锂对小胶质细胞TLR4的影响和防治POCD机制的探讨

Neuroinflammation triggerred by microglia in central nervous system(CNS) plays a key role in postoperative cognitive dysfunction (POCD),but the concrete regulatory mechanism has not yet been clear, needing further study. Toll-like receptor 4（TLR4）is very important in the activation of microglia, which acts as a central regulator of inflammation. And glycogen synthase Kinase-3β (GSK-3β) is also involved in the regulation of inflammation response. Recent study indicated that PI3K/Akt pathway may play a vital role in the regulation of inflammation responses mediated by TLR4 and GSK-3β. Our previous study indicated that prophylactic lithium can alleviate surgery-associated impairment of the spatial memory in aged rats, which is strongly associated with the reduced activities of GSK-3βand the levels of IL-1β in hippocampus through PI3K/Akt/mTORC2 pathway. And our preliminary experiment demonstrated that the expressions of TLR4 mRNA and protein in the BV-2 microglia cells under LPS stimulation were down-regulated by Lithium, meanwhile, the inflammation response was inhibited. These results are not been reported. So in this study ,we intended to observe the changes of TLR4 and PI3K/AKT expression, GSK-3β activity, inflammatory factors in microglia in vitro ; and changes of learing and memory function in the partial hepatectomy model of mouse using gene knockout、siRNA、plasmid transfection and tool drugs, so that to explore whether TLR4 and it mediated signaling pathways play a critical role in the regulation of inflammation reponse and learing and memory function . So our study will provide a new insight for better prevention and treatment of POCD in clinical.

术后认知功能障碍（POCD）与中枢炎症反应有关，小胶质细胞为主要角色，TLR4对小胶质细胞的活化至关重要。我们的工作表明术前给予氯化锂可改善大鼠术后认知功能，抑制海马内GSK-3β活性、降低IL-1β水平；还影响GSK-3β的上游事件。预实验提示锂盐可下调TLR4的表达，抑制小胶质细胞的炎症反应；这一结果未见报道。由此我们提出假说：TLR4是POCD发生中的一个重要环节，锂对GSK-3β上游的PI3K/Akt通路和TLR4的调节作用与其防治POCD的效应有关。本课题拟用基因敲除、RNA干扰、质粒转染及工具药物，分别在体外观察脂多糖刺激下，锂对小胶质细胞TLR4、PI3K/Akt、GSK-3β、炎性细胞因子的表达水平及海马神经元LTP和形态的影响；用基因敲除小鼠在手术模型上观察TLR4在POCD发生机制中的作用，探讨锂的作用环节，以验证我们的假说, 从而为防治POCD提供新策略。

我们利用动物行为学实验、ELISA、PCR和蛋白免疫印迹等技术研究锂对小胶质细胞TLR4的影响和防治POCD的机制。.首先，我们对野生型小鼠进行胫骨骨折手术，观察术后小鼠记忆能力的改变、外周和海马TLR4/MyD88信号通路的变化；并利用TLR4−/−小鼠和MyD88−/−小鼠进一步探讨TLR4对小胶质细胞的调控在小鼠术后学习记忆能力下降中的作用。结果显示：野生型小鼠术后学习记忆能力的下降，外周和海马中的TLR4/MyD88发生活化，海马区小胶质细胞明显增生活化，TLR4信号通路的活化可以促进骨髓源性巨噬细胞进入中枢，促进中枢小胶质细胞的活化，参与调控POCD的发生发展。.其次，我们将老年SD大鼠随机分为对照组、腹腔注射LiCl组、手术组、腹腔注射LiCl后行手术组，观察术后大鼠记忆能力的改变、外周和中枢炎症的改变。结果显示：术前预先给予LiCl可抑制手术导致的中枢炎症和学习记忆能力的下降；且术后术后海马中的TLR2和TLR4表达显著增加，而LiCl可抑制TLR4/ NF-κB的活化。这些结果提示，TLR4信号通路参与LiCl对神经炎症的改善；LiCl可以抑制TLR4信号通路活化，抑制术后中枢炎症和POCD的发生。.再次，我们对小鼠行胫骨骨折手术，检测小鼠术后外周和海马中S100A8和S100A9的表达；术前给予S100A8的抗体，观察其对术后中枢炎症和学习记忆能力改变的影响。另外利用TLR4−/−小鼠和MyD88−/−小鼠进一步探讨S100A8在POCD的作用与TLR4之间的关系。结果提示：小鼠胫骨骨折术后S100A8的表达受到TLR4信号通路的调控，术后升高的S100A8可以促进中枢小胶质细胞的活化，参与调控术后学习记忆能力的下降。.最后，用原代小胶质细胞离体观察验证LiCl对内毒素（LPS）致小胶质细胞活化的保护作用及其与TLR4的关系。结果发现： LiCl可以有效抑制小胶质细胞的活化，且与TLR4和PI3K/AKT信号通路相关。.综上所述，小胶质细胞表面的TLR4受体是POCD发生中发挥重要角色，锂盐除可抑制GSK-3β外，还对GSK-3β上游的PI3K/AKT通路和TLR4发挥调节作用，改善术后中枢炎症和POCD的发生。这一探索性研究有助于为防治POCD提供新思路。