基于LGR4靶点和在线定向捕获的“山茱萸-白芍”酒炙配伍治疗类风湿关节炎物质基础和作用机制研究

Chines herbal medicine processing and compatibility are the characteristics of TCM clinic dialectical pharmacy, but the researches on discussing Chinese herbal medicine processing in compatibility are few, which result in that the selection of crude drug processed drugs in clinical composition is lacking scientific theory, but only depends on experience of clinicians, restricts the development of Chinese herbal medicine processing and science of prescription. Compatibility of Fructus corni and Paeoniae Radix Alba against rheumatoid arthritis effect is verified by clinic, in preliminary study, discover Fructus corni and Paeoniae Radix Alba occurs obvious changes after processing with wine,the function of nourishing liver and kidney reinforces, and give play to the function of anti-RA through regulating LGR4 gene and protein expression, but the synergia mechanism of frying wine’s compatibility has not researched, so this project takes Fructus corni and Paeoniae Radix Alba as representative, adopts the mode of “fixed composition/selective catch of etabolized product..active detection and recognition”, starts from witro and vivo comprehensive evaluation and molecular organism level,systematically analyzes and compares the change in chemical composition, pharmacodynamic action and internal dynamic process before and after processing with wine and compatibility of Fructus corni and Paeoniae Radix Alba, illustrates the pharmacodynamic material basis of wine enhancing the anti-RA effect and LGR4 target controlling mechanism, uncovers the scientific connotation of processing and compatibility. This research’s carrying out will provide theory evidence for clinical application of wining to Fructus corni and Paeoniae Radix Alba, and provide new thought for research on Chinese herbal medicine processing and compatibility.

中药炮制与配伍是中医临床辩证用药的特色，但在配伍中探讨中药炮制机理的研究鲜有报道，导致临床组方中饮片炮制品的选择缺乏科学理论依据而只能依赖于临床医师的经验，制约了中药炮制和方剂学的发展。山茱萸与白芍配伍抗类风湿关节炎效应为临床所验证，课题组前期研究发现山茱萸和白芍酒炙后化学成分发生了显著改变，补益肝肾作用增强，并通过调节LGR4基因和蛋白表达发挥抗类风湿关节炎作用，但其酒炙配伍的增效机制尚未研究。故本项目以”山茱萸-白芍“药对为代表，采用“固有成分/代谢产物选择性捕捉-活性检识”模式，从体内外整体评价和分子生物水平入手，系统研究比较山茱萸和白芍酒炙及配伍前后化学成分、药效作用和体内动态过程的变化，阐明其酒炙增强抗类风湿关节炎作用的药效物质基础及LGR4靶向调控机制，揭示炮制配伍的科学内涵。本研究的开展将为酒炙山茱萸和白芍临床应用提供理论依据，同时为中药炮制配伍研究提供新思路。

中药炮制与配伍是中医临床辩证用药的特色，但在配伍中探讨中药炮制机理的研究鲜有报道，导致临床组方中饮片炮制品的选择缺乏科学理论依据而只能依赖于临床医师的经验，制约了中药炮制和方剂学的发展。本研究通过UPLC-TOF/MS结合糖组学技术发现山茱萸酒炙前后推测鉴定出88种成分，山茱萸炮制后化学成分发生了质和量的变化，从次生代谢物组和糖组快速筛选区分了山茱萸生制品的化学标志物，并通过对化学标志物的模拟炮制，探寻了中药炮制化学的转化机制，揭示了山茱萸“酒制”整体品质的变化规律。通过UPLC-TOF/MS分析白芍中的主要成分为芍药苷、芍药内酯苷、苯甲酰芍药苷等成分发生转化，血清中有5个原药材的移行成分，2个结构已明确，分别为儿茶素和没食子酰基芍药苷，3个芍药苷及没食子酰基芍药苷的代谢产物。山茱萸-白芍酒炙配伍前后，在正、负离子模式共鉴定135个化合物，其中山茱萸和白芍炮制前后配伍共有27个物质发生了变化。从检测结果分析，酒山茱萸对酒白芍主要成分稳定性和溶出影响较大，酒山茱萸配伍酒白芍后对自身成分存在较大转化。山茱萸和白芍酒炙后配伍对白芍的影响可能大于山茱萸。通过构建小鼠和大鼠的CIA诱导的类风湿关节炎模型，发现酒山茱萸+酒白芍配伍给药组改善RA效果最为明显，生山茱萸+生白芍配伍给药组和酒山茱萸给药组次之，生白芍给药组、酒白芍给药组和生山茱萸给药组也有一定改善趋势。山茱萸-白芍酒炙配伍前后可显著影响经典破骨细胞形成通路OPG-RANKL-RANK，上调骨保护素OPG基因和蛋白表达，下调RANKL、RANK、TRAF6 和相关下游信号因子C-SRC、AKT、Gαq、GSK-3β及炎症相关P65、NF-кB1和破骨分化相关C-FOS基因和蛋白表达，抑制OPG-RANKL-RANK-NF-κB破骨形成及炎症相关信号通路；且显著上调破骨负调控基因LGR4抑制RANKL-RANK通路活化，进一步参与调控LGR4相关RANKL-LGR4-TRAF6-C-SRC-NF-NF-κB破骨负调控途径抑制破骨细胞分化。本研究的开展将为酒炙山茱萸和白芍临床应用提供理论依据，同时为中药炮制配伍研究提供新思路。