通窍活血汤通过调节星形胶质细胞Glu-Gln循环降低Glu兴奋性毒性改善CIRI大鼠的神经功能  

Tongqiao Huoxuetang Improves Neurological Deficits in CIRI Rats by Regulating Glu-Gln Circulation to Reduce Glutamate Excitotoxicity of Astrocytes

作　　者：张立娟[1] 张蔷 娄原 姜雅楠 韩冰冰[1,2] 马珂 赵海军 王世军[1,2] 王媛 ZHANG Li-juan;ZHANG Qiang;LOU Yuan;JIANG Ya-nan;HAN Bing-bing;MA Ke;ZHAO Hai-jun;WANG Shi-jun;WANG Yuan(Shandong University of Traditional Chinese Medicine(TCM),Jinan 250355,China;Shandong Co-Innovation Center of Classic TCM Formula,Jinan 250355,China)

机构地区：[1]山东中医药大学,济南250355 [2]山东省中医经典名方协同创新中心,济南250355

出　　处：《中国实验方剂学杂志》2021年第22期31-40,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：山东省高等学校青创科技支持计划项目(2019KJK013);济南市高校院所创新团队项目(2020GXRC012);山东中医药大学青年科研创新团队(2020-54-17);山东省高校青创人才引育计划项目(鲁教人字[2019]9号-202/201)。

摘　　要：目的:观察并比较3种不同方法制备的通窍活血汤对脑缺血再灌注损伤(CIRI)大鼠的保护作用,并从星形胶质细胞谷氨酸(Glu)代谢通路探讨其作用机制。方法:选用SPF级雄性SD大鼠,以改良线栓法建立CIRI模型,将造模成功的大鼠随机分为模型组、通窍活血汤水煎组、通窍活血汤酒煎组、通窍活血汤醇提组,并设假手术组,分别给予对应药物灌胃,连续治疗7 d。通窍活血汤3个组的灌胃剂量为6.3 g·kg^(-1)·d^(-1),假手术组和模型组给予同体积生理盐水灌胃。末次灌胃结束后,以神经功能缺损评分(mNSS)检测大鼠神经功能恢复情况,苏木素-伊红(HE)染色观察缺血脑组织的形态学变化,高效液相色谱法(HPLC)检测大鼠缺血脑组织中Glu含量的变化,免疫组织荧光法检测缺血脑组织中谷氨酸转运体-1(GLT-1)和胶质纤维酸性蛋白(GFAP)及谷氨酰胺合成酶(GS)和GFAP的共表达情况;蛋白免疫印迹法(Western blot)检测缺血脑组织中GFAP,GLT-1和GS蛋白的表达情况。结果:与假手术组比较,模型组大鼠mNSS评分显著升高(P<0.01),缺血脑组织皮质大片坏死,细胞排列紊乱、边界模糊,出现细胞水肿及炎性浸润;缺血脑组织中Glu的含量显著增加(P<0.01),GLT-1与GFAP共表达及GS与GFAP共表达显著降低(P<0.01),GFAP蛋白表达显著升高(P<0.01),GLT-1,GS蛋白表达明显降低(P<0.05,P<0.01);与模型组比较,3种方法制备的通窍活血组mNSS评分均显著降低(P<0.01),大鼠缺血脑组织皮质及海马神经细胞的损伤程度均有所减轻;缺血脑组织中Glu的含量均明显减少(P<0.05,P<0.01),GLT-1与GFAP共表达均明显升高(P<0.05,P<0.01),GFAP,GLT-1蛋白表达均明显升高(P<0.05,P<0.01),通窍活血汤酒煎组、通窍活血汤醇提组GS与GFAP共表达明显增加(P<0.05,P<0.01),GS蛋白的表达显著升高(P<0.01)。与通窍活血汤水煎组比较,通窍活血汤醇提组GLT-1和GFAP共表达及GS和GFAP共表达明显增加(P<0.05),通窍活血�Objective: To observe and compare the protective effects of Tongqiao Huoxue decoction(TQHX)prepared by three methods against cerebral ischemia-reperfusion injury(CIRI),and to explore its mechanism through the glutamate(Glu)metabolic pathway in astrocytes. Method: The male SD rats of SPF grade were subjected to CIRI model induction by the modified middle cerebral artery occlusion method. The model rats were randomly divided into a model group,a sham operation group,and water-decocted,winedecocted,and alcohol-extracted TQHX(6.3 g·kg^(-1)·d^(-1))groups. The rats were treated correspondingly for 7 days.Those in the sham operation group and the model group were treated with an equal volume of normal saline by gavage. After the final treatment,the neurological function of rats was assessed by the modified neurological severity score(mNSS). Hematoxylin-eosin(HE)staining was used to observe the morphological changes of ischemic brain tissues in rats. High-performance liquid chromatography(HPLC)was used to detect glutamate(Glu)in ischemic brain tissues. The expression of glutamate transporter-1(GLT-1)and glial fibrillary acidic protein(GFAP)and co-expression of glutamine synthetase(GS)and GFAP in ischemic brain tissues were detected by immunofluorescence assay. Western blot was used to detect the protein expression of GFAP,GLT-1,and GS. Result: Compared with the sham operation group,the model group showed increased mNSS(P<0.01),large necrosis of cerebral cortex in ischemic brain tissues with disordered cell arrangement,obscure boundary,intracellular edema,and inflammatory infiltration,elevated Glu in ischemic brain tissues(P<0.01),declining GLT-1-GFAP co-expression and GS-GFAP co-expression(P<0.01),up-regulated expression of GFAP protein,and reduced protein expression of GLT-1 and GS(P<0.05,P<0.01). Compared with the model group,the TQHX groups showed decreased mNSS(P<0.01),relieved injury in the cerebral cortex and hippocampal nerve cells in ischemic brain tissues,reduced Glu expression(P<0.05,P<0.01),elevated co-expre

关 键 词：通窍活血汤 脑缺血再灌注损伤 兴奋性毒性损伤 谷氨酸 星形胶质细胞 

分 类 号：R2-0[医药卫生—中医学] R22