基于TLR4通路调控NLRP3/Caspase1轴研究中药肝豆汤对高铜诱导神经炎性损伤的保护机制

NLRP3/Caspase1 axis- mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper in brain, which may triggers neuroinflammation and subsequent neurotoxicity through TLR4-mediated NLRP3/Caspase1 axis activation in microglia. Wilson’s disease (WD) is an autosomal recessive inherited disorder of copper (Cu) metabolism, resulting in pathological accumulation of copper in many organs and tissues, predominantly in the liver and brain. Copper accumulation induced neuroinflammation and neurodegeneration in striatum and hippocampus of rodent models for WD. We reported that the TCM GanDou decoction (GDD) decrease the expression of plasma pro-inflammatory cytokines. Meanwhile, GDD protect the neuron from Cu-induced oxidative cell death in the brain of rodent models for WD. Therefore, we assume that the presence of GDD may inhibit Cu-induced neuroinflammation and subsequent neurotoxicity through decrease the activation of TLR4-mediated NLRP3/Caspase1 axis in microglia. Primary rat microglia and hippocampal neuron will be co-cultured with CuSO4 and treated with GDD to study the protection mechanism of GDD on neuronal damage induced by high copper through TLR4-mediated NLRP3/Caspase1 axis activation in microglia. Therefore, this project will provide theoretical basis of GDD for treatment of WD patient with neurological presentation. Besides, this project may provide new therapeutic targets and the research ideas for the prevention and treatment of neurodegeneration disease with Chinese medicine.

小胶质细胞内NLRP3/Caspase1轴激活引起的神经炎性损伤与多种神经遗传变性病的发生发展密切相关。研究证实，TLR4通路通过调控NLRP3/Caspase1轴促进炎症因子分泌，是高铜诱导神经炎性损伤主要因素。Wilson病（WD）是一种可治性遗传性铜代谢障碍变性疾病，临床发现难治性脑型WD的基底节、丘脑、脑干等部位存在神经元变性损伤，WD动物模型脑内铜沉积区域亦存在神经炎性损伤。前期研究发现中药肝豆汤具有抑制WD患者外周炎症因子释放，并减轻WD动物模型神经元损伤的功效，推测中药肝豆汤可能作用于TLR4通路进而调控NLRP3/Caspase1轴，减轻高铜诱导的神经炎性损伤。为此，本研究拟构建高铜环境下小胶质细胞与海马神经元共培养模型，探究肝豆汤调控神经炎性损伤的作用靶点。旨在从干预神经炎性损伤的角度为肝豆汤治疗铜损脑络提供理论依据，为中医药治疗神经遗传变性病提供新的治疗及研究思路。

NLRP3炎症小体激活引起的神经炎性损伤与多种神经遗传变性病的发生发展密切相关。TLR4通路通过调控NLRP3炎症小体促进炎症因子分泌，是高铜诱导神经炎性损伤主要因素。Wilson病（WD）是一种可治性遗传性铜代谢障碍疾病，临床发现难治性脑型WD的基底节、丘脑、脑干等部位存在神经元变性，WD动物模型脑内铜沉积区域亦存在神经炎性损伤。然而，高铜所致病理损伤与慢性小胶质细胞神经炎症的关系尚未完全阐明。前期研究发现中药肝豆汤具有抑制WD患者外周炎症因子释放，并减轻WD动物模型神经元损伤的功效。为此，本研究拟应用慢病毒包装的Nlrp3 siRNA静脉注射，通过免疫荧光、ELISA和Western blotting分析等方法论证铜沉积与神经炎症的相关性，并通过体内外实验探究肝豆汤调控神经炎性损伤的作用靶点。旨在从干预神经炎性损伤的角度为肝豆汤治疗铜损脑络提供理论依据。.研究发现，NLRP3炎症小体激活是铜诱导神经元损伤的重要途径。在WD动物模型中，cleaved-caspase-1和ASC蛋白表达增加，促进炎症介质的释放。CuCl2可激活小鼠小胶质细胞NLRP3炎症小体，导致细胞外白IL-1β和ASC的大量释放。肝豆汤可阻断小鼠小胶质细胞中CuCl2介导的炎症反应活化和胞外ASC释放。此外，口服肝豆汤可通过抑制TLR4通路阻断WD动物模型NLRP3炎症小体激活，有效缓解运动障碍、神经元变性。.NLRP3炎症小体在WD小鼠模型病理损伤中起着关键作用，其可能是减轻WD铜沉积引起神经元损伤潜在的可行治疗靶点。肝豆汤能有效抑制中枢神经系统NLRP3小体，且长期口服具有神经保护性和安全性。本研究揭示了肝豆汤治疗实验性WD的可能机制，为肝豆汤治疗铜损脑络提供新的理论依据。