DataSheet1_Integrated Metabolomics and Network Pharmacology Study on the Mechanism of Kangfuxiaoyan Suppository for Treating Chronic Pelvic Inflammatory Disease.docx

Kangfuxiaoyan suppository (KFXYS) is a commonly used traditional Chinese medicine (TCM) preparation for the treatment of chronic pelvic inflammatory disease (CPID) clinically, and its safety and effectiveness have been well verified. However, the potential mechanism remains unclear. The integrated strategy of metabolomics and network pharmacology was employed in the study to reveal the potential mechanism of KFXYS in the treatment of CPID. Our research consists of five steps. First, the effect of KFXYS in reversing uterine inflammation indexes was verified. Second, based on the comprehensive characterization of 123 chemical ingredients of KFXYS, the ingredients of KFXYS absorbed into blood were identified by UPLC-Q-TOF/MS, then ADME research was carried out on the main ingredients. Third, the differential metabolites with significant correlation to inflammatory indexes were discovered by metabolomics and correlation analysis. Fourth, the potential targets and pathways of KFXYS in treating CPID were predicted by network pharmacology based on the ingredients which had good ADME behavior. Fifth, the proteins in common pathways of metabolomics and network pharmacology were used to screen the key targets from the potential targets of network pharmacology, and the potential mechanism of KFXYS in treating CPID was clarified. As a result, KFXYS significantly reversed the uterine inflammation indexes, including IL-1 and IL-6. The ingredients absorbed into blood including matrine, sophocarpine, aloin, esculetin-O-glucuronide, 7,4′-dihydroxyisoflavone-O-glucuronide, and 4′-methoxyisoflavone-7-O-glucuronide had good ADME behavior in vivo. Among the differential metabolites, Leukotriene A4, 5-Hydroxyindoleacetic acid, Ornithine, Arginine, and PC (20:1 (11Z)/20:4 (8Z,11Z,14Z,17Z)) were significant correlation to inflammation indexes. The integration analysis of metabolomics and network pharmacology shows that KFXYS may regulate the key targets including ARG1, NOS2, NOS3, etc. We speculate that ingredients of KFXYS, such as matrine, sophocarpine, aloin etc. act on the key proteins including ARG1, NOS2, and NOS3, to exert anti-inflammatory effect.

康腹消炎栓（Kangfuxiaoyan Suppository, KFXYS）是临床常用于治疗慢性盆腔炎（chronic pelvic inflammatory disease, CPID）的中药制剂，其安全性与有效性已得到充分验证，但潜在作用机制尚未阐明。本研究采用代谢组学与网络药理学相结合的整合策略，以揭示KFXYS治疗慢性盆腔炎的潜在分子机制。本研究共分为五个步骤：其一，验证KFXYS对子宫炎症指标的逆转作用；其二，在全面表征KFXYS所含123种化学成分的基础上，通过超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）鉴定其吸收入血的活性成分，并对核心成分开展吸收、分布、代谢、排泄（ADME）特征研究；其三，通过代谢组学及相关性分析，筛选出与炎症指标显著相关的差异代谢物；其四，基于具备良好ADME特征的入血成分，通过网络药理学预测KFXYS治疗慢性盆腔炎的潜在靶点与通路；其五，选取代谢组学与网络药理学共有的通路蛋白，从网络药理学预测的潜在靶点中筛选核心靶点，进而阐明KFXYS治疗慢性盆腔炎的潜在作用机制。研究结果表明，KFXYS可显著逆转子宫炎症相关指标，包括白细胞介素-1（Interleukin-1, IL-1）与白细胞介素-6（Interleukin-6, IL-6）。其吸收入血的成分包括苦参碱、槐果碱、芦荟素、七叶亭-O-葡萄糖醛酸苷、7,4'-二羟基异黄酮-O-葡萄糖醛酸苷及4'-甲氧基异黄酮-7-O-葡萄糖醛酸苷，上述成分在体内均表现出良好的ADME特征。在差异代谢物中，白三烯A4、5-羟吲哚乙酸、鸟氨酸、精氨酸及磷脂酰胆碱PC(20:1(11Z)/20:4(8Z,11Z,14Z,17Z))与炎症指标存在显著相关性。代谢组学与网络药理学的整合分析显示，KFXYS可能通过调控精氨酸酶1（Arginase 1, ARG1）、一氧化氮合酶2（Nitric Oxide Synthase 2, NOS2）、一氧化氮合酶3（Nitric Oxide Synthase 3, NOS3）等核心靶点发挥生物学作用。本研究推测，KFXYS中的苦参碱、槐果碱、芦荟素等成分可通过作用于ARG1、NOS2及NOS3等核心蛋白，进而发挥抗炎功效。