摘要目的:评价TANK结合激酶1(TBK1)/受体相互作用蛋白激酶1(RIPK1)信号通路在老龄小鼠术后认知功能障碍(POCD)中的作用。方法:SPF级健康雄性C57BL/6小鼠50只，18月龄，体质量20~25 g，采用随机数字表法分为5组( n＝10)：对照组(C组)、POCD组、二甲基亚砜组(DMSO组)、GSK组和GSK＋Nec-1组。采用左侧胫骨骨折闭合复位内固定术制备小鼠POCD模型，术前30 min通过脑立体定位海马CA1区注射二甲基亚砜、TBK1抑制剂GSK8612和RIPK1抑制剂Nec-1(0.5 μl/侧)。于术前及术后3 d时采用场景条件性恐惧实验测定小鼠认知功能，随后麻醉小鼠处死取海马，采用蛋白质印迹法检测TBK1、RIPK1、IL-1β、TNF-α、激活蛋白1(AP-1)和巢蛋白的表达；采用实时荧光定量聚合酶链反应法检测Bcl-2、Bax和caspase-3 mRNA的表达；免疫荧光染色法观察TBK1与RIPK1分子结合情况及海马DG区神经干细胞增殖水平。 结果:与C组比较，POCD组术后3 d时僵立时间百分比降低，海马Bax mRNA、caspase-3 mRNA、RIPK1、IL-1β、TNF-α和AP-1表达上调，TBK1、Bcl-2 mRNA和巢蛋白表达下调，DG区神经干细胞增殖水平降低( P<0.05或0.01)；与POCD组比较，GSK组小鼠术后3 d时僵立时间百分比降低，海马Bax mRNA、caspase-3 mRNA、IL-1β、TNF-α和AP-1表达上调，Bcl-2 mRNA和巢蛋白表达下调，DG区神经干细胞增殖水平降低( P<0.05或0.01)；与GSK组比较，GSK＋Nec-1组小鼠术后3 d时僵立时间百分比升高，海马Bax mRNA、caspase-3 mRNA、IL-1β、TNF-α和AP-1表达下调，Bcl-2 mRNA、巢蛋白及DG区神经干细胞增殖水平上调( P<0.05或0.01)。 结论:TBK1/RIPK1信号通路参与了老龄小鼠POCD发生发展的过程。

abstractsObjective:To evaluate the role of the TANK-binding kinase 1 (TBK1)/receptor-interacting protein kinase 1 (RIPK1) signaling pathway in postoperative cognitive dysfunction (POCD) in aged mice.Methods:Fifty SPF healthy male C57BL/6 mice, aged 18 months, weighing 20-25 g, were divided into 5 groups ( n=10 each) using a table of random numbers: control group (group C), POCD group, dimethyl sulfoxide group, GSK group and GSK+ Nec-1 group. A mouse model of POCD was established by the closed reduction internal fixation of the left tibial fracture in anesthetized animals. Dimethyl sulfoxide, TBK1 inhibitor GSK8612 and RIPK1 inhibitor Nec-1 (0.5 μl/side) were stereotactically injected into the hippocampal CA1 region at 30 min before operation. Cognitive function was assessed using the contextual fear conditioning test before operation and at 3 days after operation. The mice were then anesthetized and sacrificed, and the hippocampal tissues were obtained for determination of the expression of TBK1, RIPK1, interleukin-lbeta (IL-1β), tumor necrosis factor-alpha (TNF-α), activator protein 1 (AP-1) and Nestin (by Western blot), the expression of Bcl-2, Bax and caspase-3 mRNA (by fluorescent quantitative real-time polymerase chain reaction) and for examination of TBK1/RIPK1 molecular interactions and neural stem cell proliferation in the hippocampal dentate gyrus (DG) region (by immunofluorescent staining). Results:Compared with C group, the percentage of freezing time was significantly decreased at 3 days after operation, the expression of Bax mRNA, caspase-3 mRNA, RIPK1, IL-1β, TNF-α and AP-1 was up-regulated, the expression of TBK1, Bcl-2 mRNA and Nestin was down-regulated, and the proliferation of neural stem cells in the hippocampal DG region was decreased in POCD group ( P<0.05 or 0.01). Compared with POCD group, the percentage of freezing time was significantly decreased at 3 days after operation, the expression of Bax mRNA, caspase-3 mRNA, IL-1β, TNF-α and AP-1 was up-regulated, the expression of TBK1, Bcl-2 mRNA and Nestin was down-regulated, and the proliferation of neural stem cells in the hippocampal DG region was decreased in GSK group ( P<0.05 or 0.01). Compared with GSK group, the percentage of freezing time was significantly increased at 3 days after operation, the expression of Bax mRNA, caspase-3 mRNA, IL-1β, TNF-α and AP-1 was down-regulated, the expression of TBK1, Bcl-2 mRNA and Nestin was up-regulated, and the proliferation of neural stem cells in the hippocampal DG region was increased in GSK+ Nec-1 group ( P<0.05 or 0.01). Conclusions:The TBK1/RIPK1 signaling pathway is involved in the pathogenesis of POCD in aged mice.