BACKGROUND G-protein-coupled estrogen receptor (GPER/GPR30) is a novel membrane-associated estrogen receptor that can induce rapid kinase signaling in various cells. Activation of GPER can prevent hippocampal neuronal cell death following transient global cerebral ischemia (GCI), although the mechanisms remain unclear. In the current study, we sought to address whether GPER activation exerts potent anti-inflammatory effects in the rat hippocampus after GCI as a potential mechanism to limit neuronal cell death. METHODS GCI was induced by four-vessel occlusion in ovariectomized female SD rats. Specific agonist G1 or antagonist G36 of GPER was administrated using minipump, and antisense oligonucleotide (AS) of interleukin-1β receptor antagonist (IL1RA) was administrated using brain infusion kit. Protein expression of IL1RA, NF-κB-P65, phosphorylation of CREB (p-CREB), Bcl2, cleaved caspase 3, and microglial markers Iba1, CD11b, as well as inflammasome components NLRP3, ASC, cleaved caspase 1, and Cle-IL1β in the hippocampal CA1 region were investigated by immunofluorescent staining and Western blot analysis. The Duolink II in situ proximity ligation assay (PLA) was performed to detect the interaction between NLRP3 and ASC. Immunofluorescent staining for NeuN and TUNEL analysis were used to analyze neuronal survival and apoptosis, respectively. We performed Barnes maze and Novel object tests to compare the cognitive function of the rats. RESULTS The results showed that G1 attenuated GCI-induced elevation of Iba1 and CD11b in the hippocampal CA1 region at 14 days of reperfusion, and this effect was blocked by G36. G1 treatment also markedly decreased expression of the NLRP3-ASC-caspase 1 inflammasome and IL1β activation, as well as downstream NF-κB signaling, the effects reversed by G36 administration. Intriguingly, G1 caused a robust elevation in neurons of a well-known endogenous anti-inflammatory factor IL1RA, which was reversed by G36 treatment. G1 also enhanced p-CREB level in the hippocampus, a transcription factor known to enhance expression of IL1RA. Finally, in vivo IL1RA-AS abolished the anti-inflammatory, neuroprotective, and anti-apoptotic effects of G1 after GCI and reversed the cognitive-enhancing effects of G1 at 14 days after GCI. CONCLUSIONS Taken together, the current results suggest that GPER preserves cognitive function following GCI in part by exerting anti-inflammatory effects and enhancing the defense mechanism of neurons by upregulating IL1RA.

中文翻译：

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G蛋白偶联的雌激素受体激活在整体性脑缺血后上调海马中的IL-1受体拮抗剂：对神经元自卫的影响。

背景技术G蛋白偶联雌激素受体（GPER / GPR30）是一种新型的膜相关雌激素受体，可以在各种细胞中诱导快速激酶信号传导。GPER的激活可预防短暂性全脑缺血（GCI）后海马神经元细胞死亡，尽管其机制尚不清楚。在当前的研究中，我们试图解决GCI激活后GPER激活是否在大鼠海马中发挥有效的抗炎作用，作为限制神经元细胞死亡的潜在机制。方法通过四管闭塞法在去卵巢雌性SD大鼠中诱导GCI。使用微型泵施用GPER的特定激动剂G1或拮抗剂G36，并使用脑输液试剂盒施用白介素-1β受体拮抗剂（IL1RA）的反义寡核苷酸（AS）。IL1RA，NF-κB-P65，通过免疫荧光染色和海马CA1区研究了CREB（p-CREB），Bcl2，裂解的caspase 3和微胶质标记Iba1，CD11b以及炎症小体组分NLRP3，ASC，裂解的caspase 1和Cle-IL1β的磷酸化。蛋白质印迹分析。进行Duolink II原位邻近连接测定（PLA），以检测NLRP3与ASC之间的相互作用。用NeuN和TUNEL分析的免疫荧光染色分别分析神经元存活和凋亡。我们进行了Barnes迷宫和Novel对象测试，以比较大鼠的认知功能。结果结果显示，再灌注14天后，G1减弱了GCI诱导的海马CA1区Iba1和CD11b升高，这种作用被G36阻断。G1处理还显着降低了NLRP3-ASC-caspase 1炎性小体的表达和IL1β激活，以及下游NF-κB信号传导，这种作用被G36给药逆转。有趣的是，G1在神经元中引起了众所周知的内源性抗炎因子IL1RA的强烈升高，而G36处理则逆转了这种情况。G1还增强了海马中的p-CREB水平，这是一种已知增强IL1RA表达的转录因子。最后，体内IL1RA-AS取消了GCI后G1的抗炎，神经保护和抗凋亡作用，并在GCI后14天逆转了G1的认知增强作用。结论综合起来，