中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (7): 1221-1229.doi: 10.4103/1673-5374.251571

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

GPER激动剂G1可抑制脑缺血再灌注后内质网应激引起的细胞凋亡

  

  • 出版日期:2019-07-15 发布日期:2019-07-15
  • 基金资助:

    国家自然科学基金（81560175，81260159）

GPER agonist G1 suppresses neuronal apoptosis mediated by endoplasmic reticulum stress after cerebral ischemia/reperfusion injury

Zi-Wei Han 1, 2 , Yue-Chen Chang 1, 2, , Ying Zhou 1, 2 , Hang Zhang 2, 3 , Long Chen 1, 2 , Yang Zhang 1, 2 , Jun-Qiang Si 1, 2 , Li Li 1, 2, 4   

  1. 1 Department of Physiology, Medical College of Shihezi University, Shihezi, Xinjiang Uygur Autonomous Region, China
    2 Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University School of Medicine, Shihezi, Xinjiang Uygur Autonomous Region, China
    3 Affiliated Teng Zhou Central People’s Hospital, Jining Medical University, Jining, Shandong Province, China
    4 Department of Physiology, Jiaxing College of Medicine, Jiaxing, Zhejiang Province, China
  • Online:2019-07-15 Published:2019-07-15
  • Contact: Li Li, PhD, lily7588@163.com; Jun-Qiang Si, PhD, sijunqiang@shzu.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81560175, 81260159 (both to LL).

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摘要：

已有研究显示，内质网应激途径的激活与脑缺血再灌注损伤密切相关。因此实验选取内质网应激介导的细胞凋亡途径中的关键蛋白GRP78，Caspase-12及CHOP来研究内质网应激在G蛋白偶联雌激素受体(GPER)产生脑保护过程中的潜在机制。实验将雌性SD大鼠卵巢切除(OVX)，再采用大脑中动脉栓塞法制备去势脑缺血再灌注损伤大鼠模型(OVX+I/R)，分别于缺血再灌注后即刻经侧脑室注射100μg/kg的雌激素E2 (OVX+I/R+E2)，100μg/kg的GPER激动剂G1(OVX+I/R+G1)。采用Longa评分法检测各组神经行为学变化，(1)采用TTC染色检测各组脑梗死体积变化，采用尼氏染色法观察各组脑组织形态学改变，采用TUNEL染色检测各组神经元凋亡发现，相比于OVX+I/R组，E2和G1干预后大鼠神经功能明显改善，脑梗死体积缩小，脑组织正常尼氏小体数量显著增多，海马区凋亡神经元减少；(2)为检测内质网应激相关蛋白表达水平及分布情况，采用免疫荧光检测caspase12的分布及其表达、采用PCR和Western-blot技术分别检测GRP78、Caspase-12和CHOP的mRNA以及蛋白表达显示，相比于OVX+I/R组，E2和G1处理可显著降低GRP78，CHOP和caspase-12 mRNA及蛋白的表达水平；(3)然而于术前30 min经侧脑室注射100 μg/kg的GPER拮抗剂G15 (OVX+I/R+E2+G15)可以消除E2对脑缺血再灌注损伤的影响；(4)上述数据证实，E2以及GPER能够抑制内质网应激相关蛋白的表达，减轻海马区神经元凋亡，从而改善脑缺血再灌注损伤造成的功能障碍。

orcid: 0000-0001-8591-0676 (Li Li)
           0000-0001-6704-2115 (Jun-Qiang Si)

关键词: 脑缺血再灌注损伤, 雌激素, G蛋白偶联雌激素受体, G1, G15, 内质网应激, GRP78, Caspase-12, CHOP, 神经元凋亡, 神经再生

Abstract:

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfu¬sion (I/R) injury. In this study, three key proteins in the endoplasmic reticulum stress pathway (glucose-regulated protein 78, caspase-12, and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor. Female Sprague-Dawley rats received ovariectomy (OVX), and then cerebral I/R rat models (OVX + I/R) were established by middle cerebral artery occlusion. Immediately after I/R, rat models were injected with 100 μg/kg E2 (OVX + I/R + E2), or 100 μg/kg G protein-coupled estrogen receptor agonist G1 (OVX + I/R + G1) in the lateral ventricle. Longa scoring was used to detect neurobehavioral changes in each group. Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining. Morphological changes in neurons were observed by Nissl staining. Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group, neurological function was remarkably improved, infarct volume was reduced, number of normal Nissl bodies was dramatically increased, and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention. To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum, caspase-12 distribution and expression were detected by immunofluorescence, and mRNA and protein levels of glucose-regulated protein 78, caspase-12, and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay. The results showed that compared with the OVX + I/R group, E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78, C/EBP homol¬ogous protein, and caspase-12. However, the G protein-coupled estrogen receptor antagonist G15 (OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury. These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus, thereby improving dysfunction caused by cere¬bral I/R injury. Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. SHZ A2017-171) on February 27, 2017.

Key words: nerve regeneration, cerebral ischemia/reperfusion injury, estrogen, G protein-coupled estrogen receptor, G1, G15, endoplasmic reticulum stress, glucose-regulated protein 78, caspase-12, C/EBP homologous protein, neuronal apoptosis, neural regeneration