基于滑膜液微透析-生物标志物的藏医药浴对RA“干黄水”机制研究

Rheumatoid arthritis (RA) is a dominant disease which belonging to "Zhenbubing" of Tibetan medicine. Heat and "huangshui" are the causes and closely related to metabolic markers. Tibetan medicine Wuwei Ganlu is the classic basis foundation for the treatment of "Zhenbubing" in Tibetan medicine, and it has exact effect on "zhenbubing" caused by "huangshui" disorder due to the "Sanyin" imbalance. Our previous research（NSFC 81260672）shows that the Tibetan medicine bath is associated with the regulation of gene expression on the JAK-STAT, MAPK, and NF-κB signaling pathways in RA rat model synovial cells. Therefore, this topic proposes a hypothesis of "Microdialysis - biomarkers can reveal the influence of the effective components of the Wuwei Ganlu bath on the metabolic profile of RA in real time and dynamically". A microdialysis technique would be used to collect the synovial fluid samples of the knee joints of the Tibetan medicine Wuwei Ganlu Bath before and after treatment in an animal model of RA rats. The endogenous metabolite profiles and drug absorption profiles will be detected and established by UPLC-Q-TOF/MS technology. According to the analysis of metabolic pathways to elucidate, the mechanism of action of Tibetan medicine bath to prevent and treat RA would be explained. Applying data mining technology to backtrack to find out the chemical composition information associated with biomarkers in the spectrum of drug metabolism, and clarify the "Gan Huangshui" mechanism of Tibetan medicine bath to prevent and treat RA.

类风湿性关节炎（RA）属于藏医“真布病”范畴，是藏医特色优势病种，热邪与“黄水”沉着是病缘，与代谢标志物密切相关。藏药五味甘露浴是藏医外治“真布病”的经典基础方，对“三因”失调致“黄水”紊乱引起的“真布病”疗效确切。本课题组前期研究（NSFC 81260672）表明：藏医药浴对RA模型大鼠滑膜细胞JAK-STAT、MAPK和NF-κB等信号通路上基因表达的调控与RA炎症相关。故本课题提出“微透析-生物标志物可以实时、动态地揭示藏药五味甘露浴有效组分对RA的代谢轮廓的影响”。采用RA大鼠动物模型，采集藏药五味甘露浴治疗前后的膝关节滑膜液微透析样本，采用UPLC-Q-TOF/MS技术进行检测，建立内源性代谢物谱及药物吸收成分谱，根据代谢通路分析，阐明藏医药浴防治RA的作用机制，应用数据挖掘技术逆向追踪，找出药物代谢成分谱中与生物标志物相关联的化学组分信息，阐明藏医药浴防治RA的“干黄水”机制。

类风湿性关节炎（RA）属于藏医“真布病”范畴，是藏医特色优势病种，热邪与“黄水”沉着是病缘，与代谢标志物密切相关。故本课题提出“生物标志物的变化可以揭示藏药五味甘露浴有效组分对RA的代谢轮廓的影响”。我们建立了CIA大鼠模型给予藏药浴治疗，结果藏药五味甘露药浴颗粒（WGMG）组明显减轻大鼠足趾肿胀度以及脾脏系数（P<0.01），说明WGMG可以改善RA关节的炎症反应，治疗效果佳。采用UPLC-Q-TOF/MS技术分析WGMG全方的化学成分，采集治疗前后动物血清、膝关节滑膜液进行成分分析，与香豆酸、阿魏酸等16个对照品进行比对，鉴定出化学成分药物中有89个，血清中有28个，滑膜液中有21个，均为复方中原型成分。采用代谢组学PCA、PLS-DA等分析方法，发现WGMG组与CIA组大鼠体内物质代谢存在差异性。筛选出胆酸、半胱氨酸等9个潜在生物标志物，WGMG抗RA可能与改善谷胱甘肽代谢、戊糖磷酸途径、半胱氨酸和蛋氨酸代谢通路等有关。采用网络药理学获得WGMG化学成分和RA疾病靶点并整合，得到山奈酚、反式阿魏酸等10个相关的活性成分，主要作用于TNF、AKT1和MAPK8等15个关键靶点，涉及MAPK、NF-κB、IL-17和Jak-STAT等18条内分泌调节、疼痛等相关的信号通路。建立了藏药刺柏的质量标准草案，收载于2021年8月1日实施的《四川省藏药材标准》。并对刺柏中槲皮苷、穗花杉双黄酮和竹柏双黄酮A等化学成分进行薄层鉴别和含量测定。采用ELISA、免疫组化、Western Blot、RT-PCR检测发现WGMG通过下调NLRP3通路上NF-κB、TNF-α、IL-1β、IL-18等致炎因子及上调抗炎因子IL-4，有效改善了RA的关节滑膜增生与炎症。WGMG抗RA的作用机制与抑制NLRP3过度激活，达到消肿止痛、改善RA关节软骨与骨的目的相关。综上，本课题完成了内源性代谢物谱及药物吸收成分谱，根据代谢通路分析，找出了药物代谢成分谱中与生物标志物相关联的化学组分信息，基本阐明了藏医药浴防治RA的“干黄水”机制，证明了其对“三因”失调致“黄水”紊乱引起的“真布病”疗效确切。此外，藏药刺柏的质量标准草案的建立对其质量控制具有一定的参考价值。