针刺对缺血性脑损伤神经血管单元功能的影响及机制研究
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摘要
随着人口老龄化的进展，缺血性脑血管病已成为严重危害人类健康的疾病之一。针刺具有安全、简便、经济等优点，不仅在我国接受针灸治疗的脑卒中病人有很高的比例，在国外接受针灸治疗的中风患者人数亦在逐年增多，针刺疗法已经逐渐成为世界范围内中风的常规治疗方法。
研究发现，脑缺血的病理生理变化十分复杂，涉及到星形胶质细胞、微血管内皮细胞及神经元损伤及功能失调，并因此提出“神经血管单元”的概念。为研究针刺治疗中风的机制，有必要从神经血管单元角度对其进行进一步探讨，这将为其机制研究提供一个全新的思路，为其临床的应用提供坚实的实验基础。
目的：电针MCAO大鼠内关穴、曲池穴，研究针刺对星形胶质细胞激活的影响，探讨针刺调节脑微循环血流量及能量代谢的作用机制，为针刺治疗缺血性脑中风的神经血管单元机制研究提供一定的理论和实验基础。
方法：线栓法建立MCAO大鼠模型，通过神经行为学评分、微循环学、组织形态学、免疫组织化学、实时定量PCR、Western blot等方法，观察连续针刺14天对MCAO大鼠神经功能恢复、软脑膜微循环血流量，缺血脑组织形态改变、脑组织谷氨酸含量、胶质纤维酸性蛋白（GFAP）、代谢性谷氨酸受体5（mGluR5）mRNA、花生四烯酸细胞色素P450表氧化酶2C11（CYP2C11）mRNA表达的影响，探讨针刺通过活化星形胶质细胞调节微血管血流量的作用及机制；通过观察针刺对MCAO大鼠乳酸及丙酮酸含量、脑组织GFAP蛋白的表达、葡萄糖转运体1（GLUT1）、单羧酸转运体MCT1、单羧酸转运体MCT2的表达的影响，从神经血管单元功能角度探讨针刺通过激活星形胶质细胞调节脑组织微循环血流量及能量代谢的作用及机制。应用SPSS19.0统计学软件进行数据统计及结果分析。
结果：针刺内关穴、曲池穴能够提高MCAO大鼠神经体征评分，提高脑组织中GFAP、MCT1、MCT2及GLUT1蛋白的表达；上调GFAP mRNA、mGluRs mRNA、CYP2C11mRNA的表达。针刺内关穴、曲池穴可以有效减少MCAO大鼠脑组织中过量的谷氨酸。
结论：针刺可激活星形胶质细胞，一方面可促进mGluRs及CYP2C11mRNA的表达，增加大鼠软脑膜微循环血流量；另一方面，可以促进无氧糖酵解产物乳酸的利用，增强缺血脑组织对葡萄糖的摄取，以满足神经元在脑缺血时的能量需求；针刺对缺血脑组织神经血管单元成员的功能具有调节作用，这是其治疗缺血性脑中风的机制之一。
With the progress of population aging, ischemic neurovascular disease has becomeone of the diseases that seriously endanger human health. Because of the advantage ofacupuncture in safety, convenience and economy, the number of stroke patients receivingacupuncture treatment in foreign countries is increasing year by year while there is evenmuch more high proportion of stroke patients who accept acupuncture and moxibustiontherapy in China. Therefore, acupuncture has gradually become a routine treatment ofstroke worldwide. It was found in studies that cerebral ischemia has complicatedpathophysiological changes which involve the structural damages and malfunction of cellsincluding astrocytes, microvascular endothelial cells and neurons. So the concept ofneurovascular unit was introduced. Therefore, it is necessary to investigate further themechanisms of acupuncture treatment for stroke from the perspective of neurovascularunit. This would provide a totally new idea for the mechanism research of acupuncturetherapy of stroke and the experiment foundation for the clinical application.
Objective: To investigate the effects and mechanisms of acupuncture on the cerebralmicrocirculatory flow and energy metabolism, the influence of acupuncture on activationof astrocytes by electro-acupuncture at Neiguan and Quchi, which will provide a totallynew idea for the mechanism research of acupuncture therapy of stroke and experimentfoundation for the clinical application。
Methods: Focal cerebral ischemia rat model was established using suture-occludedmethod. The effect of acupuncture on the recovery of nerve function damage, theactivation of astrocyte, pial microcirculatory blood flow, the content of brain glutamate,the mRNA expression of mGluRs and CYP2C11was observed after acupuncture for14days with the methods of neurological behavior score, microcirculation,immunohistochemistry, real-time quantitative PCR and western blot analysis. Through theabove research, the effects and the mechanisms of acupuncture on the astrocytes of MCAO rat in regulating the cerebral microcirculatory blood flow was investigated. Theeffect of acupuncture on the content of lactate and pyruvic acid, the expression of GFAP,GLUT1, MCT1and MCT2was observed to study the effects and the mechanisms ofacupuncture on the astrocytes of MCAO rat in regulating brain energy metabolism. Theresults were analyzed with software SPSS19.0.
Results: The acupuncture at Neiguan and Quchi significantly improved the recoveryof the nerve function damage in MCAO rat. The pial microcirculatory blood flow and thecontent of pyruvic acid in ischemic cerebral tissue rose, and the expression of GFAP,GLUT1, and GFAP mRNA, GLUT1mRNA, mGluRs mRNA, CYP2C11mRNA wasup-regulated. The acupuncture at Neiguan and Quchi drastically reduced the excessiveglutamate and lactate in ischemic tissue in MCAO rat.
Conclusion: Astrocytes can be activated by the acupuncture at Neiguan and Quchi,which could promotes the expression of mGluRs and CYP2C11mRNA leading to theincrease of the pial microcirculatory blood flow. On the other hand, the activation of theastrocytes promotes the use of the lactate in ischemic tissue leading to the decrease of it.In brief, acupuncture could regulate the function of neurovascular unit, which may be oneof the mechanisms that the acupuncture can improve ischemic stroke.
研究发现，脑缺血的病理生理变化十分复杂，涉及到星形胶质细胞、微血管内皮细胞及神经元损伤及功能失调，并因此提出“神经血管单元”的概念。为研究针刺治疗中风的机制，有必要从神经血管单元角度对其进行进一步探讨，这将为其机制研究提供一个全新的思路，为其临床的应用提供坚实的实验基础。
目的：电针MCAO大鼠内关穴、曲池穴，研究针刺对星形胶质细胞激活的影响，探讨针刺调节脑微循环血流量及能量代谢的作用机制，为针刺治疗缺血性脑中风的神经血管单元机制研究提供一定的理论和实验基础。
方法：线栓法建立MCAO大鼠模型，通过神经行为学评分、微循环学、组织形态学、免疫组织化学、实时定量PCR、Western blot等方法，观察连续针刺14天对MCAO大鼠神经功能恢复、软脑膜微循环血流量，缺血脑组织形态改变、脑组织谷氨酸含量、胶质纤维酸性蛋白（GFAP）、代谢性谷氨酸受体5（mGluR5）mRNA、花生四烯酸细胞色素P450表氧化酶2C11（CYP2C11）mRNA表达的影响，探讨针刺通过活化星形胶质细胞调节微血管血流量的作用及机制；通过观察针刺对MCAO大鼠乳酸及丙酮酸含量、脑组织GFAP蛋白的表达、葡萄糖转运体1（GLUT1）、单羧酸转运体MCT1、单羧酸转运体MCT2的表达的影响，从神经血管单元功能角度探讨针刺通过激活星形胶质细胞调节脑组织微循环血流量及能量代谢的作用及机制。应用SPSS19.0统计学软件进行数据统计及结果分析。
结果：针刺内关穴、曲池穴能够提高MCAO大鼠神经体征评分，提高脑组织中GFAP、MCT1、MCT2及GLUT1蛋白的表达；上调GFAP mRNA、mGluRs mRNA、CYP2C11mRNA的表达。针刺内关穴、曲池穴可以有效减少MCAO大鼠脑组织中过量的谷氨酸。
结论：针刺可激活星形胶质细胞，一方面可促进mGluRs及CYP2C11mRNA的表达，增加大鼠软脑膜微循环血流量；另一方面，可以促进无氧糖酵解产物乳酸的利用，增强缺血脑组织对葡萄糖的摄取，以满足神经元在脑缺血时的能量需求；针刺对缺血脑组织神经血管单元成员的功能具有调节作用，这是其治疗缺血性脑中风的机制之一。
With the progress of population aging, ischemic neurovascular disease has becomeone of the diseases that seriously endanger human health. Because of the advantage ofacupuncture in safety, convenience and economy, the number of stroke patients receivingacupuncture treatment in foreign countries is increasing year by year while there is evenmuch more high proportion of stroke patients who accept acupuncture and moxibustiontherapy in China. Therefore, acupuncture has gradually become a routine treatment ofstroke worldwide. It was found in studies that cerebral ischemia has complicatedpathophysiological changes which involve the structural damages and malfunction of cellsincluding astrocytes, microvascular endothelial cells and neurons. So the concept ofneurovascular unit was introduced. Therefore, it is necessary to investigate further themechanisms of acupuncture treatment for stroke from the perspective of neurovascularunit. This would provide a totally new idea for the mechanism research of acupuncturetherapy of stroke and the experiment foundation for the clinical application.
Objective: To investigate the effects and mechanisms of acupuncture on the cerebralmicrocirculatory flow and energy metabolism, the influence of acupuncture on activationof astrocytes by electro-acupuncture at Neiguan and Quchi, which will provide a totallynew idea for the mechanism research of acupuncture therapy of stroke and experimentfoundation for the clinical application。
Methods: Focal cerebral ischemia rat model was established using suture-occludedmethod. The effect of acupuncture on the recovery of nerve function damage, theactivation of astrocyte, pial microcirculatory blood flow, the content of brain glutamate,the mRNA expression of mGluRs and CYP2C11was observed after acupuncture for14days with the methods of neurological behavior score, microcirculation,immunohistochemistry, real-time quantitative PCR and western blot analysis. Through theabove research, the effects and the mechanisms of acupuncture on the astrocytes of MCAO rat in regulating the cerebral microcirculatory blood flow was investigated. Theeffect of acupuncture on the content of lactate and pyruvic acid, the expression of GFAP,GLUT1, MCT1and MCT2was observed to study the effects and the mechanisms ofacupuncture on the astrocytes of MCAO rat in regulating brain energy metabolism. Theresults were analyzed with software SPSS19.0.
Results: The acupuncture at Neiguan and Quchi significantly improved the recoveryof the nerve function damage in MCAO rat. The pial microcirculatory blood flow and thecontent of pyruvic acid in ischemic cerebral tissue rose, and the expression of GFAP,GLUT1, and GFAP mRNA, GLUT1mRNA, mGluRs mRNA, CYP2C11mRNA wasup-regulated. The acupuncture at Neiguan and Quchi drastically reduced the excessiveglutamate and lactate in ischemic tissue in MCAO rat.
Conclusion: Astrocytes can be activated by the acupuncture at Neiguan and Quchi,which could promotes the expression of mGluRs and CYP2C11mRNA leading to theincrease of the pial microcirculatory blood flow. On the other hand, the activation of theastrocytes promotes the use of the lactate in ischemic tissue leading to the decrease of it.In brief, acupuncture could regulate the function of neurovascular unit, which may be oneof the mechanisms that the acupuncture can improve ischemic stroke.
引文
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