BACKGROUND Inflammasome-mediated neuroinflammation plays an important role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of the TGR5 receptor has been shown to be neuroprotective in a variety of neurological diseases. This study aimed to investigate the effects of the specific synthetic TGR5 agonist, INT-777, in attenuating NLRP3-ASC inflammasome activation and reducing neuroinflammation after SAH. METHODS One hundred and eighty-four male Sprague Dawley rats were used. SAH was induced by the endovascular perforation. INT-777 was administered intranasally at 1 h after SAH induction. To elucidate the signaling pathway involved in the effect of INT-777 on inflammasome activation during EBI, TGR5 knockout CRISPR and PKA inhibitor H89 were administered intracerebroventricularly and intraperitoneally at 48 h and 1 h before SAH. The SAH grade, short- and long-term neurobehavioral assessments, brain water content, western blot, immunofluorescence staining, and Nissl staining were performed. RESULTS The expressions of endogenous TGR5, p-PKA, and NLRP3-ASC inflammasome were increased after SAH. INT-777 administration significantly decreased NLRP3-ASC inflammasome activation in microglia, reduced brain edema and neuroinflammation, leading to improved short-term neurobehavioral functions at 24 h after SAH. The administration of TGR5 CRISPR or PKA inhibitor (H89) abolished the anti-inflammation effects of INT-777, on NLRP3-ASC inflammasome, pro-inflammatory cytokines (IL-6, IL-1β, and TNF-a), and neutrophil infiltration at 24 h after SAH. Moreover, early administration of INT-777 attenuated neuronal degeneration in hippocampus on 28 d after SAH. CONCLUSIONS INT-777 attenuated NLRP3-ASC inflammasome-dependent neuroinflammation in the EBI after SAH, partially via TGR5/cAMP/PKA signaling pathway. Early administration of INT-777 may serve as a potential therapeutic strategy for EBI management in the setting of SAH.

中文翻译：

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INT-777 通过 TGR5/cAMP/PKA 信号通路减轻大鼠蛛网膜下腔出血后 NLRP3-ASC 炎症小体介导的神经炎症

背景炎性体介导的神经炎症在蛛网膜下腔出血（SAH）后的早期脑损伤（EBI）的发病机制中起重要作用。TGR5受体的激活已被证明在多种神经疾病中具有神经保护作用。本研究旨在研究特异性合成 TGR5 激动剂 INT-777 在减轻 SAH 后 NLRP3-ASC 炎症小体活化和减少神经炎症方面的作用。方法使用184只雄性Sprague Dawley大鼠。SAH是由血管内穿孔引起的。在 SAH 诱导后 1 小时鼻内给予 INT-777。为了阐明 INT-777 在 EBI 期间对炎症小体激活的影响所涉及的信号通路，TGR5 敲除 CRISPR 和 PKA 抑制剂 H89 在 SAH 前 48 小时和 1 小时进行脑室内和腹膜内给药。进行了 SAH 分级、短期和长期神经行为评估、脑水含量、蛋白质印迹、免疫荧光染色和 Nissl 染色。结果SAH后内源性TGR5、p-PKA和NLRP3-ASC炎性体的表达增加。INT-777 给药显着降低了小胶质细胞中 NLRP3-ASC 炎症小体的激活，减少了脑水肿和神经炎症，导致 SAH 后 24 小时的短期神经行为功能得到改善。TGR5 CRISPR 或 PKA 抑制剂 (H89) 的施用消除了 INT-777 对 NLRP3-ASC 炎性体、促炎细胞因子（IL-6、IL-1β 和 TNF-a）和中性粒细胞浸润的抗炎作用在 SAH 后 24 小时。此外，在 SAH 后 28 天，早期给予 INT-777 可减轻海马神经元变性。结论 INT-777 减轻 SAH 后 EBI 中的 NLRP3-ASC 炎症小体依赖性神经炎症，部分通过 TGR5/cAMP/PKA 信号通路。INT-777 的早期给药可作为 SAH 环境中 EBI 管理的潜在治疗策略。