基于PI3K/AKT/Nrf2-NLRP3炎性小体途径研究右美托咪定干预慢性应激致海马损伤的作用机制

Chronic stress can cause adverse effects on the multi-system organ function, studies have shown that it has a short-term or permanent damage on the nervous system and greater damage to the cognitive abilities of animals, even affect across generations. Our previous study found that dexmedetomidine (DEX) can significantly reduce inflammatory to alleviate the hippocampal injury induced by chronic stress. Therefore, after established the model of chronic stress, using Western Blotting, transmission electron microscopy, confocal laser scanning microscope, etc to observe the changes of behavior, hormone levels, hippocampal histology and proteins which related inflammatory and anti-inflammatory pathway after DEX pretreatment in rats，to explore the protective effect of DEX on hippocampus injury induced by chronic stress. In vitro, using CRISPR/Cas9 genome editing technologies to knockout NLRP3, Nrf2, and its downstream target genes HO-1 and NQO1 in the model of dexamethasone induced hippocampal neuronal injury, clarifying the mechanism of DEX by activating the PI3K/AKT/Nrf2 signaling pathway and with Nrf2 as the key site inhibit the NLRP3 inflammatory. It provide new ideas for prevention hippocampal injury caused by chronic stress, and then to further expands the clinical use of DEX, in the meantime, provides theoretical basis for revealing the molecular mechanism of its pharmacodynamics effects.

慢性应激可对动物机体多系统器官功能造成不良的影响，相关研究表明其可对动物神经系统产生短期或永久的损伤，对动物认知能力产生较大的损害，甚至可跨代产生影响。前期研究发现在慢性应激致大鼠海马损伤模型中DEX可明显减轻炎症反应从而起到有效的保护作用。因此课题组建立大鼠慢性应激模型，应用免疫印迹、透射电镜、激光共聚焦等技术，通过对DEX干预后大鼠的行为学、激素水平、海马形态、炎症及抗炎通路相关蛋白等的检测，探究DEX对慢性应激致海马损伤的保护作用；并构建地塞米松诱导海马神经细胞损伤模型，利用CRISPR/Cas9基因编辑技术敲除NLRP3和Nrf2及其下游靶基因HO-1和NQO1，阐明DEX通过激活PI3K/AKT/Nrf2信号通路，以Nrf2为关键位点抑制NLRP3炎性小体的作用机制，为防治慢性应激引起的海马损伤提供新思路，进一步拓展DEX的临床用途，为揭示其药效作用的分子机制提供理论依据。

慢性应激可对动物机体多系统器官功能造成不良的影响，相关研究表明其可对动物神经系统产生短期或永久的损伤，对动物认知能力产生较大的损害，甚至可跨代产生影响。课题组前期研究发现在慢性应激致大鼠海马损伤模型中DEX可明显减轻炎症反应从而起到有效的保护作用，但其确切的保护作用机制尚不明确。本项目的主要研究内容：1.建立大鼠慢性应激模型，进行DEX干预。从行为学、激素水平、海马组织细胞形态及炎症因子表达等方面，验证DEX对慢性应激致海马损伤的保护作用。2.重点从抑制NLRP3炎性小体表达的角度，探究DEX对慢性应激致海马损伤的保护作用机制；通过体外试验敲除NLRP3基因（CRISPR/Cas9基因编辑技术）及抑制抗炎通路关键靶基因（PI3K抑制剂），明确NLRP3炎性小体在应激致海马损伤中的作用及DEX抑制NLRP3炎性小体表达的相关作用机制及关键靶位。本研究阐明DEX通过激活PI3K/Akt/Nrf2信号通路，减轻海马小胶质细胞过度激活，抑制NLRP3炎症小体过度表达，阻断炎症因子的趋化，恢复海马小胶质细胞稳态，从而对海马损伤起到保护作用。研究结果将为防治慢性应激引起的海马损伤提供新思路，进一步拓展DEX的临床用途，为揭示其药效作用的分子机制提供理论依据。