Zhang Li-Fang, Zhou Zhi-Wei, Wang Zhen-Hai, Du Yan-Hui, He Zhi-Xu, Cao Chuanhai, Zhou Shu-Feng
Department of Neurology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People's Republic of China ; Department of Pharmaceutical Science, College of Pharmacy, University of South Florida, Tampa, FL, USA.
Department of Pharmaceutical Science, College of Pharmacy, University of South Florida, Tampa, FL, USA.
Drug Des Devel Ther. 2014 Dec 24;9:241-72. doi: 10.2147/DDDT.S71106. eCollection 2015.
There is an increasing prevalence of Alzheimer's disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-β (Aβ) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β), Erk1/2, PI3K, Akt, Tau, Wnt3α, β-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aβ40/42 and Aβ42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3β, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells. Coff or Caff, plus Mel downregulated Wnt3α expression but upregulated β-catenin. However, Coff or Caff plus Mel did not significantly alter the production of T helper cell (Th)1-related interleukin (IL)-12 and interferon (IFN)-γ and Th2-related IL-4 and IL-10 in N2a/APP cells. The autophagy of cells was not affected by the combinations. Taken together, combination of Caff or Coff, before treatment with Mel elicits an additive antiamyloidogenic effects in N2a/APP cells, probably through inhibition of Aβ oligomerization and modulation of the Akt/GSK3β/Tau signaling pathway.
阿尔茨海默病（AD）的患病率日益上升，已成为一个公共卫生问题。然而，AD发病机制的潜在机制尚未完全明确，目前的治疗药物在AD患者中无法产生可接受的疗效。先前的动物研究表明，咖啡（Coff）、咖啡因（Caff）和褪黑素（Mel）对AD具有有益作用。在AD中观察到昼夜节律紊乱，而时间疗法对AD已显示出有前景的效果。在本研究中，我们研究了Coff或Caff加Mel的组合对神经母细胞瘤细胞系-2a（N2a）/淀粉样前体蛋白（APP）细胞中淀粉样β蛋白（Aβ）生成是否产生协同/相加作用以及相关的可能机制。细胞用Coff或Caff处理，有或没有联合Mel，采用三种不同的时间方案。在方案1中，细胞在白天用Coff或Caff处理12小时，然后在夜间用Mel处理12小时。对于方案2，细胞从上午7点至次日上午7点用Coff或Caff加Mel处理24小时。在方案3中，细胞用Coff或Caff加Mel按照方案1或2连续处理5天。使用酶联免疫吸附测定（ELISA）试剂盒测定细胞外Aβ40/42和Aβ寡聚体水平。通过蛋白质免疫印迹法检测糖原合酶激酶3β（GSK3β）、细胞外信号调节激酶1/2（Erk1/2）、磷脂酰肌醇3激酶（PI3K）、蛋白激酶B（Akt）、微管相关蛋白tau（Tau）、Wnt3α、β-连环蛋白和核因子E2相关因子2（Nrf2）的表达和/或磷酸化水平。结果表明，方案1产生相加的抗淀粉样蛋白生成作用，细胞外Aβ40/42和Aβ42寡聚体水平显著降低。方案2未产生显著效果，与方案1相比，方案三显示出较小的抗淀粉样蛋白生成作用。Coff或Caff加Mel通过Nrf2途径降低N2a/APP细胞中的氧化应激。Coff或Caff加Mel抑制N2a/APP细胞中GSK3β、Akt、PI3K p55和Tau的磷酸化，但增强PI3K p85和Erk1/2的磷酸化。Coff或Caff加Mel下调Wnt3α表达但上调β-连环蛋白。然而，Coff或Caff加Mel未显著改变N2a/APP细胞中辅助性T细胞（Th）1相关的白细胞介素（IL）-12和干扰素（IFN）-γ以及Th2相关的IL-4和IL-10的产生。细胞的自噬不受这些组合的影响。综上所述，在使用Mel治疗之前，Caff或Coff的组合在N2a/APP细胞中引发相加的抗淀粉样蛋白生成作用，可能是通过抑制Aβ寡聚化和调节Akt/GSK3β/Tau信号通路。
