小胶质细胞TLR4/MyD88信号通路在氢气抗肠缺血再灌注脑损伤中的作用及机制

We have confirmed that intestinal ischemia/reperfusion (I/R) can induce brain injury via enhancing microglial activation. TLR4 plays a crucial role in microglial activation, and TLR4/MyD88 signaling pathway is involved in organ damages. Previous studies have shown that hydrogen has neuroprotection with anti-oxidant and anti-inflammatory, but the exact mechanisms are unclear.Our preliminary experiments showed that TLR4 expressed in activated microglia after intestinal I/R; hydrogen could significantly reduce the activation of microglia and the expression of TLR4. Therefore, above results strongly suggest that the protective effect of hydrogen may be related to its regulation on microglial activity and TLR4/MyD88 signaling pathway. Accordingly, on the basis of previous studies, the project combining with cell model will be performed to further explore the effect of hydrogen on TLR4/MyD88 signaling pathway and its downstream molecules, such as NF -kappa B, TNF-α, with immunofluorescence staining, RNA interference, ELISA, TUNEL staining techniques. The aim of the project is to obtain reliable evidence on neuroprotection of hydrogen via microglial TLR4/MyD88 signaling pathway after intestinal I/R. The project is expected to clarify the mechanisms of intestinal I/R-induced brain injury and the neuroprotection of hydrogen. It can provide adequate scientific basis for new drug development and clinical application of hydrogen.

我们已证实肠缺血/再灌注(I/R)能通过增强脑中小胶质细胞活性而损伤脑。TLR4对小胶质细胞的活化至关重要，TLR4/MyD88信号通路参与了器官损伤。研究表明氢气具有脑保护作用，与抗氧化及抗炎等有关，但具体机制不清。我们预实验显示：TLR4在肠I/R脑中活化的小胶质细胞上表达；氢气可明显降低小胶质细胞活性和TLR4表达。上述结果强烈提示氢气脑保护作用可能与其调控小胶质细胞活性及TLR4/MyD88信号通路有关。据此，本项目拟在前期研究基础上，结合细胞模型，应用免疫荧光染色、RNA干扰、ELISA、TUNEL染色等技术，进一步观察氢气对TLR4/MyD88信号通路及其下游分子NF-κB、TNF-α等的影响，旨在获得小胶质细胞TLR4/MyD88信号通路在氢气抗肠I/R脑损伤中的可靠证据。本项目有望阐明肠I/R所致脑损伤及氢气脑保护的新机制，为氢气的新药开发及临床应用提供充分的科学依据。

氢气（H2）是一种无色、无味的气体，具有抗炎、抗氧化及抗凋亡活性。而肠缺血/再灌注(I/R)大鼠脑内有炎症、氧化应激反应及凋亡发生，提示抗炎药物和抗氧化剂有可能成为治疗肠I/R所致器官损伤的药物。本项目重点研究氢气在肠I/R所致脑损伤中的作用及其对小胶质细胞活性及TLR4/MyD88 信号通路的调控。利用夹闭肠系膜上动脉90min方法建立大鼠肠I/R模型，观察氢气对肠I/R大鼠生存率、运动和认知功能的影响。此外，利用H&E染色、免疫组织化学和western blot等方法检测H2对肠I/R大鼠脑损伤、小胶质细胞标志物（Iba 1）表达、细胞凋亡以及TLR4/MyD88信号通路的影响。结果表明不同浓度H2（2%及4%）均能改善认知功能，减轻脑损伤，提高生存率。同时证实肠I/R大鼠血NSE及S100β含量、脑中小胶质细胞活化、NF-κB、TLR4及MyD88表达量增加。免疫荧光双标结果提示活化的小胶质细胞可能是TLR4及MyD88的主要来源。H2能抑制脑中小胶质细胞活化、TLR4、MyD88及NF-κB表达。ELISA结果发现肠I/R能诱导脑组织炎性因子IL-1β、IL-6及TNF-α含量增加，同时也表明氢气能抑制脑组织炎性因子产生。进一步研究证实脑组织中ROS和MDA含量增加，SOD活性下降， H2可通过抑制氧自由基产生，发挥抗氧化损伤作用。此外，H2降低促凋亡蛋白caspase-3表达量，抑制细胞凋亡, 从而发挥预防和治疗肠I/R所致脑损伤的作用。. 利用小胶质细胞和神经元共培养体系，证实了肠I/R大鼠血清通过激活小胶质细胞，从而对神经元造成损伤。采用RNA 干扰、western blot、ELISA及TUNEL等方法探讨H2对神经细胞的保护作用。细胞免疫染色及细胞活力分析结果发现H2能抑制血清介导的小胶质细胞激活诱发的神经元损伤。此外，H2通过抑制小胶质细胞TLR4/NF-κB信号通路的激活，进而减少下游炎症因子TNF-α、IL-1β和IL-6的产生。进一步实验也发现H2能减少细胞外ROS的产生，抑制氧自由基的生成及神经元的凋亡。这些结果表明H2可能是通过调节小胶质细胞活性，而抑制炎症反应及氧化应激所致的神经元损伤。本研究对于阐明小胶质细胞介导的炎症反应和氧化损伤在肠I/R 脑损伤中具有重要意义，而且为H2的新药开发及临床应用提供了理论依据。