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Ghrelin在缺血预处理抗大鼠海马神经元缺血再灌损伤中的作用
The role of ghrelin in the protection of ischemic preconditioning against ischemia/reperfusion injury in rat hippocampal neurons

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DOI:
作者:
刘亚君，陈筱南，刘克敬，陈连璧
LIU Yajun, CHEN Xiaonan, LIU Kejing, CHEN Lianbi
作者单位:
中国医学科学院微循环研究所，北京100005；山东省医学科学院附院，济南250013；山东大学医学院， 济南250012；
Institute of Microcirculation, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005; Affiliated Hospital, Shandong Academy of Medical Sciences, Jinan 250013; Institute of Physiology, School of Medicine, Shandong University, Jinan 250012,China
关键词:
缺血预处理；缺血再灌注损伤；海马；神经元；Ghrelin；UCP2
Ischemic preconditioning; Ischemia/reperfusion injury; Hippocampus; Neurons; Ghrelin; UCP2
摘要:
目的：探讨ghrelin在脑缺血预处理(ischemic preconditioning, IPC)保护海马神经元缺血再灌注损伤中的作用及其与线粒体解偶联蛋白-2（uncoupling protein 2, UCP2）表达的关系。 方法：用四血管阻断法（4 VO）建立全脑缺血模型，将大鼠随机分为对照组（CON组）、缺血再灌组（I/R组）、缺血预处理＋缺血再灌组（IPC＋I/R组）、缺血再灌＋生理盐水组（I/R＋N组）、缺血再灌＋ghrelin组（I/R+G组）和对照＋生理盐水组（CON＋N组）。用ELISA法检测缺血再灌注后血清中ghrelin水平；大鼠注射ghrelin后，HE染色观察海马CA1区神经元的组织形态学变化，免疫组化测定海马CA1区Bcl-2表达，RT-PCR法检测海马UCP2 mRNA表达。 结果：ELISA法检测结果显示，致死性缺血再灌24 h后，IPC+I/R组血浆ghrelin 浓度较对照组和I/R组明显升高（p<0.001），并且持续72 h（p<0.001）。I/R组与对照组各对应时间点ghrelin 水平均无显著变化。HE染色结果显示，注射ghrelin后，CON＋N组、I/R＋N组和I/R+G组存活细胞数分别为251.3 ± 8.7、28.5±7.3和179.3±11.5/mm，I/R+G组存活神经元数目明显多于I/R＋N组（p<0.001），但明显少于对照组（p<0.001）。免疫组化结果显示，CON＋N组、I/R＋N组和I/R+G组大鼠海马CA1区神经元Bcl-2蛋白表达均不明显；RT-PCR结果显示，I/R+G组海马CA1区有大量UCP2 mRNA表达，明显多于CON+N组和I/R＋N组（p< 0.001）。 结论：缺血预处理能促进ghrelin释放，并通过ghrelin上调海马CA1区神经元UCP2的表达，减轻缺血再灌注损伤。
AIM: To explore the role and mechanism of ghrelin in the neuroprotection of IPC against ischemia/reperfusion injury of hippocampal neurons and the relationship between ghrelin and UCP2. METHODS: A rat 4-vessel global ischemia model was used. Rats were divided into control group (CON group), ischemia/ reperfusion group (I/R group), ischemic preconditioning+ischemia/reperfusion group (IPC+I/R group), ischemia/ reperfusion+ghrelin group (I/R+G group), ischemia/ reperfusion+saline group (I/R+N group) and control+saline group (CON+N group). The ghrelin levels in serum at 24 h, 48 h and 72 h after ischemia/reperfusion were measured by ELISA. The morphological changes of neurons in hippocampal CA1 region were examined by HE staining. The expression of Bcl-2 of the neurons in the same region was detected by immunohistochemistry and the expression of UCP2 mRNA of hippocampal neurons was assayed by RT-PCR. RESULTS: At 24 h after lethal ischemia, the ghrelin level in serum in IPC+I/R group was increased and significantly higher than that in CON group and I/R group (p<0.001). The high level continued to 72 h after lethal ischemia (p<0.001). In I/R group, the ghrelin level in serum was constant, and not significantly different compared with the CON group. After administration of ghrelin, the numbers of surviving neurons in the CON+N group, I/R+N group and I/R+G group were 251.3±8.7, 28.5±7.3 and 179.3±11.5/mm respectively. The number of surviving neurons in I/R+G group was greater than that in I/R+N group (p<0.001) and less than that in CON+N group (p<0.001). The expression of Bcl-2 in the neurons of hippocampal CA1 region in CON+N group, I/R+N group and I/R+G group was not obvious. RT-PCR showed a signignificantly stronger expression of UCP2 mRNA in I/R+G group than in CON+N group and I/R+N group (p<0.001). CONCLUSION: Ischemia preconditioning may promote the release of ghrelin, which may upregulate the expression of UCP2 in the neurons of the hippcampal CA1 region and alleviate the ischemia/reperfution injury.

 

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