GPR30激活后下调P53-PUMA信号抑制全脑缺血诱导的海马神经元线粒体凋亡途径

GPR30 activation inhibits mitochondrial apoptosis via down-regulating P53-PUMB signaling in the hippocampal CA1 region following global cerebral ischemia

目的采用GPR30特异激动剂、拮抗剂作为工具药,已证实GPR30激活具有抗神经炎症和抗凋亡作用。但具体机制尚不清楚。文章旨在研究揭示G蛋白耦联雌激素受体30(GPR30)激活对脑缺血后海马CA1区神经元凋亡的作用并进一步阐明其对P53-PUMA信号的影响。方法采用大鼠四动脉结扎全脑缺血模型,选择性诱导海马CA1区神经元延迟性死亡。雌性SD大鼠随机分为:假手术(Sham)组,缺血/再灌注(Ischemia/Reperfusion,I/R)1d、3d、7d组,GPR30特异激动剂G1处理组(I/R 1d+G1,I/R 3d+G1),P53特异抑制剂Pifithrin-α(Pifi-α)处理组(Pifi-α,I/R 3d+Pifi-α,I/R 7d+Pifi-α),GPR30特异拮抗剂G36处理组(I/R 7d+Pifi-α+G36)。于大鼠切除双侧卵巢同时皮下埋置G1或G36微量释放泵,至实验结束;缺血前侧脑室注射P53抑制剂Pifi-α。Western Blot、免疫组化检测蛋白水平;TUNEL法检测海马CA1区神经元凋亡、NeuN染色观察神经元生存情况。结果激光扫描共聚焦显微镜下观察结果显示,I/R 7d组海马CA1区NeuN阳性染色细胞数量(7.500±2.320)较假手术组(61.000±2.671)减少(P<0.01),但TUNEL阳性细胞数量(73.667±5.296)增多较假手术组(3.667±1.174),差异有统计学意义(P<0.01)。与I/R 7d组相比,Pifi-α处理组海马CA1区NeuN阳性细胞数(58.143±4.783)增加(P<0.01),而TUNEL阳性细胞数(23.571±3.545)减少(P<0.01)。与I/R 7d+Pifi-α组相比,I/R 7d+Pifi-α+G36组NeuN阳性细胞数量(46.143±4.426)减少(P<0.01),而TUNEL阳性细胞数(46.143±3.985)增加(P<0.01)。Western blot结果显示,与假手术组相比,GCI后1d、3d海马CA1区P53磷酸化水平均升高(P<0.05)。与缺血/再灌注相应时间点相比,用G1激活GPR30受体降低了缺血/再灌注诱导的P53磷酸化水平(P<0.01)。免疫组化结果显示,与假手术组相比,缺血再灌注3d组海马CA1区p-P53免疫荧光强度增强,且与DAPI荧光共定位;而G1处理组类似于假手术组,显示海马CA1区p-P53免疫荧光强度较I/R 3d组降低。Western blot分析结果显示,与假手术组相比较,I/R 3d组海马CA1区PUMA水平明显升高,Bcl2水平降低(P<0.01);与I/R 3d组比较,I/R 3d+Pifi-α组PUMA蛋白降低(P<0.01),而Bcl2水平升高(P<0.01)。结论激活GPR30具有显著的神经保护作用,其作用机制与下调P53-PUMA信号通路、抑制线粒体凋亡密切相关。

Objective This study was to reveal the anti-apoptotic role of G protein-coupled estrogen receptor(GPR30)activation and further to elucidate the effect on P53-PUMA signaling in the hippocampal CA1 region following global cerebral ischemia(GCI).Methods GCI was induced by rat four vascular occlusion that selectively led to delayed neuronal death of the hippocampal CA1 region.The female SD rats were randomly divided into the following groups:Sham group,ischemia/reperfusion group(I/R 1d,I/R 3d,I/R 7d),GPR30 agonist G1-treatment group(I/R 1d+G1,I/R 3d+G1),P53 inhibitor Pifithrin-α(Pifi-α)-treated group(Pifi-α,I/R 3d+Pifi-α,I/R 7d+Pifi-α),and GPR30 antagonist G36-treated group(I/R 7d+Pifi-α+G36).G1 or G36 was administrated using a mini-pump planted subcutaneously beginning at ovariectomy surgery in female rats until the end of the experiments.Pifi-αwas administrated before ischemia via intracerebroventricular injection.Western blot and immunohistochemistry were used to detect protein expression.TUNEL was used to investigate neuronal apoptosis.And NeuN staining was used to observe neuronal survival in the hippocampal CA1 region.Results Compared to Sham group,I/R 7d resulted in a decrease in the number of NeuN-positive cells(reflecting survival neurons)in the hippocampal CA1 region,and Pifi-αtreatment reversed the decrease(P<0.05).By contrast,I/R 7d enhanced the number of TUNEL-positive cells(reflecting apoptosis cells),compared to the Sham group,whereas Pifi-αtreatment decreased TUNEL-positive cells induced by GCI(P<0.05).G36-administration almost abolished the neuroprotective role caused by Pifi-α,showing an attenuation of NeuN+cells,and an enhancement of TUNEL+cells in the hippocampal CA1 region(P<0.05).Compared to the Sham group,I/R 1d and I/R 3d enhanced P53 phosphorylation(activation)and PUMA protein level(P<0.05)with a decrease of Bcl2 protein level in the hippocampus,while G1-treatment reversed the changes induced by GCI(P<0.05).The protein level of P53 had no difference in all groups.Furthermore,Pifi-αadministration reduced PUMA protein level and elevated anti-apoptotic protein Bcl2,compared to I/R 3d of reperfusion after GCI(P<0.05).Conclusion GPR30 activation exerts neuroprotective role via P53-PUMA signaling pathway and mitigates mitochondria-dependent apoptosis in the hippocampal CA1 region following GCI.