电针百会、足三里穴对脑缺血大鼠ET-1、内皮素受体A、B 、MMP2及VEGF-A表达的影响

Ischemic stroke is one of the leading causes of morbidity and mortality worldwide. The destruction of the BBB is closely connected with the brain edema after CIRI. ET-1、endothelin-A receptor、endothelin-B receptor、MMP2 and VEGF-A have been linked to the permeabilization of the BBB and the deteriorating cerebral edema in CIRI. SD rats were randomly divided into 3 groups, the sham group (S), the model group (M), and the electro-acupuncture group (EA). The M and the EA groups were divided into 24h and 48h timepoints, each subgroup contain 8 rats. Followed by 2h ischemia and 24 h and 48 h reperfusion, neurological function was assessed. The brains were harvested for infarct size estimation by TTC staining. The expression of ET-1、endothelin-A receptor、endothelin-B receptor、MMP2 and VEGF-A were determined by Western blot, immunohistochemistry and qPCR. The serum level of ET-1、MMP2、VEGF-A were determined by ELISE. We aim to further explore the mechanism of EA at GV20 and ST36 on cerebral edema caused by CIRI. EA would be an alternative and complementary therapeutic strategy for clinical treatment of stroke.

缺血性脑卒中的发病率和致残率均较高。脑水肿是缺血再灌注损伤的重要机制之一。血脑屏障的破坏和脑水肿密切相关。内皮素-1（ET-1）、内皮素受体A、内皮素受体B 、基质金属蛋白酶-2（MMP2）及血管内皮生长因子-A(VEGF-A)对维持脑缺血再灌注损伤后血脑屏障完整性起重要作用。本研究探讨电针百会和足三里组穴对脑缺血再灌注损伤大鼠ET-1、内皮素受体A、内皮素受体B、MMP2和VEGF-A的影响。将大鼠随机分成假手术组、模型组、电针组。模型和电针组按再灌注时间分为24h和48两个亚组，每亚组大鼠8只。在规定时间对各组大鼠神经功能评分和脑TTC 染色。Western Blot法、免疫组化法、qPCR法检测脑组织ET-1、内皮素受体A 、内皮素受体B、MMP2、VEGF-A表达；ELISA法检测ET-1、MMP2、VEGF-A在外周血清表达。探讨电针是否通过影响内皮素引起的脑水肿通路改善脑水肿。

研究背景：脑缺血性疾病是目前严重威胁人类健康的疾病之一。脑水肿和细胞凋亡是脑缺血再灌注损伤（CIRI）后的主要并发症。内皮素-1（ET-1）、内皮素受体B 、基质金属蛋白酶-2（MMP2）及血管内皮生长因子-A(VEGF-A)对维持CIRI后血脑屏障完整性进而改善CIRI起重要作用。EPO介导的JAK2/STAT3信号转导通路对改善脑缺血再灌注损伤后的细胞凋亡有影响。电针百会、足三里穴在CIRI中有重要的作用。.研究内容：制备线栓法大鼠脑缺血模型，根据再灌注后时间分为不同时间点。在脑缺血后2h及处死前采用Ludmila Belayev12分评分法、前肢踩空实验、网屏实验、Longa评分和大鼠粘附移除时间实验评价各组大鼠的神经功能缺损情况。TTC染色检测各组大鼠脑梗死体积，HE染色检测缺血脑组织组织形态学的改变。Tunel 法检测脑组织细胞凋亡情况；ELISA法检测血清ET-1以及VEGF-A表达；免疫组织化学染色和Western blot法检测脑缺血大鼠ET-1, 内皮素受体B, MMP2，VEGF-A，EPO/EpoR，JAK2/STAT3的表达；采用实时荧光定量 PCR 法检测脑缺血大鼠脑组织ET-1, 内皮素受体B, MMP2, VEGF-A, EPO/EpoR及JAK2/STAT3 mRNA的表达。免疫荧光法双标法检测大鼠脑组织中VEGF-A和星形胶质细胞共表达的情况。免疫荧光染色观察 NeuN 及 GFAP 表达；免疫荧光双染法检测大鼠脑组织中JAK2和神经元，JAK2和星型胶质细胞共表达的情况。免疫荧光法观察EPO+ 细胞和TUNEL+ 细胞共表达情况。.重要结果：研究结果显示，电针可以通过调节大鼠ET-1相关通路的分子表达水平减少细胞水肿，提高EPO介导的JAK2/STAT3蛋白和mRNA水平减少细胞凋亡，进而多途径、多靶点减轻脑缺血再灌注损伤大鼠神经功能缺损和缺血性脑损伤。 .科学意义：为针刺治疗脑血管病提供了客观依据。