抗發炎相關化合物 VB-037 及甘草次酸 在帕金森氏症細胞模式上之治療潛力
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2018
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帕金森氏症為僅次於阿茲海默氏症之第二常見的神經退化性疾 病，主要症狀有手足顫抖、僵硬、動作緩慢、站立不穩等，患者黑質 多巴胺神經元大量減少，並出現包含 α-Synuclein (簡稱 α-Syn)蛋白的 路易氏體。α-Syn 蛋白本質上無特定結構，易形成不溶性的纖維及聚 集。近年研究發現微膠細胞參與的免疫與發炎反應，與帕金森氏症致 病機制相關，且細胞外的 α-Syn 蛋白，會刺激微膠細胞產生前發炎激 素及活性氧化物。為探究 α-Syn 刺激的發炎反應，本研究以大腸桿菌 BL21 表現 His 標籤的 α-Syn 蛋白，並以親和性色層純化之。2 μg/μl 濃度的單體 α-Syn 於 37°C 培養箱搖晃一週後，Thioflavin T 螢光分析 及共軛焦顯微鏡觀察，皆確認了時間依的 α-Syn 纖維的形成。小鼠 BV-2 微膠細胞以製備的 α-Syn 纖維處理後，前發炎因子一氧化氮(NO) 及 Iba1 上升，喹啉衍生物 VB-037 及五環的三萜系化合物甘草次酸 (glycyrrhetic acid) [中藥脹果甘草(Glycyrrhiza inflata)及芍藥甘草湯 (Shaoyao Gancao Tang)活性成分甘草酸(glycyrrhizin)之代謝產物]，可 有效抑制 BV-2 微膠細胞生成的 NO、Iba1 及成熟的介白素-1β (IL-1β)。小鼠發炎抗體矩陣及西方墨點/酵素免疫分析法/即時聚合酶 鏈反應驗證，進一步揭示介白素-1α (IL-1α)、介白素-1β、腫瘤壞死因 子-α (TNF-α)、干擾素-γ (IFN-γ)、顆粒球巨噬細胞群落刺激因子 (GM-CSF)、介白素-6 (IL-6)及白血球生長激素(G-CSF)，在 α-Syn 纖 維刺激發炎的小鼠 BV-2 微膠細胞中表現增加，且 VB-037 及甘草次 酸前處理能有效的降低這些前發炎因子的表現與釋放。為檢測抗發炎性的 VB-037 及甘草次酸在帕金森氏症的治療潛能，建立 A53T α-Syn-GFP 誘導表現及 α-Syn 纖維依聚集形成的人類神經母細胞瘤 SH-SY5Y 細胞，並以佛波酯 TPA 誘導多巴胺神經元指標酪胺酸烴酶 (Tyrosine hydroxylase)表現的神經分化，ProteoStat 染色及西方墨點/ 點漬法試驗顯示，VB-037 及甘草次酸的處理能有效的降低 α-Syn 聚 集形成。此研究提供新的應用觀點，來助益於抑制 α-Syn 刺激神經發 炎的帕金森氏症治療藥物開發。
關鍵:帕金森氏症、α-Synuclein、神經發炎、VB-037、甘草次酸
Parkinson’s disease (PD) is an age-related neurodegenerative disorder that ranks only second behind Alzheimer’s disease in prevalence. PD is characterized by resting tremor, rigidity and bradykinesia, in addition to the loss of dopaminergic (DA) neurons in substantia nigra and the presence of α-synuclein (α-Syn)-containing Lewy bodies. α-Syn is an intrinsically unstructured protein prone to forming insoluble fibrils and aggregates. Recently, it is becoming evident that immune response engaged by microglia actively contributes to the pathogenesis of PD and extracellular α-Syn increases the production of pro-inflammatory mediators in microglia. To examine the capacity of α-Syn to stimulate inflammation, His-tagged α-Syn was expressed in Escherichia coli BL21 and purified by affinity chromatography. Monomeric α-Syn in 2 μg/μl concentration was incubated at 37°C with continuous shaking for a week and a time-dependent fibrillation of α-Syn was confirmed by biochemical thioflavin T fluorescence assay and confocal microscope examination. Increased production of nitric oxide (NO) and Iba1 was observed after addition of α-Syn fibrils and pretreatment with a quinoline compound VB-037 and a pentacyclic triterpenoid glycyrrhetic acid (a metabolite of glycyrrhizin, an active constituent of Chinese herbal medicine Glycyrrhiza inflata and Shaoyao Gancao Tang) suppressed the NO production, Iba1 expression and IL-1β maturation in mouse BV-2 microglia. Mouse inflammation antibody array and western/ELISA/real-time PCR confirmation further revealed increased expression of IL-1α, IL-1β, TNF-α, IFN-γ, GM-CSF, IL-6 and G-CSF in α-Syn-inflamed BV-2 cells and both VB-037 and glycyrrhetic acid pretreatment effectively reduced the expression and release of these pro-inflammatory mediators. To examine the therapeutic potential of anti-inflammatory compounds VB-037 and glycyrrhetic acid in PD, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) up-regulated tyrosine hydroxylase (TH, DA neuronal marker) and neuronal differentiation in human neuroblastoma SH-SY5Y cells with inducible A53T α-Syn-GFP expression and α-Syn fibril-dependent aggregate formation were established. By ProteoStat stain and western/dot blot examination, VB-037 and glycyrrhetic acid effectively reduced α-Syn aggregation. The study offers new viewpoints of application to benefit drug development for α-Syn-stimulated neuroinflammation in PD. Keywords: Parkinson’s disease, α-synuclein, neuroinflammation, VB-037, glycyrrhetic acid
Parkinson’s disease (PD) is an age-related neurodegenerative disorder that ranks only second behind Alzheimer’s disease in prevalence. PD is characterized by resting tremor, rigidity and bradykinesia, in addition to the loss of dopaminergic (DA) neurons in substantia nigra and the presence of α-synuclein (α-Syn)-containing Lewy bodies. α-Syn is an intrinsically unstructured protein prone to forming insoluble fibrils and aggregates. Recently, it is becoming evident that immune response engaged by microglia actively contributes to the pathogenesis of PD and extracellular α-Syn increases the production of pro-inflammatory mediators in microglia. To examine the capacity of α-Syn to stimulate inflammation, His-tagged α-Syn was expressed in Escherichia coli BL21 and purified by affinity chromatography. Monomeric α-Syn in 2 μg/μl concentration was incubated at 37°C with continuous shaking for a week and a time-dependent fibrillation of α-Syn was confirmed by biochemical thioflavin T fluorescence assay and confocal microscope examination. Increased production of nitric oxide (NO) and Iba1 was observed after addition of α-Syn fibrils and pretreatment with a quinoline compound VB-037 and a pentacyclic triterpenoid glycyrrhetic acid (a metabolite of glycyrrhizin, an active constituent of Chinese herbal medicine Glycyrrhiza inflata and Shaoyao Gancao Tang) suppressed the NO production, Iba1 expression and IL-1β maturation in mouse BV-2 microglia. Mouse inflammation antibody array and western/ELISA/real-time PCR confirmation further revealed increased expression of IL-1α, IL-1β, TNF-α, IFN-γ, GM-CSF, IL-6 and G-CSF in α-Syn-inflamed BV-2 cells and both VB-037 and glycyrrhetic acid pretreatment effectively reduced the expression and release of these pro-inflammatory mediators. To examine the therapeutic potential of anti-inflammatory compounds VB-037 and glycyrrhetic acid in PD, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) up-regulated tyrosine hydroxylase (TH, DA neuronal marker) and neuronal differentiation in human neuroblastoma SH-SY5Y cells with inducible A53T α-Syn-GFP expression and α-Syn fibril-dependent aggregate formation were established. By ProteoStat stain and western/dot blot examination, VB-037 and glycyrrhetic acid effectively reduced α-Syn aggregation. The study offers new viewpoints of application to benefit drug development for α-Syn-stimulated neuroinflammation in PD. Keywords: Parkinson’s disease, α-synuclein, neuroinflammation, VB-037, glycyrrhetic acid
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帕金森氏症, α-Synuclein, 神經發炎, VB-037, 甘草次酸, Parkinson’s disease, α-synuclein, neuroinflammation, VB-037, glycyrrhetic acid