Guangzhou Key Laboratory of Formula-Pattern of Traditional Chinese Medicine, Formula-Pattern Research Center, School of Traditional Chinese Medicine, Jinan University, Guangzhou, 510632, China.
College of Pharmacy and International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education, Jinan University, Guangzhou, 510632, PR China.
J Ethnopharmacol. 2021 Oct 28;279:114396. doi: 10.1016/j.jep.2021.114396. Epub 2021 Jul 8.
The overall therapeutic effect of traditional Chinese medicine formulae (TCMF) was achieved by the interactions of multiple components with multiple targets. However, current pharmacology research strategies have struggled to identify effective substance groups and encountered challenges in elucidating the underlying mechanisms of TCMF.
In this study, a comprehensive strategy was proposed and applied to elucidate the interactions of the multiple components that underlie the functions of the famous TCMF: Xian-Ling-Gu-Bao (XLGB) capsule on bone metabolism in vivo and to elucidate the molecular mechanisms underlying the effects of XLGB on bone cells, especially on osteoblasts.
The efficacy of XLGB in the protection against bones loss in ovariectomized (OVX) rats was confirmed by Micro-CT analysis. The anti-osteoporosis mechanism involved in the systemic regulatory actions of XLGB was elucidated by transcriptome sequencing analysis on bone marrow mesenchymal stem cells isolated from OVX rats. Moreover, the components absorbed in XLGB-treated plasma were characterized by mass spectrometry analysis, and subsequently, a standardized preparation process of drug-containing plasma was established. The synergistic osteogenic effect of the multiple components in plasma was investigated by a combination and then knockout of components using pre-osteoblast MC3T3-E1 cells. In order to decipher the underlying mechanism of XLGB, the targets of the absorbed components on bone were predicted by target prediction and network pharmacology analysis, then several interactions were validated by biochemical and cell-based assay.
A total of 18 genes, including HDC, CXCL1/2, TNF, IL6 and Il1b, were newly found to be the major target genes regulated by XLGB. Interestingly, we found that a combination of the three absorbed components, i.e. MSP, rather than their single form at the same concentration, stimulated the formation of calcified nodules in MC3T3-E1 cells, suggesting a synergistic effect of these components. Besides, target prediction and experimental validation confirmed the binding affinity of corylin and icaritin for estrogen receptor α and β, the inhibitory activity of isobavachin and isobavachalcone on glycogen synthase kinase-3β, and the inhibitory activity of isobavachalcone on cathepsin K. The cell-based assay further confirmed the result of the biochemical assay. A network that integrated absorbed components of XLGB-targets-perturbation genes-pathways against osteoporosis was established.
Our current study provides a new systemic strategy for discovering active ingredient groups of TCM formulae and understanding their underlying mechanisms.
中药方剂（TCMF）的整体治疗效果是通过多种成分与多个靶点的相互作用实现的。然而，目前的药理学研究策略在识别有效物质组方面遇到了困难，并在阐明 TCMF 的潜在机制方面遇到了挑战。
本研究提出并应用了一种综合策略，以阐明著名中药方剂仙灵骨葆（XLGB）胶囊在体内对骨代谢的多种成分的相互作用，并阐明 XLGB 对骨细胞，特别是成骨细胞的作用的分子机制。
通过 Micro-CT 分析证实了 XLGB 对去卵巢（OVX）大鼠骨丢失的保护作用。通过对 OVX 大鼠骨髓间充质干细胞进行转录组测序分析，阐明了 XLGB 全身调节作用涉及的抗骨质疏松机制。此外，通过质谱分析鉴定了 XLGB 处理后血浆中吸收的成分，并随后建立了含药血浆的标准化制备工艺。使用预成骨 MC3T3-E1 细胞对血浆中的多种成分进行组合和敲除，研究其在体内的协同成骨作用。为了解释 XLGB 的潜在机制，通过靶预测和网络药理学分析预测了吸收成分在骨上的靶标，然后通过生化和基于细胞的测定验证了几个相互作用。
共发现 18 个基因，包括 HDC、CXCL1/2、TNF、IL6 和 Il1b，它们是 XLGB 主要调控的靶基因。有趣的是，我们发现三种吸收成分的组合，即 MSP，而不是它们以相同浓度的单一形式，刺激 MC3T3-E1 细胞形成钙化结节，这表明这些成分具有协同作用。此外，靶预测和实验验证证实了 corylin 和 icaritin 对雌激素受体 α 和 β 的结合亲和力，isobavachin 和 isobavachalcone 对糖原合酶激酶-3β 的抑制活性，以及 isobavachalcone 对组织蛋白酶 K 的抑制活性。基于细胞的测定进一步证实了生化测定的结果。建立了一个整合了 XLGB 吸收成分-靶标-骨质疏松途径干扰基因的网络。
本研究为发现中药方剂的有效成分组并了解其潜在机制提供了一种新的系统策略。
