星形胶质细胞TRPM2通道在LPS致脑损伤中的作用及机制研究

Sepsis associated encephalopathy (SAE) is a diffuse cerebral dysfunction resulted by systemic inflammatory response.SAE is the most common form of encephalopathy in critical ill patients with poor prognosis. Astrocytes not only constitute the basic skeleton of the central nervous system, and is involved in the conduction of the central nervous system information and processing, important participant in the functional activity of the central nervous system. Biphasic effect of lipopolysaccharide can lead to the astrocytes activation or apoptosis, and activation of astrocytes on neuronal protection and toxicity. Calcium homeostasis and abnormal calcium signaling may be a key factor in the activation of astrocytes. The type of transient receptor potential M2 (TRPM2) channel is a non-selective calcium channel that is highly expressed in the brain. The project intends to observe hippocampus dentate gyrus neuron apoptosis and activation of astrocytes in the LPS induced brain damage model. We are to detect hippocampal slice synaptic function with patch-clamp, detect TRPM2/AIF/EndoG pathway gene and protein expression, and observe changes in cognitive function. Further, the mechanism of TRPM2/TLR-4/NF-κBp65 pathway in LPS induced brain damage will be demonstrated. Both in vitro and in vivo data point to the role of astrocytes as both major source and target of LPS induced brain damage signaling. In this study, understanding the astroglial inflammatory response occurring in brain tissue may provide new strategies for targeting astrocyte-mediated LPS induced brain damage. Inhibition of cell TRPM2 channel activation may improve the survival rate of astrocytes. Lack of specific agonists or blockers is still very limited understanding of the TRPM2 channel, therefore the introduction of TRPM2-/- mice establish LPS sepsis model to study astrocytes TRPM2 LPS induced brain damage mechanism , can provide the basis for the treatment of septic encephalopathy.

脓毒性脑病是感染后全身炎症反应所致的弥漫性大脑功能障碍和意识改变，是病人早期死亡及中后期认知功能障碍的独立危险因素。但脓毒性脑病的病因和病理机制非常复杂，确切机制尚未完全明确。星形胶质细胞在中枢神经系统信息的传导与处理及损伤的修复发挥重要作用。抑制星形胶质细胞TRPM2通道活性可提高其生存率,推测星形胶质细胞TRPM2通道的诱导表达与星形胶质细胞的凋亡密切相关。本项目拟在建立脓毒性脑病模型基础上，观察野生型和TRPM2基因敲除小鼠海马齿状回神经细胞凋亡及星形胶质细胞活化规律，膜片钳检测海马脑片突触功能，检测TRPM2/AIF/EndoG通路上下游分子表达特点，观察小鼠认知功能改变；并通过离体星形胶质细胞培养，论证TRPM2/TLR-4/NF-κBp65通路在免疫损伤中的作用机制。这将为探索星形胶质细胞TRPM2通道在脓毒性脑病中的作用及发病机制提供理论依据，对脑损伤康复具有重要临床意义。

脓毒性脑病(sepsis-associated encephalopathy, SAE)是感染后全身炎症反应所致的弥漫性大脑功能障碍和意识改变,是多脏器功能不全综合征( multiple organdysfunction syndrome, MODS)的重要组成部分及患者预后不良的独立危险因素。SAE的病因和病理机制非常复杂,以白细胞浸润、神经元细胞变性坏死、星型胶质细胞和小胶质细胞激活为标志的炎症反应起到了重要的作用。其中星形胶质细胞在中枢神经系统损伤后呈现保护或毒性作用的"双相"性及活化的时空性特点和SAE的临床表现有很好的契合性。瞬时感受器电位M2型(Transient Receptor Potential 2 Channels, TRPM2)通道是位于细胞膜上的一种多功能钙离子通透性的非选择性阳离子通道,通过调节胞内钙离子浓度可以调节氧化应激或再灌注等病理损伤中的细胞死亡。TRPM2通道可能参与调节了LPS诱导的星形胶质细胞的活化和凋亡。研究发现脓毒症在小鼠脑内的炎症改变及星形胶质细胞增殖和胶质化的时间规律，TRPM2在LPS致炎小鼠脑内星形胶质细胞增殖和胶质化的调节作用及参与BNIP3/AIF/EndoG介导LPS致炎小鼠海马区神经细胞的凋亡；以及通过星形胶质细胞培养及TRPM2-siRNA转染，进一步研究LPS是否可以诱导星形胶质细胞的适应性表达，以及TRPM2-siRNA转染是否可以有效抑制LPS诱导的体外星形胶质细胞的活化和致炎性细胞因子TNF-α、IL-1β、IL-6的分泌。从而明确星形胶质细胞TRPM2通道与脓毒性脑病的关系和分子机制，期待为防治脓毒症及脓毒性脑病的治疗及远期康复提供理论依据。