School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, China.
School of Life Sciences, Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, China.
J Ethnopharmacol. 2021 Oct 5;278:114283. doi: 10.1016/j.jep.2021.114283. Epub 2021 Jun 30.
Gegen Qinlian Decoction (GQD) (including: Puerariae lobatae (Willd.) Ohwi, radix; (short for Gengen) Glycyrrhiza uralensis Fisch., root and rhizome (short for Gancao), honeyed; Coptis chinensis Franch., rhizome (short for Huanglian); Scutellaria baicalensis Georgi, radix, boiled (short for S. baicalensis) has been widely used to treat inflammatory bowel disease (IBD) and colorectal cancer (CRC). To explore compatibility mechanism of GQD could be of advantage to investigate the complex principle of TCM, which might be conducive to the exploration of the modernization of TCM.
In this study, a strategy based on system pharmacology was constructed to uncover the multi-target regulation and compatibility mechanism of GQD on the Wnt signaling pathways.
The pharmacological network of GQD was constructed by TCMSP, DAVID, Uniprote database. The cell growth inhibitory effects of puerarin (PUE), wogonin (WOG), berberine (BER), and glycyrrhetinic acid (GLY) on SW480 cells were assessed using CCK-8 assay. The multi-target regulation and compatibility mechanism of combination PUE with GLY were examined by RNA-seq, HPLC-QQQ/MS, qRT- PCR and Western blot analysis.
Network pharmacology analysis indicated that PUE, WOG, BER and GLY were the active components in GQD and had a synergistic effect on the targets of the Wnt signaling pathway. Additionally, pharmacological experiments revealed that WOG, BER, and GLY inhibited activity of colorectal cancer (CRC) cell lines SW480 cells, and that PUE only exhibited effective antitumour activity when combined with GLY. CTNNB1, CCND1 and SMAD4 were identified as synergistic targets inhibited by PUE-GLY. Moreover, PUE-GLY could influence the Wnt signaling pathway by upregulating GSK3B and downregulating CTNNB1 synergistically. It also showed that GLY could effectively increase the intracellular content of PUE based on HPLC-QQQ/MS analysis, and this process was achieved by influencing the targets of the membrane's pathway, such as cell adhesion molecules, focal adhesion, and tight junctions.
GLY was revealed a multi-target mechanism, which could downregulate CTNNB1 as the active component and intervene in membrane proteins (CDH1, CADM1, ITGB2, ICAM1, ITGA1) as 'guide' in the formulae. Moreover, the mechanism of synergistic antitumour action of PUE (the active component of Monarch drug) and GLY (the active component of Guide drug) on the Wnt signaling pathway was explored systematically. It was a promising breakthrough for elucidating the scientific connotation of the compatibility of TCM formulae and provide a valuable and practicable methodology for clarifying the mechanisms of TCM.
葛根芩连汤（GQD）（包括：葛根、甘草、黄连；黄芩）已广泛用于治疗炎症性肠病（IBD）和结直肠癌（CRC）。探索 GQD 的配伍机制有助于研究中药的复杂原理，这可能有助于探索中药的现代化。
本研究构建了一种基于系统药理学的策略，以揭示 GQD 对 Wnt 信号通路的多靶点调节和配伍机制。
通过 TCMSP、DAVID、Uniprote 数据库构建 GQD 的药理网络。用 CCK-8 法评估葛根素（PUE）、汉黄芩素（WOG）、小檗碱（BER）和甘草酸（GLY）对 SW480 细胞的细胞生长抑制作用。通过 RNA-seq、HPLC-QQQ/MS、qRT-PCR 和 Western blot 分析研究 PUE 与 GLY 联合的多靶点调节和配伍机制。
网络药理学分析表明，PUE、WOG、BER 和 GLY 是 GQD 中的活性成分，对 Wnt 信号通路的靶点具有协同作用。此外，药理实验表明，WOG、BER 和 GLY 抑制结直肠癌细胞系 SW480 细胞的活性，而 PUE 仅与 GLY 联合时才表现出有效的抗肿瘤活性。CTNNB1、CCND1 和 SMAD4 被鉴定为 PUE-GLY 协同抑制的靶标。此外，PUE-GLY 可以通过协同上调 GSK3B 和下调 CTNNB1 来影响 Wnt 信号通路。基于 HPLC-QQQ/MS 分析，还表明 GLY 可以通过影响细胞膜途径的靶标（如细胞黏附分子、黏着斑和紧密连接），有效增加 PUE 的细胞内含量。
揭示了 GLY 的多靶点机制，作为活性成分，它可以下调 CTNNB1，并作为“向导”干预配方中的膜蛋白（CDH1、CADM1、ITGB2、ICAM1、ITGA1）。此外，系统探讨了 PUE（君药活性成分）和 GLY（臣药活性成分）对 Wnt 信号通路协同抗肿瘤作用的机制。这为阐明中药方剂配伍的科学内涵提供了有希望的突破，为阐明中药的机制提供了一种有价值和实用的方法。
