β-羟丁酸对LPS诱发的帕金森病模型的神经保护作用及其机制

发布时间：2018-09-14 07:16

【摘要】：越来越多的证据表明神经炎症在帕金森病（Parkinson’s disease, PD）发生发展中起到重要作用。小胶质细胞是神经炎症发生过程中主要的效应细胞，其过度活化后能产生一些促炎酶类（iNOS和COX-2）和促炎细胞因子类（TNF-α、IL-1β和IL-6）。这些炎性介质能损伤周围的多巴胺（dopamine, DA）神经元，最终导致其变性死亡。因此，抑制小胶质细胞过度活化可能是阻止PD进一步发展的潜在的治疗措施。β-羟丁酸（β-hydroxybutyric acid, BHBA）是氨基酸和脂肪酸代谢重要的中间代谢物，其像葡萄糖一样能为大脑提供能量，特别是哺乳期几乎能替代葡萄糖成为大脑的主要能量来源。另外，BHBA在PD中也能起到神经保护作用，但是其机制尚不明确。有研究表明BHBA在巨噬细胞和单核细胞中，通过其受体GPR109A抑制LPS诱导的炎症。所以，我们推测BHBA在PD中可能通过GPR109A受体抑制神经炎症，从而起到神经保护作用。因此，本实验建立了体外细胞炎症模型、LPS诱导的PD体外细胞模型和体内动物模型，系统研究了BHBA在炎症介导的PD模型中的神经保护作用及其机制。在小胶质细胞系BV-2和原代小胶质细胞的细胞炎症模型中发现，BHBA能显著抑制LPS诱导的iNOS、COX-2、TNF-α、IL-1β和IL-6蛋白和mRNA表达的增加。用PTX阻断或siRNA沉默GPR109A能抑制这种效应的发生。Western blotting结果显示BHBA在小胶质细胞系BV-2细胞中能降低LPS诱导的IκB-α降解和NF-κB核转位，然而对ERK1/2、p38-MAPK和JNK的磷酸化水平没有影响；BHBA在原代小胶质细胞中能抑制LPS诱导的NF-κB磷酸化水平，siRNA沉默GPR109A能抑制这种效应。这些结果说明BHBA在小胶质细胞中通过GPR109A/NF-κB信号途径抑制LPS诱导的炎症。为了进一步研究，BHBA是否能通过其抑炎作用对PD起到缓解或治疗作用，我们首先在LPS诱导的PD细胞模型中初步研究了其对DA神经元的保护作用及其机制。结果发现BHBA能抑制LPS在中脑神经-胶质细胞中诱导的DA神经元变性，且这种抑制作用具有浓度依赖性。BHBA的这种神经保护作用是通过抑制小胶质细胞活化来完成的，因为BHBA在中脑神经-胶质细胞中能显著抑制LPS诱导的iNOS、COX-2、TNF-α、IL-1β和IL-6的产生。进一步研究发现BHBA在LPS诱导的PD动物模型中能减弱阿扑吗啡诱导的旋转行为；能浓度依赖性抑制LPS诱导的纹状体中DA和3,4-二羟基苯乙酸（3,4-Dihydroxyphenylacetic acid, DOPAC）的减少；能浓度依赖性抑制LPS诱导的DA神经元减少，并且能抑制LPS诱导的酪氨酸羟化酶（tyrosine hydroxylase,TH）的减少。另外，，BHBA也能浓度依赖性抑制LPS诱导的中脑黑质区小胶质细胞的活化。以上结果表明，BHBA在小胶质细胞中通过GPR109A介导的信号通路起到抑炎作用；BHBA在PD细胞和动物模型中能通过抑制小胶质细胞介导的神经炎症，来改善LPS诱导的运动功能障碍和保护DA神经元。因此，GPR109A介导的信号通路可能是治疗PD的潜在靶点。
[Abstract]:More and more evidences show that neuroinflammation plays an important role in the development of Parkinson's disease (PD). Microglia are the main effector cells in the process of neuroinflammation. Overactivation of microglia can produce some inflammatory enzymes (iNOS and COX-2) and inflammatory cytokines (TNF-a, IL-1beta and IL-6). Sexual mediators can damage peripheral dopamine (DA) neurons and eventually lead to their degeneration and death. Therefore, inhibition of microglia over-activation may be a potential therapeutic measure to prevent the further development of PD. Beta-hydroxybutyric acid (BHBA) is an important intermediate metabolite in amino acid and fatty acid metabolism, such as glucose. It also provides energy for the brain, especially during lactation, which almost replaces glucose as the main energy source. In addition, BHBA also plays a neuroprotective role in PD, but its mechanism is not clear. Studies have shown that BHBA inhibits LPS-induced inflammation in macrophages and monocytes through its receptor GPR109A. It is speculated that BHBA may play a neuroprotective role in PD by inhibiting neuroinflammation through GPR109A receptor. In this study, we established in vitro cell inflammation model, LPS-induced PD cell model in vitro and in vivo animal model, and systematically studied the neuroprotective effect and mechanism of BHBA in inflammation-mediated PD model.
BHBA significantly inhibited LPS-induced increase of iNOS, COX-2, TNF-a, IL-1beta and IL-6 protein and mRNA expression in microglial cell line BV-2 and primary microglial cell inflammation models. Blockade of GPR109A by PTX or silencing of GPR109A with siRNA inhibited this effect. Western blotting results showed that BHBA could inhibit the expression of iNOS, COX-2, TNF-a, IL-1beta and IL-6 protein and mRNA in microglial cell line BV BHBA could inhibit LPS-induced NF-kappa B phosphorylation in primary microglia, and GPR109A silencing could inhibit this effect. These results suggest that BHBA can suppress LPS-induced NF-kappa B phosphorylation in microglia through GPR109A/NF-kappa B signaling pathway. No. 2 pathway inhibits LPS induced inflammation.
To further investigate whether BHBA can alleviate or treat PD by its anti-inflammatory effect, we first studied the protective effect of BHBA on DA neurons in LPS-induced PD cell model and its mechanism. This neuroprotective effect of BHBA is achieved by inhibiting microglial activation in a concentration-dependent manner, because BHBA significantly inhibits LPS-induced iNOS, COX-2, TNF-a, IL-1beta and IL-6 production in mesencephalic neuroglial cells.
Further studies have shown that BHBA can attenuate apomorphine-induced rotation in LPS-induced PD animal models, inhibit LPS-induced reduction of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum in a concentration-dependent manner, and inhibit LPS-induced decrease of DA neurons in a concentration-dependent manner. In addition, BHBA also inhibited LPS-induced microglia activation in substantia nigra in a concentration-dependent manner.
These results suggest that BHBA plays an anti-inflammatory role in microglia through GPR109A-mediated signaling pathway; BHBA improves LPS-induced motor dysfunction and protects DA neurons by inhibiting microglia-mediated neuroinflammation in PD cells and animal models. Potential targets.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.5;R-332

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