电针对局灶性脑缺血大鼠海马微血管内皮细胞转运功能的影响
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摘要
目的:
通过研究针刺对局灶性脑缺血大鼠海马CA1区微循环灌流量、葡萄糖代谢、微血管内皮细胞转运相关蛋白表达的影响,研究针刺对微血管内皮细胞转运功能的影响,探讨针刺治疗缺血性脑中风的机制。
方法:
1.Wistar大鼠200只随机分为假手术对照组、模型组、针刺内关组和针刺地机组,各组再随机分为第15天和第30天观察组,共八个组。采用穿线阻塞大脑中动脉法建立局灶性脑缺血大鼠模型(MCAO)。
2.造模后第15、30天应用激光多普勒微循环血流分析仪,检测各组大鼠针刺后海马CA1区微循环血流量的变化。
3.脑组织匀浆检测葡萄糖、丙酮酸、乳酸、Ca~(2+)的含量和谷氨酸浓度。
4.HE染色观察脑组织神经细胞的形态学变化并计算缺血侧海马CA1区神经元坏死率。
5.免疫组织化学测定葡萄糖转运蛋白1(Glucose transporter 1,GLUT-1)及P-糖蛋白(P-Glycoprotein,P-gp)在MCAO大鼠缺血侧海马CA1区微血管内皮细胞的表达。
6.蛋白质印迹(Western blotting)方法测定GLUT-1及P-gp在脑组织内的相对含量。
结果:
1.本实验采用穿线阻塞大脑中动脉法成功建立了局灶性脑缺血大鼠模型(MCAO)。
(1)针刺内关组MACO大鼠海马CA1区的微循环血流量明显高于同时间段模型组和针刺地机组,具有显著性差异。
(2)针刺内关组大鼠脑组织乳酸含量、谷氨酸浓度、Ca~(2+)的含量明显降低,葡萄糖和丙酮酸的含量明显升高,具有显著性差异。
(3)针刺内关组大鼠缺血侧海马CA1区神经元的死亡率明显低于同时间段模型组大鼠,具有显著性差异。
2.免疫组化法测定GLUT-1和P-gp在MCAO大鼠缺血侧海马CA1区微血管内皮细胞的表达数及积分光密度值(IOD),结果显示针刺内关组明显高于模型组和同时间段针刺地机组,差异具有显著性。
3.Western blotting测定GLUT-1在MCAO大鼠脑组织的蛋白相对含量,结果显示针刺内关组明显高于模型组和同时间段针刺地机组,具有显著性差异;P-gp在MCAO大鼠脑组织蛋白的相对含量为阴性。
结论:
针刺可促进MCAO大鼠海马CA1区微循环血流量和微血管内皮细胞GLUT-1和P-gp的蛋白表达,提高脑缺血后微血管内皮细胞的转运功能,从而改善脑组织能量代谢,减轻脑缺血后神经细胞的损伤,促进MCAO大鼠脑损伤的恢复。
Objective: To elucidate the mechanism of electroacupuncture in treating cerebral ischemic disease, it is studied that the influence of acupuncture at Neiguan on mocrocirculatory blood flow of hippocampus and transporting function of microvascular endothelial cells in rats with focal cerebral ischemic.
Methods: Cerebral ischemia animal models were induced by middle cerebral artery occlusion with thread ligature in Wistar rats. These rats were randomly divided into eight groups: sham operation groups, model groups, Neiguan groups and Diji groups. Electroacupuncture was applied for fifteenth days and thirtieth days after MCAO. The change of microcirculatory blood flow in hippocampal CA1 area was monitored by laser-Doppler Flow meter (LFD )on the fifteenth,thirtyth days after MCAO respectively in each group. Morphological changes of hippocampal neurons were observed by H.E. The content of Glucose,PyruvicAcid,Lactic Acid,Ca~(2+) and Glutamate in brain tissue was analyzed with specialized measuring kits. The expression of GLUT-1 and P-gp in hippocampal CA1 area was observed with the method of Immuno-histochemical and Western Blotting.
Results: Microcirculatory blood flow in hippocampal CA1 region of MCAO rats was obviously increased after electroacupuncture at Neiguan acupoint, this effects were more obveriously than the effects in Diji group. The rate of necrosis neurons in hippocampal CA1 region of MCAO rats in Neiguan group was decreased more obviously than that in other groups. The content of Lactic Acid,Ca~(2+) and Glutamate in brain tissue were decreased after acupuncturing at Neiguan . The content of Pyruvic Acid and Glucose was increased in Neiguan groups. Expressing of GLUT-1 and P-gp at the endothelia cells in hippocampal CA1 region of MCAO rats was increased after electroacupuncture. Western blotting showed that the protein contents of GLUT-1 in brains of MCAO rats went up after electroacupuncture. However, P-gp contents were negtively.
Conclusions: Microcirculatory blood flow in hippocampal CA1 region of MCAO rats could be increased by acupuncture. The rate of necrosis neurons in hippocampal CA1 region could be decreased through electroacupuncture. Expression of GLUT-1 and P-gp protein in hippocampal CA1 area of MCAO rats could be increased by acupuncture. Transporting function of microvascular endothelial cells could be improved and neuronal injury was relieved after cerebral ischemia by means of electroacupuncture.
通过研究针刺对局灶性脑缺血大鼠海马CA1区微循环灌流量、葡萄糖代谢、微血管内皮细胞转运相关蛋白表达的影响,研究针刺对微血管内皮细胞转运功能的影响,探讨针刺治疗缺血性脑中风的机制。
方法:
1.Wistar大鼠200只随机分为假手术对照组、模型组、针刺内关组和针刺地机组,各组再随机分为第15天和第30天观察组,共八个组。采用穿线阻塞大脑中动脉法建立局灶性脑缺血大鼠模型(MCAO)。
2.造模后第15、30天应用激光多普勒微循环血流分析仪,检测各组大鼠针刺后海马CA1区微循环血流量的变化。
3.脑组织匀浆检测葡萄糖、丙酮酸、乳酸、Ca~(2+)的含量和谷氨酸浓度。
4.HE染色观察脑组织神经细胞的形态学变化并计算缺血侧海马CA1区神经元坏死率。
5.免疫组织化学测定葡萄糖转运蛋白1(Glucose transporter 1,GLUT-1)及P-糖蛋白(P-Glycoprotein,P-gp)在MCAO大鼠缺血侧海马CA1区微血管内皮细胞的表达。
6.蛋白质印迹(Western blotting)方法测定GLUT-1及P-gp在脑组织内的相对含量。
结果:
1.本实验采用穿线阻塞大脑中动脉法成功建立了局灶性脑缺血大鼠模型(MCAO)。
(1)针刺内关组MACO大鼠海马CA1区的微循环血流量明显高于同时间段模型组和针刺地机组,具有显著性差异。
(2)针刺内关组大鼠脑组织乳酸含量、谷氨酸浓度、Ca~(2+)的含量明显降低,葡萄糖和丙酮酸的含量明显升高,具有显著性差异。
(3)针刺内关组大鼠缺血侧海马CA1区神经元的死亡率明显低于同时间段模型组大鼠,具有显著性差异。
2.免疫组化法测定GLUT-1和P-gp在MCAO大鼠缺血侧海马CA1区微血管内皮细胞的表达数及积分光密度值(IOD),结果显示针刺内关组明显高于模型组和同时间段针刺地机组,差异具有显著性。
3.Western blotting测定GLUT-1在MCAO大鼠脑组织的蛋白相对含量,结果显示针刺内关组明显高于模型组和同时间段针刺地机组,具有显著性差异;P-gp在MCAO大鼠脑组织蛋白的相对含量为阴性。
结论:
针刺可促进MCAO大鼠海马CA1区微循环血流量和微血管内皮细胞GLUT-1和P-gp的蛋白表达,提高脑缺血后微血管内皮细胞的转运功能,从而改善脑组织能量代谢,减轻脑缺血后神经细胞的损伤,促进MCAO大鼠脑损伤的恢复。
Objective: To elucidate the mechanism of electroacupuncture in treating cerebral ischemic disease, it is studied that the influence of acupuncture at Neiguan on mocrocirculatory blood flow of hippocampus and transporting function of microvascular endothelial cells in rats with focal cerebral ischemic.
Methods: Cerebral ischemia animal models were induced by middle cerebral artery occlusion with thread ligature in Wistar rats. These rats were randomly divided into eight groups: sham operation groups, model groups, Neiguan groups and Diji groups. Electroacupuncture was applied for fifteenth days and thirtieth days after MCAO. The change of microcirculatory blood flow in hippocampal CA1 area was monitored by laser-Doppler Flow meter (LFD )on the fifteenth,thirtyth days after MCAO respectively in each group. Morphological changes of hippocampal neurons were observed by H.E. The content of Glucose,PyruvicAcid,Lactic Acid,Ca~(2+) and Glutamate in brain tissue was analyzed with specialized measuring kits. The expression of GLUT-1 and P-gp in hippocampal CA1 area was observed with the method of Immuno-histochemical and Western Blotting.
Results: Microcirculatory blood flow in hippocampal CA1 region of MCAO rats was obviously increased after electroacupuncture at Neiguan acupoint, this effects were more obveriously than the effects in Diji group. The rate of necrosis neurons in hippocampal CA1 region of MCAO rats in Neiguan group was decreased more obviously than that in other groups. The content of Lactic Acid,Ca~(2+) and Glutamate in brain tissue were decreased after acupuncturing at Neiguan . The content of Pyruvic Acid and Glucose was increased in Neiguan groups. Expressing of GLUT-1 and P-gp at the endothelia cells in hippocampal CA1 region of MCAO rats was increased after electroacupuncture. Western blotting showed that the protein contents of GLUT-1 in brains of MCAO rats went up after electroacupuncture. However, P-gp contents were negtively.
Conclusions: Microcirculatory blood flow in hippocampal CA1 region of MCAO rats could be increased by acupuncture. The rate of necrosis neurons in hippocampal CA1 region could be decreased through electroacupuncture. Expression of GLUT-1 and P-gp protein in hippocampal CA1 area of MCAO rats could be increased by acupuncture. Transporting function of microvascular endothelial cells could be improved and neuronal injury was relieved after cerebral ischemia by means of electroacupuncture.
引文
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