背景:酒精性肝病(ALD)是与饮酒相关的最严重和不可逆的肝损伤。Puerariae和SemenHoveniae是消除酒精作用的中药(TCM)。许多研究表明,两种药材的结合具有治疗ALD的增强作用。
目的:本研究的目的是评估葛花-海葵药对的药理作用,阐明其在酒精诱导的BRL-3A细胞治疗中的作用机制,并通过谱效关系研究揭示药物对中发挥药理作用的有效成分。
方法:首先,MTT测定,ELISA,荧光探针分析,通过检测药效学指标和相关蛋白表达,采用Westernblot方法研究该药物对在酒精诱导的BRL-3A细胞中的作用机制。其次,建立了不同比例的药物对和不同溶剂提取样品的化学色谱图的HPLC方法。然后,主成分分析,采用皮尔逊双变量相关分析和灰色关联分析来建立药效学指标与HPLC色谱图之间的谱-效应相关性。此外,通过HPLC-MS方法鉴定了体内原型成分及其代谢产物。
结果:葛花-精液药物对显著增加细胞活力,降低ALT的活性,AST,TC和TG,减少TNF-α的产生,IL-1β,IL-6、MDA和ROS,增加了SOD和GSH-Px的活性,与酒精诱导的BRL-3A细胞相比,CYP2E1的蛋白表达降低。药物对通过上调磷酸-PI3K的水平来调节PI3K/AKT/mTOR信号通路,磷酸-AKT和磷酸-mTOR。此外,光谱-效应关系研究的结果表明,P1(绿原酸),P3(daidzin),P4(6″-O-木糖酰-缩水甘油),P5(缩水甘油),P6(未知),P7(未知),P9(未知),P10(6″-O-木糖酰-tectoridin),P12(tectoridin)和P23(未知)可以被认为是治疗ALD的药物对的主要成分。此外,6″-O-xylosyl-tectoridin,tectoridin,Daidzin,6″-O-木糖酰缩水甘油和缩水甘油可被吸收到血液中,并在大鼠体内表现出明显的代谢和排泄行为。
结论:在这项研究中,初步研究并揭示了葛花-海子对酒精诱导的BRL-3A细胞的肝保护作用和药理机制。通过光谱-效应关系研究,潜在的药效学成分,如大豆苷,6″-O-木糖酰-缩水甘油,6″-O-xylosyl-tectoridin,缩水甘油,和tectoridin通过调节PI3K/AKT/mTOR信号通路对酒精诱导的氧化应激和炎症发挥药理作用。本研究为揭示ALD治疗的药效物质基础和药理作用机制提供了实验依据和数据支持。此外,它为探索复杂中药生物活性的主要有效成分提供了有力的手段。
BACKGROUND: Alcoholic liver disease (ALD) is the most serious and irreversible liver damage associated with alcohol consumption. Flos Puerariae and Semen Hoveniae are traditional Chinese medicines (TCM) for dispelling the effects of alcohol. Many studies have shown that the combination of two medicinal materials has the enhanced effect of treating ALD.
OBJECTIVE: The aim of this
study is to assess the pharmacological effects of Flos Puerariae-Semen Hoveniae medicine pair, to elucidate its action mechanism in the treatment of alcohol-induced BRL-3A cells, and to reveal the active ingredients in the medicine pair that exerted pharmacological effects by spectrum-effect relationship
study.
METHODS: Firstly, MTT assays, ELISA, fluorescence probe analysis, and Western blot were employed to
study the underlying mechanisms of the medicine pair in alcohol-induced BRL-3A cells by examining pharmacodynamic indexes and related protein expression. Secondly, HPLC method was established for chemical chromatograms of the medicine pair with different ratios and the sample extracted by different solvents. Then, principal component analysis, pearson bivariate correlation analysis and grey relational analysis were applied for development of the spectrum-effect correlation between pharmacodynamic indexes and HPLC chromatograms. Moreover, prototype components and their metabolites in vivo were identified by the HPLC-MS method.
RESULTS: Flos Puerariae-Semen Hoveniae medicine pair remarkably increased cell viability, decreased the activity of ALT, AST, TC and TG, reduced the generation of TNF-α, IL-1β, IL-6, MDA and ROS, increased the activity of SOD and GSH-Px, reduced protein expression of CYP2E1, compared with alcohol-induced BRL-3A cells. The medicine pair modulated the PI3K/AKT/mTOR signaling pathways by up-regulating the levels of phospho-PI3K, phospho-AKT and phospho-mTOR. Also, the results of the spectrum-effect relationship
study showed that P1 (chlorogenic acid), P3 (daidzin), P4 (6″-O-xylosyl-glycitin), P5 (glycitin), P6 (unknown), P7 (unknown), P9 (unknown), P10 (6″-O-xylosyl-tectoridin), P12 (tectoridin) and P23 (unknown) can be considered as the main components of the medicine pair in the treatment of ALD. Furthermore, 6″-O-xylosyl-tectoridin, tectoridin, daidzin, 6″-O-xylosyl-glycitin and glycitin can be absorbed into the blood and showed clear metabolic and excretion behaviors in rats.
CONCLUSIONS: In this
study, the hepatoprotective effects and the pharmacology mechanism of Flos Puerariae-Semen Hoveniae medicine pair in alcohol-induced BRL-3A cells were initially investigated and revealed. Through the spectrum-effect relationship study, the potential pharmacodynamic constituents such as daidzin, 6″-O-xylosyl-glycitin, 6″-O-xylosyl-tectoridin, glycitin, and tectoridin exert pharmacological effects on alcohol-induced oxidative stress and inflammation by modulating the PI3K/AKT/mTOR signaling pathways. This study provided experimental basis and data support for revealing the pharmacodynamic substance basis and pharmacology mechanism in the treatment of ALD. Moreover, it provides a robust mean of exploring the primary effective components responsible for the bioactivity of complicated TCM.