Department of Intensive Care Unit, HuaShan Hospital, Fudan University, Shanghai 200040, China; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai 200032, China.
Department of Intensive Care Unit, HuaShan Hospital, Fudan University, Shanghai 200040, China.
Exp Neurol. 2021 Jan;335:113504. doi: 10.1016/j.expneurol.2020.113504. Epub 2020 Oct 13.
Survivors of sepsis must often endure significant cognitive and behavioral impairments after discharge, but research on the relevant mechanisms and interventions remains lacking. TGR5, a member of the class A GPCR family, plays an important role in many physiological processes, and recent studies have shown that agonists of TGR5 show neuroprotective effects in a variety of neurological disorders. To date, no studies have assessed the effects of TGR5 on neuroinflammatory, cognitive, or behavioral changes in sepsis models.
A total of 267 eight-week-old male Sprague-Dawley rats were used in this study. Sepsis was induced via cecal ligation and puncture (CLP). All animals received volume resuscitation. The rats were given TGR5 CRISPR oligonucleotide intracerebroventricularly 48 h before CLP surgery. INT-777 was administered intranasally 1 h after CLP, and the cAMP inhibitor, SQ22536, was administered intracerebroventricularly 1 h after CLP. Survival rate, bodyweight change, and clinical scores were assessed, and neurobehavioral tests, western blot, and immunofluorescence staining were performed. The cognitive function of rats was measured using the Morris water maze during 15-20 days after CLP.
The expression of TGR5 in the rat hippocampus was upregulated, and peaked at 3 days after CLP. The survival rate of rats after CLP was less than 50%, and the growth rate, in terms of weight, was significantly decreased. While INT-777 treatment did not improve these changes, the treatment did reduce the clinical scores of rats at 24 h after CLP. On day 15 and later, the surviving mice completed a series of behavioral tests. CLP rats showed spatial and memory deficits and anxiety-like behaviors, but INT-777 treatment significantly improved these effects. Mechanistically, immunofluorescence analysis showed that INT-777 treatment reduced the number of microglia in the hippocampus, neutrophilic infiltration, and the expression of inflammatory factors after CLP in rats. Moreover, INT-777 treatment significantly increased the expression of TGR5, cAMP, p-PKA, and p-CREB, but downregulated the expression of IL-1β, IL-6, and TNF-α. CRISPR-mediated TGR5 knockdown and SQ22536 treatment abolished the neuroprotective effects of TGR5 activation after CLP.
This study demonstrates that INT-777 treatment reduced neuroinflammation and microglial cell activation, but improved cognitive impairment in the experimental sepsis rats. TGR5 has translational potential as a therapeutic target to improve neurological outcomes in sepsis survivors.
脓毒症幸存者在出院后常常会经历显著的认知和行为障碍，但相关机制和干预措施的研究仍很缺乏。TGR5 是 A 类 GPCR 家族的成员，在许多生理过程中发挥着重要作用，最近的研究表明，TGR5 的激动剂在多种神经疾病中具有神经保护作用。迄今为止，尚无研究评估 TGR5 对脓毒症模型中神经炎症、认知或行为变化的影响。
本研究共使用了 267 只 8 周龄雄性 Sprague-Dawley 大鼠。通过盲肠结扎和穿孔（CLP）诱导脓毒症。所有动物均接受容量复苏。CLP 手术前 48 小时，通过脑室内给予 TGR5 CRISPR 寡核苷酸。CLP 后 1 小时，经鼻给予 INT-777，CLP 后 1 小时，经脑室内给予 cAMP 抑制剂 SQ22536。评估生存率、体重变化和临床评分，并进行神经行为测试、western blot 和免疫荧光染色。CLP 后 15-20 天，通过 Morris 水迷宫测量大鼠的认知功能。
TGR5 在大鼠海马中的表达上调，在 CLP 后 3 天达到峰值。CLP 后大鼠的生存率低于 50%，体重增长率明显下降。虽然 INT-777 治疗不能改善这些变化，但确实降低了 CLP 后 24 小时大鼠的临床评分。在第 15 天及以后，幸存的小鼠完成了一系列行为测试。CLP 大鼠表现出空间和记忆缺陷以及焦虑样行为，但 INT-777 治疗显著改善了这些效应。机制上，免疫荧光分析表明，INT-777 治疗减少了 CLP 后大鼠海马中小胶质细胞的数量、中性粒细胞浸润和炎症因子的表达。此外，INT-777 治疗显著增加了 TGR5、cAMP、p-PKA 和 p-CREB 的表达，而降低了 IL-1β、IL-6 和 TNF-α的表达。CRISPR 介导的 TGR5 敲低和 SQ22536 治疗消除了 TGR5 激活对 CLP 后神经保护作用。
本研究表明，INT-777 治疗减轻了实验性脓毒症大鼠的神经炎症和小胶质细胞激活，但改善了认知障碍。TGR5 作为一种治疗靶点，具有改善脓毒症幸存者神经功能结局的转化潜力。
