拟载脂蛋白E-141012肽对脑血管痉挛小鼠代谢型谷氨酸受体/细胞外信号调节激酶途径的调节作用

Role of apoE-mimetic peptide1410 in the regulatory efficacy of metabtropic glutamale receptor/extracellular signal regulated kinase pathway during cerebral vasospasm in mice

目的探讨脑血管痉挛(CVS)后细胞外信号调节激酶(ERK)途径的作用机制,及重组载脂蛋白E(apoE)拟肽对脑血管痉挛损伤的保护作用。方法采用非开颅血管内线栓法制备小鼠蛛网膜下腔出血(SAH)并脑血管痉挛模型。将134只健康雄性ICR小鼠随机分为4组:假手术组、模型对照组、拮抗剂组,apoE治疗组。ApoE治疗组将拟apoE-1410以无菌磷酸盐缓冲液溶解后,经尾静脉注射0.6mg/kg,术前30min开始第1次,每12h1次。拮抗剂组于SAH后10 min侧脑室注射生理盐水或LY367385(500nmol/L)5μl,术后行神经功能评分。分别在SAH后6、24、48h 3个时相点取脑组织标本,在光镜和电镜下观察脑组织病理变化,采用反转录-聚合酶链式反应(RT-PCR)检测各组代谢型谷氨酸受体1(mG1uR1)mRNA的表达变化;免疫印迹法(Western blotting)检测mGluR1、磷酸化细胞外信号调节激酶(ERK1/2)蛋白的表达;用原位缺口末端标记法(TUNEL)检测神经细胞凋亡情况。结果与假手术组比较,模型对照组小鼠神经功能评分显著降低,随脑血管痉挛时间延长,各组小鼠mGluR1 mRNA、mGluR1和磷酸化ERK1/2蛋白均有不同程度增加,凋亡细胞增多,神经细胞出现变性坏死、细胞器数量减少。与模型对照组比较,拮抗剂组和apoE治疗组小鼠神经功能评分增加,mGluR1 mRNA、mGluR1、磷酸化ERK1/2蛋白表达均有不同程度下调,神经细胞凋亡数目减少,应用apoE1410拟肽减轻了脑组织形态学和超微结构损伤。结论脑血管痉挛后mGluR1的表达增强可通过激活ERK信号途径诱导神经细胞凋亡,apoE拟肽对脑血管痉挛损伤具有抗损坏作用。

Objective To investigate mechanisms of the extracellular signal-regulated kinase signaling pathway, and the protective effect of apoE-mimetic peptidel410 after severe cerebral vasospasm （CVS）in mice. Methods A SAH model was established by endovascular perforation without opening cranium. 134 male ICR mice were randomly divided into four groups： sham operation group, model control group, LY367385 group and apoE group. Ten minutes after SAH, 5 μl of LY367385 were microinjected into the lateral cerebral ventricle in LY367385 group. Before 30 rain of operation mice were injected with apoE-mimetic peptidel410（0. 6 mg/kg）via the tail vein and repeated every 12 hours for three days in apoE group. The neurological behavioral function was evaluated. At different time points （6, 24, 48 hours） after operation, changes in brain tissue were observed with light and electron microscopy. The expression of mGluR1 mRNA was detected using RT-PCR, Western blotting assay was used to examine mGluR1 and p-ERK1/2 expression. The number of apoptotic cells was determined by TUNEL method. Results In model control group, the neurological function scores were significantly lower, and the mGluR1 mRNA, mGluR1, p-ERK1/2 level and the number of apoptosis cells were significantly enhanced. Some neurons displayed histopathologie changes of necrosis, and organelles decreased. Mice treated with LY367385 or apoE-mimetie peptidel410 had reduction in mGluR1 mRNA, mGluR1, ERK1/2 activity, and neuronal apoptosis, apoE-mimetie peptidel410 notably relieved the neurological scores, and improved the uhrastructural changes. Conclusion The increased expression of mGluRl in hippoeampus may induce apoptosis by activation of ERK1/2 signaling after CVS. LY367385 has a good therapeutic effect on severe CVS.