1 and GS in CA1 area of hippocampus increased first and then decreased, and the expression of GLT1 and GS in drug group was higher than that in model group at the same time point (P 0.01).
After cerebral ischemia, the expression of PACAP in hippocampal CA1 region of BYHWD group was significantly higher than that of model group (P 0.01), while the expression of GLT1 and GS in hippocampus of BYHWD + PACAP (6-38) group was weaker than that of BYHWD group, and the concentration of glutamate in BYHWD + PACAP (6-38) group was higher than that in BYHWD + PACAP (6-38) group. Group YHWD is strong.
Conclusion:
Buyang Huanwu Decoction can protect neurons by reducing glutamate concentration after ischemia through PACAP-mediated GLTl and GS expression in astrocytes.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R277.7
本文编号：2241040
[Abstract]:Objective:
Cerebrovascular disease (cvd) is a kind of disease that seriously affects human health. globally, CVD is one of the main diseases causing death and disability, especially ischemic cerebrovascular disease (ICVD). the main pathogenesis of icVD mainly includes energy depletion, acid depletion. Poisoning, oxygen free radical production, damage, nitric oxide and nitric oxide synthase, calcium overload, inflammatory factors, apoptosis regulators, excitatory amino acids (EAA). EAA is one of the most important pathogenesis, which can cause other mechanisms. Glutamate (Glu) is the central nervous system. Buyang Huanwu Decoction (BYHWD) has been reported to reduce the concentration of extracellular glutamate, and glutamate transporter-1 (GLT-1) or GLT-1 (glutamate transporter-1, GLT-1) is the most abundant excitatory amino acid in astrocytes. In this study, immunohistochemistry and Western-blot were used to explore whether Buyang Huanwu decoction could increase the expression of GLT1 and GS protein in astrocytes after cerebral ischemia, thereby reducing the concentration of extracellular glutamate. We hope to provide more experimental evidence for the treatment of cerebral ischemia by buyanghuan Five Decoction.
Method:
1. the effect of buyanghuanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were divided into model group (1d, 3d, 5d, 7d) and drug group (1d, 3d, 5d, 7d) by middle cerebral artery occlusion (MCAO). The rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day, and the rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day. The expression of GLT1 and glial fibrillary acidic protein (GFAP), GS and GFAP were detected by fluorescence double labeling. The pictures were observed under microscope and analyzed by ImageJ.
2. the mechanism of Buyang huanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were randomly divided into sham operation group, model group, BYHWD group, pituitary adenylate cyclase activating polypeptide (6-38), PACAP (6-38) group (BYHWD + PACAP (6-38), 18 rats in each group were injected into tail vein 2 hours after ischemia. The rats in sham operation group, model group and BYHWD group were given 1 ml normal saline, BYHWD + PACAP (6-38) were given PACAP (6-38) (1.49 mmol/ml). In addition, the rats in each group were given 2 ml normal saline after 2 h of ischemia and 24 h of reperfusion respectively. HWD+PACAP(6-38) group was given Buyang Huanwu Decoction(16g/kg/time,twice/day) for 3 days. Animals were taken within 12 hours after the last intragastric administration. Three of them were taken from each group to prepare frozen sections and the expression of barium PACAP38.GLT1,GS and GFAP were detected by immunohistochemistry. The expression of barium GLT1, GS and GFAP in hippocampus of ischemic brain was detected by Western blot and the concentration of glutamate was detected by ultraviolet spectrophotometry.
Result:
After cerebral ischemia, the expression of GLT1 and GS in CA1 area of hippocampus increased first and then decreased, and the expression of GLT1 and GS in drug group was higher than that in model group at the same time point (P 0.01).
After cerebral ischemia, the expression of PACAP in hippocampal CA1 region of BYHWD group was significantly higher than that of model group (P 0.01), while the expression of GLT1 and GS in hippocampus of BYHWD + PACAP (6-38) group was weaker than that of BYHWD group, and the concentration of glutamate in BYHWD + PACAP (6-38) group was higher than that in BYHWD + PACAP (6-38) group. Group YHWD is strong.
Conclusion:
Buyang Huanwu Decoction can protect neurons by reducing glutamate concentration after ischemia through PACAP-mediated GLTl and GS expression in astrocytes.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R277.7
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