中藥複方製劑芍藥甘草湯和Coumarin-chalcone衍生物在Tau蛋白易聚集表現之阿茲海默氏症細胞模式中之療效

Abstract

阿茲海默氏症(Alzheimer’s disease, AD)為一種進行性且不可逆的神經退化性疾病，受損的記憶和認知能力會逐漸失常最終造成身體功能的完全喪失。AD病人腦中最主要的特徵為不正常的Amyoild β與Tau蛋白聚集形成的老化斑塊(Senile plaque)和神經纖維纏結(Neurofibrillary tangles)。神經纖維纏結主要組成是高度磷酸化的Tau蛋白，而Tau蛋白的病變不僅在AD病人，也在許多Tauopathy中見到。在大腦中由於錯誤折疊形成的蛋白堆積，引發許多病理事件，例如氧化壓力、神經發炎，或是與神經細胞功能相關的訊息傳遞障礙，降低神經細胞存活。本篇研究主要是利用人類胚胎腎細胞HEK-293和神經纖維瘤母細胞SH-SY5Y，表現易聚集特性的ΔK280 TauRD片段蛋白，作為評估中藥製劑芍藥甘草湯(SG-Tang)以及Coumarin-chalcone衍生物LM-021、LM-031和LMDS-1~4化合物，抑制蛋白聚集、抗氧化和神經保護性的情形。芍藥甘草湯製劑，由甘草與白芍以等比例製成，除了可有效降低以LPS/IFN-γ刺激後活化的BV-2微膠細胞所釋出的NO、TNF-α、IL-1β及IL-6等前驅發炎細胞因子外，並可抑制由於誘導表現ΔK280 TauRD蛋白而上升的BAD、BID、CASP3、CASP8和CYCS表現，來達到Tau蛋白聚集抑制和神經保護性。其中LM-021除了本身有化學伴護活性可直接抑制ΔK280 TauRD的聚集外，還可透過PKA、CaMKII、ERK等路徑活化CREB和下游的BDNF、BCL2表現，來展現其神經保護之功能。接著在ΔK280 TauRD-DsRed SH-SY5Y細胞中，觀察到LM-031可提升HSPB1伴護蛋白表現以降低Tau蛋白的錯誤折疊，以及藉由活化NRF2/NQO1/GCLC和CREB調控之BDNF/AKT/ERK/BCL2路徑，來達到抑制細胞凋亡和促進細胞存活之效果。最後，因LM-031可提升CREB依存的BDNF表現，利用虛擬篩選找出LM-031類似化合物LMDS-1~4，並進一步以TRKB與ligand作用的d5-domain (PDB 1hcf)，以分子模擬計算LM-031類似化合物與此蛋白片段的結合構形。細胞實驗結果顯示，LMDS-1和LMDS-2可經由TRKB/ERK和TRKB/PI3K/AKT訊息路徑，增加CREB磷酸化及其下游BDNF、BCL2基因表現。總結來說，本研究結果顯示芍藥甘草湯的抗氧化和抗發炎活性，可抑制神經細胞凋亡，在Tau蛋白聚集的細胞中展現其神經保護的能力，而Coumarin-chalcone衍生物LM-031、LM-021、LMDS-1、LMDS-2，具有活化HSPB1、NRF2及/或TRKB的作用，來降低Tau蛋白的錯誤摺疊、抑制細胞凋亡和促進神經細胞存活。以上這些研究結果顯示了芍藥甘草湯和Coumarin-chalcone衍生物，應用在AD治療上的潛能。Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease that affects memory and cognitive decline gradually and finally loss the ability in body motion. Unusual amyloid β protein aggregates and neurofibrillary tangles are found in the brains of AD patients. The neurofibrillary tangles are consisted of hyperphosphorylated Tau proteins, and this pathology hallmark exists in not only AD but also other tauopathies brains. The misfolded protein deposits cause series of events such as accumulation of oxidative stress, neuronal inflammation, and impairment of cell signaling to reduce neuronal survival. In this study pro-aggregator Tau (ΔK280 TauRD)-expressing human 293/SH-SY5Y cells were used to evaluate formulated Chinese herbal medicine Shaoyao Gancao Tang (SG-Tang) and coumarin-chalcone derivatives LM-021, LM-031 and LMDS-1~4 for their effects in anti-aggregative, anti-oxidative, and neuroprotective activities. SG-Tang, made of P. lactiflora and G. uralensis at 1:1 ratio), reduced NO, TNF-α, IL-1β and IL-6 in LPS/IFN-γ-activated mouse BV-2 microglia. Also, SG-Tang down-regulated BAD, BID, CASP3, CASP8 and CYCS expression for neuroprotection in ΔK280 TauRD-DsRed SH-SY5Y cells. The chemical chaperone activity of LM-021 inhibited ΔK280 TauRD aggregation in vitro. And LM-021 activated CREB-mediated BDNF and BCL2 gene expression through PKA, CaMKII and ERK for neuroprotection. In addition, another LM compound LM-031, not only upregulated HSPB1 chaperone to reduce Tau misfolding, but also activated NRF2/NQO1/GCLC and CREB-dependent BDNF/AKT/ERK/BCL2 pathways. Through these mechanisms, LM-031 suppress apoptosis and promote neuronal survival in ΔK280 TauRD-DsRed SH-SY5Y cells. As LM-031 upregulating CREB-dependent BDNF expression, virtual screening was conducted to obtain LM-031 analogs LMDS-1~4, followed by docking computation with TRKB d5 domain (PDB 1hcf). Among them, LMDS-1 and LMDS-2 activated TRKB/ERK and TRKB/PI3K/AKT signaling to increase CREB phosphorylation and downstream BDNF and BCL2 gene expression. In conclusion, SG-Tang displays neuroprotection by exerting anti-oxidative and anti-inflammatory activities to suppress neuronal apoptosis, and coumarin-chalcone derivatives LM-031, LM-021, LMDS-1 and LMDS-2 target HSPB1, NRF2 and/or TRKB to reduce Tau misfolding, suppress apoptosis and promote neuron survival. The study results shed light on the potential application of SG-Tang and these coumarin-chalcone derivatives in therapeutics of AD.