基础医学与临床 ›› 2021, Vol. 41 ›› Issue (3): 398-403.

• 研究论文 • 上一篇    下一篇

大鼠脑缺血-再灌注后G蛋白偶联受体84的表达升高

赵翀翀,郭文婷,蔡宏斌,王欢   

  1. 兰州大学第二医院
  • 收稿日期:2020-02-24 修回日期:2020-06-30 出版日期:2021-03-05 发布日期:2021-03-01
  • 通讯作者: 王欢 E-mail:huanwang806@163.com

Increased expression of G protein-coupled receptor 84 after cerebral ischemia-reperfusion in rats

  • Received:2020-02-24 Revised:2020-06-30 Online:2021-03-05 Published:2021-03-01

摘要: 目的 探讨G蛋白偶联受体84 (GPR84)在大鼠脑缺血/再灌注(I/R)后的表达及其功能。方法 构建大鼠大脑中动脉阻断(MCAO)模型,将大鼠分为假手术组(sham)和脑缺血2 h后再灌注12 h组(I/R 12 h)、24 h组(I/R 24 h)、48 h组(I/R 48 h)、72 h组(I/R 72 h);体外培养大鼠原代皮质神经元,将细胞分成对照组(Con)、氧糖剥夺1 h复氧复糖24 h组(OGD/R 1h)和氧糖剥夺2 h复氧复糖24 h组(OGD/R 2h); GPR84小干扰RNA(siRNA)转染神经元,将细胞分为对照组(Con)、氧糖剥夺1 h复氧复糖24 h组(OGD/R 1 h)、神经元转染干扰对照后氧糖剥夺1 h复氧复糖24 h组(NC+OGD/R 1 h)和神经元转染GPR84 siRNA后氧糖剥夺1 h复氧复糖24 h组(GPR84 siRNA +OGD/R 1 h);Western blot检测GPR84的表达;免疫荧光检测脑组织中GPR84蛋白的定位;DNA 断裂的原位末端标记 (TUNEL)检测神经元的凋亡比例。结果 与假手术组相比,大鼠脑I/R后各时间点GPR84蛋白水平明显升高(P < 0.01)。GPR84主要定位于神经元和小胶质细胞,星形胶质细胞几乎不表达,并且神经元中GPR84的表达在脑I/R后明显提高。与对照组相比,OGD/R 1 h和OGD/R 2 h组神经元中GPR84蛋白的表达均明显提高(P < 0.01),并且GPR84干扰可以明显抑制氧糖剥夺后复氧复糖诱导的GPR84表达上调。与对照组相比,OGD/R组神经元凋亡明显提高(P < 0.01);与NC+OGD/R 1 h 组相比,GPR84 siRNA +OGD/R 1 h组神经元凋亡明显减少。结论 脑I/R诱导的GPR84表达升高在脑缺血性损伤中发挥着重要作用。

关键词: GPR84, 神经元, 脑缺血, 凋亡

Abstract: Objective To determine the expression and function of G protein-coupled receptor 84 (GPR84) in rat cerebral ischemia/reperfusion(I/R). Methods The rat middle cerebral artery occlusion (MCAO) models were performed, and rats were then divided into sham group, cerebral ischemia for 2 h and reperfusion for 12 h group (I/R 12 h), 24 h group (I/R 24 h), 48 h group (I/R 48 h), 72 h group (I/R 72 h); rat primary cortical neurons were isolated and cultured in vitro, neurons were then divided into control group (Con), oxygen-glucose deprivation (OGD) for 1 h and recovery for 24 h group (OGD/R 1 h), OGD for 2 h and recovery for 24 h group (OGD/R 2 h); neurons were transfected with GPR84 small interfering RNA (siRNA), cells were divided into Con group, OGD/R 1 h group, neurons pretreated with negative control and exposed to OGD for 1 h and recovery for 24 h group (NC+OGD/R 1 h), neurons pretreated with GPR84 siRNA and exposed to OGD for 1 h and recovery for 24 h group (GPR84 siRNA+OGD/R 1 h). Western blot was used to determine the expression of GPR84 protein. Immunofluorescence assay was used to analyze the localization of the immunofluorescence signals of GPR84 in cerebral ischemic tissues after MCAO. Terminal dexynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used to detect the percentage of apoptotic neurons. Results Compared with the sham group, the expression of GPR84 protein was significantly up-regulated at each time point after cerebral I/R (P < 0.01). In addition, GPR84 was mainly localized in neurons and microglia, while little GPR84 was found in astrocytes. Neurons in the MCAO group exhibited higher levels of GPR84 expression than those in the sham group. GPR84 knockdown significantly inhibited the increase of GPR84 expression induced by OGD/R. Compared with the control group, neurons in the OGD/R 1 h group exhibited higher apoptotic cells percentage. Compared with the NC+OGD/R 1 h group, the percentage of apoptotic neurons in GPR84 siRNA +OGD/R 1 h group was significantly decreased. Conclusions These results suggest that the elevated GPR84 expression induced by cerebral I/R may play an important role in cerebral ischemia injury.

Key words: GPR84, neuron, cerebral ischemia, apoptosis

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