■认知障碍是神经系统中威尔逊病(WD)的严重临床表现。Gandouling(GDL)是安徽中医药大学第一附属医院的医院制剂。先前的研究发现GDL对WD的认知障碍具有改善作用。
目的:我们旨在探索WD认知功能障碍的分子水平调控机制,并提供证据支持GDL作为治疗WD认知障碍的有希望的候选药物。我们发现在WD基因突变模型中,C3He-Atp7Btx-J/J(tx-j)小鼠脑组织中GSK3β显著上调,GDL的单体组分能与GSK3β良好结合。因此,在这项工作中,我们用了行为测试,苏木精和伊红(H&E),Nissl和TUNEL染色,通过透射电子显微镜(TEM)观察超微结构形态,亚硫酸氢盐测序(BSP),RT-qPCR,蛋白质印迹,免疫荧光,网络药理学,分子对接,和相关方法研究GDL在tx-j小鼠和HT22细胞中的作用,以阐明GDL在WD认知障碍中的作用。
结果:在这项研究中,MWM,NOR,H&E,NisslTUNEL和TEM结果表明GDL能促进学习记忆功能的修复,改善海马神经元的形态损伤,并保持线粒体的完整性。在HT22细胞实验中,CCK-8方法显示GDL增加了铜过载细胞模型的活力。研究发现GSK3β可能是GDL通过网络药理学治疗WD认知障碍的靶点。Westernblot和qRT-PCR结果证实GDL显著增加DNMT1、Nrf2和HO-1中蛋白和mRNA的表达。BSP显示Wilson组GSK3β启动子甲基化程度低于对照组,腹腔注射地西他滨后,GSK3β的启动子甲基化进一步降低,和GDL可以改善这种病理。
结论:GDL通过诱导GSK3β启动子甲基化显示出保护作用,激活WD中的Nrf2/GSK3β信号通路。
UNASSIGNED: Cognitive impairment is a serious clinical manifestation of Wilson\'s disease (WD) in the nervous system. Gandouling (GDL) is a hospital preparation of the First Affiliated Hospital of Anhui University of Chinese Medicine. Previous studies have found that GDL has an ameliorative effect on cognitive impairment in WD.
OBJECTIVE: We aimed to explore the molecular-level regulatory mechanisms underlying cognitive impairment in WD, and provide evidence supporting GDL as a promising candidate drug for the treatment of cognitive impairment in WD. We found that GSK3β was significantly up-regulated in the brain tissue of C3He-Atp7Btx-J/J (tx-j) mice in the WD gene mutant model, and the monomer components of GDL could combine well with GSK3β. Therefore, in this work, we used Behavioral tests, Hematoxylin and eosin (H&E), Nissl and Terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling(TUNEL) staining, Ultrastructural morphological observation by Transmission electron microscopy (TEM), bisulfite sequencing (BSP), Quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, immunofluorescence, network pharmacology, molecular docking, and related methods to study the effects of GDL in tx-j mice and HT22 cell to clarify the effect of GDL on cognitive impairment in WD.
RESULTS: In this study, MWM, NOR, H&E, Nissl TUNEL and TEM results showed that GDL could promote the repair of learning and memory function, improve the morphological damage to hippocampal neurons, and maintain mitochondria integrity. In the HT22 cell experiment, the CCK-8 method showed that GDL increased the viability of copper-overloaded cell models. The study found that GSK3β may be a target of GDL for the treatment of WD cognitive impairment through network pharmacology. Western blot and qRT-PCR results confirmed that GDL significantly increased the expression of proteins and mRNA in DNMT1, Nrf2, and HO-1. BSP showed that GSK3β promoter methylation was lower in the Model group than in the control group, and the promoter methylation of GSK3β was further reduced after intraperitoneal injection with decitabine, and GDL could ameliorate this pathology.
CONCLUSIONS: GDL demonstrates a protective role by inducing GSK3β promoter methylatio, regulating the GSK3β/Nrf2 signaling pathway in WD.