开心散与生脉散抗阿尔茨海默症“同病异治”作用机制网络药理学分析与中枢神经免疫调控效用验证＊

目的 基于网络药理学方法研究开心散（KXS）与生脉散（SMS）“同病异治”抗阿尔茨海默症作用机制，并通过整体动物与离体细胞实验进行验证。方法 通过数据库搜集筛选开心散与生脉散复方所含药味化学成分并预测其潜在靶点，构建复方-中药-成分-靶点网络。筛选复方共有与优势靶点，构建蛋白互作网络并进行拓扑分析，进行KEGG通路和GO功能富集分析。基于网络药理学分析获得的开心散与生脉散共性与个性的生物学GO分析结果，分别建立小鼠海马Aβ注射拟阿尔茨海默症痴呆小鼠模型与小鼠小胶质BV2细胞炎性因子损伤模型，检测相应指标，采用行为学结合生化指标分析验证网络药理学分析结果。结果 筛选出开心散与生脉散复方共有成分有109个，共有靶点490个，抗AD潜在共性调控靶点375个。开心散抗AD潜在优势调控靶点92个，生脉散抗AD潜在优势调控靶点90个。淀粉样蛋白代谢与转运、cAMP与AMPK信号通路、突触功能调控等为开心散与生脉散抗AD潜在共性调控信号通路与生物学事件。NF-κB等为开心散抗AD优势调控信号通路与生物学事件；趋化因子、胆碱能突触等为生脉散抗AD优势调控信号通路与生物学事件。验证实验结果显示开心散与生脉散能够有效改善Aβ海马区注射小鼠认知功能障碍，降低小鼠海马和小胶质细胞的炎性因子表达。结论 开心散与生脉散能通过共有及各自优势靶点发挥抗AD作用。     

HPLC/DAD/ESI-MS~n方法鉴定开心散的化学成分及其体内代谢产物(英文)

为了研究开心散的化学成分及体内代谢产物, 采用Agilent SB-C18色谱柱, 利用离子阱质谱及高分辨飞行质谱对开心散提取物及口服开心散后的大鼠血浆样品进行分析。从开心散提取物中共鉴定了39个化学成分, 包括11个人参皂苷, 14个远志皂苷, 5个远志多糖, 8个远志寡糖酯及1个远志口山酮, 其中15个化合物通过与对照品对照确定, 未检测到茯苓及石菖蒲的成分。大鼠灌胃给予开心散提取物后 (7 g/kg), 从血浆检测到10个人参皂苷和18个远志成分。本研究表明, 人参皂苷、远志皂苷及糖酯很可能是开心散的主要有效成分。     

开心散药效物质基础和药理作用机制的研究进展

开心散始载于唐代孙思邈的《备急千金要方》,由人参、茯苓、远志、石菖蒲4味药按3∶3∶2∶2的比例组成。开心散发挥药理作用的物质基础主要涉及人参皂苷、远志皂苷、远志糖酯和远志酮,而茯苓、石菖蒲的活性成分是否为开心散的药效成分的研究较少,研究范围较局限;开心散药理作用的研究主要集中在抗抑郁作用、抗老年痴呆、改善学习记忆能力、抗疲劳和镇静安神等方面,涉及机制包括神经系统、免疫系统和内分泌系统。对开心散的药效物质基础、药理作用及其机制进行较为系统的阐述,以期为日后开心散的临床应用提供一定的思路。     

开心散配伍比例对慢性压力应激抑郁小鼠皮层与海马神经营养因子系统调控的影响

目的?基于慢性压力应激抑郁小鼠模型,考察不同配伍比例的开心散对皮层与海马神经营养因子调控的影响,探讨开心散治疗抑郁症的作用机制。方法?采用一系列慢性应激压力构建小鼠抑郁症模型,给予不同配伍比例的开心散,应用ELISA法分别检测小鼠大脑皮层与海马中神经生长因子NGF与脑源性神经营养因子BDNF含量,利用qPCR与Western blot技术检测皮层与海马神经营养因子受体TrkA、TrkB、TrkC的表达。结果?不同配伍比例的开心散均能提升抑郁小鼠的糖水饮用量,延长强迫游泳时间,表现出显著的抗抑郁作用,其中以D-652配伍比例效用最佳。同时提升NGF、BDNF含量与相关受体的表达。结论?开心散调控神经营养因子系统是其抗抑郁的重要作用机制。      

中药复方开心散调控慢性压力应激小鼠海马炎性细胞因子水平抗抑郁作用机制研究＊

目的 探究中药复方开心散调控中枢神经炎症系统抗抑郁的作用机制。方法 制备开心散不同配伍比例提取物,采用小鼠慢性应激压力（CUMS）模型,通过糖水偏嗜实验、悬尾实验和强迫游泳实验评价开心散不同配伍比例的抗抑郁功效。采用ELISA法测定受试动物海马组织促炎物质如脂多糖（LPS）、IL-1β、IL-6、TNF-α的含量。采用免疫组织化学染色法观察受试动物海马区小胶质细胞形态。进而采用LPS促进小鼠小胶质永生化细胞（BV2）炎性因子表达细胞模型,采用ELISA法评价开心散抑制炎症因子表达的作用,采用Western blotting法考察NF-κB信号通路的调控情况。结果 开心散不同配伍比例能显著上调模型小鼠糖水偏嗜率,缩短小鼠悬尾和强迫游泳不动时间,以D-652效用最强。该配伍比例通过抑制NF-κB的入核和抑制NF-κB磷酸化等环节,下调BV2细胞中炎症因子IL-1β、IL-6、TNF-α的表达水平。结论 对中枢神经炎症的调控是中药复方开心散抗抑郁的重要作用环节。     

The effect of Acorus gramineus on the bioavailabilities and brain concentrations of ginsenosides Rg1, Re and Rb1 after oral administration of Kai-Xin-San preparations in rats

Aim of the study

To investigate the effect of Acorus gramineus (AG), a supposed ‘delivering servant’ according to traditional Chinese medicine principles governing multi-herb formula preparation and formulation, on facilitating the uptake of ginsenosides Rg1, Re and Rb1 to the brain after oral administration of Kai-Xin-San (KXS) preparations.

Materials and methods

Ginseng extracts or KXS with or without AG were administered to rats for pharmacokinetic study and mice for behaviour tests at a dose of 3 g ginseng per kg. The concentrations of ginsenosides in plasma and brain were determined by an LC-MS/MS method, whilst the effects of preparations on spatial learning were evaluated using the Morris water maze test.

Results

KXS in the presence of AG tended to significantly reverse the learning impairment induced by scopolamine. The presence of AG in the KXS formula led to increases in the initial absorption rate and extent of Rg1 and Re in terms of Cmax1 and AUC_0-3 h compared to KXS without AG. Although KXS were found to increase the bioavailabilities and brain concentrations of ginsenosides relative to ginseng extract, the brain-to-plasma AUC_0-12 h ratios appeared not to be affected.

Conclusions

The results suggested that the presence of AG in the KXS formula promoted the initial absorption of ginsenosides Rg1 and Re in the gastrointestinal tract, but unlikely affected the brain-to-plasma AUC ratios.     

中药复方开心散调控神经营养因子抗抑郁物质基础与作用机制研究

目的：观察开心散调控神经营养因子抗抑郁的物质基础与作用机制。方法：制备开心散大孔树脂乙醇洗脱部位,采用悬尾和强迫游泳评价抗抑郁效果。采用ELISA法测定海马中神经生长因子（NGF）与脑源性神经营养因子（BDNF）的含量。利用大鼠星型胶质瘤C6细胞系和大鼠肾上腺嗜铬瘤PC12细胞系,评价不同乙醇洗脱部位促进NGF与BDNF表达和促进PC12细胞分化的作用。结果：开心散各效用部位均能显著缩短小鼠悬尾与强迫游泳不动时间,其中70%乙醇洗脱部位抗抑郁效用最强,该部位还能通过cAMP信号通路上调C6细胞中NGF与BDNF的表达。10%乙醇洗脱部位促PC12细胞分化的能力最强。人参皂苷是促进神经营养因子表达的主要效用成分。结论：开心散调控神经营养因子是其抗抑郁效用的重要作用机制。     

运用网络药理学探讨开心散治疗抑郁症的作用机制

目的本研究旨在运用网络药理学方法,预测开心散活性成分的抗抑郁药理作用机制。方法通过TCMSP、ETCM获取开心散的成分和靶点,运用Genecard、OMIM筛选抑郁症相关的蛋白,通过STRING构建蛋白相互作用网络,使用Cytoscape进行靶点的拓扑分析、MCODE聚类分析,使用R语言进行GO和KEGG通路富集分析,最后使用MOE软件进行分子对接验证。结果网络分析表明,开心散中25个活性成分与其发挥抗抑郁作用密切相关,该机制涉及到51个靶点(包括ACHE、HTR3A、DRD2等)以及24条通路;分子对接结果显示化合物aposcopolamine、perlolyrine、hederagenin与PPARG、ACHE、SLC6A4、DRD2靶点均具有较好的结合能力。结论开心散可能主要通过神经活性受体配体相互作用、羟色胺突触、cAMP通路等相关靶标起到抗抑郁的功效,该研究为一步揭示开心散治疗抑郁症的潜在活性成分及其可能的作用机制提供了思路。     

High-throughput screening for amyloid-β binding natural small-molecules based on the combinational use of biolayer interferometry and UHPLC−DAD-Q/TOF-MS/MS

Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease (AD). In this study, a novel high-throughput screening (HTS) method aims at screening the small-molecules with amyloid-β (Aβ) binding affinity from natural medicines, based on the combinational use of biolayer interferometry (BLI) and ultra-high-performance liquid chromatography coupled with diode-array detector and quadrupole/time-of-flight tandem mass spectrometry (UHPLC?DAD-Q/TOF-MS/MS) has been firstly developed. Briefly, the components in natural medicines disassociated from biotinylated Aβ were collected to analyze their potential Aβ binding affinity by UHPLC?DAD-Q/TOF-MS/MS. Here, baicalein was confirmed to exhibit the highest binding affinity with Aβ in Scutellaria baicalensis. Moreover, polyporenic acid C (PPAC), dehydrotumulosic acid (DTA), and tumulosic acid (TA) in Kai-Xin-San (KXS) were also identified as potent Aβ inhibitors. Further bioactivity validations indicated that these compounds could inhibit Aβ fibrillation, improve the viability in Aβ-induced PC-12 cells, and decrease the Aβ content and improve the behavioral ability in Caenorhabditis elegans. The molecular docking results confirmed that PPAC, DTA, and TA possessed good binding properties with Aβ. Collectively, the present study has provided a novel and effective HTS method for the identification of natural inhibitors on Aβ fibrillation, which may accelerate the process on anti-AD drugs discovery and development.