脓毒症时VDAC影响单核/巨噬细胞代谢重编程的分子机制及积雪草酸调控作用

Activation of macrophages play an important role in sepsis induced systemic inflammatory response syndrome, but its mechnisms remain to be elucidated. Previous studies have shown that metabolic reprogramming occurs during the activation of macrophages whereas glycolytic enzymes promote the release of proimflammatory factors. The binding of voltage dependent anion channel(VDAC) and hexokinase(HK) can induce metabolic reprogramming, suggesting that it may play the central role in sepsis. Our previous studies have found that asiatic acid(AA) can bind to VDAC directly to maintain the normal mitochondrial function. Since AA was reported to have anti-inflammatory effects, we thus hypothesize that metabolic reprogramming promotes inflammation regulated by VDAC in sepsis, and that AA develop anti-inflammatory effects through preventing the binding of VDAC and HK. In this project, we employ CLP mice and LPS-stimulated macrophages as sepsis models to examine the dynamic state of metabolic reprogramming in inflammation regulated by VDAC and its relative regulatory effects under the intervention of AA. Therefore, we aim to determine the relationship between inflammation and metabolic reprogramming and the regulaiton of VDAC, as well as the novel targets of AA during sepsis. Our findings will enhance the development of new therapeutic strategy for and the treatment of sepsis.

巨噬细胞过度活化是脓毒症时全身免疫反应综合症的重要事件，但其调控机制尚未完全阐明。已有报道巨噬细胞活化时发生了能量代谢重编程,同时糖酵解酶活性的提高会引起促炎性因子的释放。电压依赖性阴离子通道(VDAC)与已糖激酶(HK)的结合会引起代谢重编程，从而有可能在脓毒症中发挥重要作用。我们的前期研究发现积雪草酸(AA)能够与VDAC直接结合，稳定线粒体功能，同时也有人发现AA具有抗炎作用。我们进一步推测脓毒症时VDAC会通过影响能量代谢来促进炎症反应，而AA可以阻止VDAC与HK的结合，从而发挥抗炎作用。本研究采用CLP脓毒症动物模型和LPS刺激的巨噬细胞模型，以AA干预，研究能量代谢重编程对炎症反应的调节作用，尤其是VDAC与HK的结合对糖酵解反应的动态调控，旨在探明脓毒症时炎性反应与能量代谢之间的相互关系以及VDAC在其中的作用，明确AA调节脓毒症的作用对象与机制，为脓毒症的治疗提供新思路。

项目背景：巨噬细胞过度活化是脓毒症及类风湿关节炎等疾病免疫反应综合症的重要事件，但其调控机制尚未完全阐明。已有报道巨噬细胞活化时发生了能量代谢重编程,同时糖酵解酶活性的提高会引起促炎性因子的释放。电压依赖性阴离子通道(VDAC)与已糖激酶(HK)的结合会引起代谢重编程，从而有可能在脓毒症中发挥重要作用。积雪草酸（AA）具有抗炎作用，但其机制尚未阐明。主要研究内容：采用小鼠骨髓巨噬细胞（BMDM），LPS/IFN-γ刺激其活化，实时荧光定量PCR检测糖酵解酶相关基因表达情况；免疫共沉淀法检测VDAC与HK的结合；采用糖酵解抑制剂2-DG或者阻断VDAC与HK结合的抑制剂6-磷酸葡萄糖处理BMDM后，ELISA法检测炎症因子的释放。采用LPS刺激的RAW264.7细胞或者小鼠，ELISA法检测细胞上清或血清中的炎症因子；HE染色检测小鼠组织病理切片，荧光定量PCR法和western blot法检测细胞或组织notch相关分子；Chip法检测Notch与IL-6启动子的结合。收集类风湿关节炎患者外周血单个核细胞，荧光定量PCR法检测糖酵解相关基因表达。重要结果：LPS/IFN-γ刺激巨噬细胞4-24 h 后，Glut1、HK、PKM等糖酵解相关酶基因表达水平上调。同时糖酵解抑制剂2-DG抑制炎症因子IL-1β的生成，但对其他细胞因子如TNF-α和IL-6无影响；LPS/IFN-γ刺激巨噬细胞24 h后，VDAC可以与HK结合，而抑制剂6-磷酸葡萄糖可以抑制炎症因子IL-1β的生成，但对其他细胞因子如TNF-α和IL-6无影响；AA通过抑制Notch信号通路对抗LPS刺激的巨噬细胞和脓毒症小鼠；积雪草酸通过上调Notch信号通路抑制内毒素所致小鼠急性肾损伤作用；类风湿关节炎患者外周血单个核细胞中糖酵解表达上调。科学意义：本研究发现了能量代谢重编程对炎症反应的调节作用，尤其是VDAC与HK的结合对炎性反应的调控，同时发现AA调控Notch信号的抗炎作用新机制。本研究还发现类风湿关节炎患者外周血单个核细胞中糖酵解相关基因高表达。本研究为脓毒症及类风湿关节炎发病机理提供了新思路。