Mechanically ventilated patients suffering critical illness are at high risk of developing neurocognitive impairments. Angiotensin type 2 receptor (AGTR2) has been demonstrated to be anti-inflammatory and neuroprotective. The present study thus aimed to investigate whether AGTR2 can alleviate cerebral dysfunction in mice subjected to cochallenge with lipopolysaccharide (LPS) and mechanical ventilation (MV), and to reveal the underlying mechanism. We utilized a mice model that received a single injection of LPS (1 mg/kg, intraperitoneally) followed 2 h later by MV (10 ml/kg, lasting for 2 h). Pretreatment with the AGTR2 pharmacological agonist C21 (0.03, 0.3, and 3 mg/kg, intraperitoneally, once daily, lasting for 10 days). Locomotor activity and behavioral deficits were evaluated 24 h post-MV by open-field and fear-condition tests. Brain hippocampus and prefrontal cortex tissues were collected for immunofluorescence staining and western blotting to evaluate the resulting impacts on microglia, including morphological traits, functional markers, synaptic engulfment, superoxide production, and signaling molecules. Compared with vehicle-control, pre-administrated C21 reduced the branch endpoints and length of microglia processes in a dose-dependent manner in mice subjected to LPS/MV. The neuroprotective effect of AGTR2 was behaviorally confirmed by the improvement of memory decline in LPS/MV-treated mice following C21 pretreatment. In addition to morphological alterations, C21 reduced microglial functional markers and reduced microglial-dendrite contact and microglial engulfment of synaptic protein markers. In terms of the underlying molecular mechanism, AGTR2 stimulation by C21 leads to activation of protein phosphatase 2A, which subsequently mitigates microglial PKCδ and NF-κB activation, and inhibites NOX2-derived ROS production. The AGTR2 agonist C21 alleviates behavioral deficits in those mice subjected to LPS/MV, via mechanisms that involve reactive microglia and abnormal synaptic plasticity in NOX2-derived ROS and the PKCδ-NFκB pathway.

Graphical Abstract

中文翻译：

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2 型血管紧张素受体药理学激动剂通过减少神经炎症和小胶质细胞吞噬树突棘来缓解神经认知缺陷

患有危重疾病的机械通气患者出现神经认知障碍的风险很高。2 型血管紧张素受体 (AGTR2) 已被证明具有抗炎和神经保护作用。因此，本研究旨在探讨 AGTR2 是否可以减轻脂多糖（LPS）和机械通气（MV）联合攻击小鼠的脑功能障碍，并揭示其潜在机制。我们使用的小鼠模型接受单次 LPS 注射（1 mg/kg，腹腔注射），2 小时后注射 MV（10 ml/kg，持续 2 小时）。用AGTR2药理学激动剂C21（0.03、0.3和3 mg/kg，腹腔注射，每日一次，持续10天）进行预处理。MV 后 24 小时通过旷场和恐惧条件测试评估运动活动和行为缺陷。收集大脑海马和前额皮质组织进行免疫荧光染色和蛋白质印迹，以评估对小胶质细胞的影响，包括形态特征、功能标记、突触吞噬、超氧化物产生和信号分子。与载体对照相比，在接受 LPS/MV 的小鼠中，预施用 C21 以剂量依赖性方式减少了小胶质细胞突起的分支端点和长度。AGTR2 的神经保护作用通过 C21 预处理后 LPS/MV 治疗小鼠记忆力下降的改善得到了行为证实。除了形态学改变之外，C21 还减少了小胶质细胞功能标记，并减少了小胶质细胞-树突接触和小胶质细胞对突触蛋白标记的吞噬。就潜在分子机制而言，C21 刺激 AGTR2 会导致蛋白磷酸酶 2A 激活，从而减轻小胶质细胞 PKCδ 和 NF-κB 的激活，并抑制 NOX2 衍生的 ROS 产生。AGTR2 激动剂 C21 通过涉及 NOX2 衍生 ROS 和 PKCδ-NFκB 通路中的反应性小胶质细胞和异常突触可塑性的机制，减轻接受 LPS/MV 的小鼠的行为缺陷。

图形概要