Yeqing Chen, Xinsheng Fan, Liping Zhou, Fangyuan Hu, Pengli Wang
College of Integrated Chinese and Western Medicine, College of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
College of Integrated Chinese and Western Medicine, College of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Phytomedicine. 2022 Jun;100:154040. doi: 10.1016/j.phymed.2022.154040. Epub 2022 Mar 11.
The Shuangshen Pingfei formula (SSPF), a classic Chinese medicine derivative formula, has been shown to exert therapeutic effects on idiopathic pulmonary fibrosis (IPF). However, the quality control compounds of SSPF remain unclear.
To select and confirm Q-markers of SSPF based on network pharmacology, cytobiology, animal-based pharmacodynamics, and phytochemical and pharmacokinetic analyses.
A compound-target network was constructed based on previous research. In addition, high-degree compounds of SSPF were chosen as potential Q-marker candidates. Animal and cytological experiments were performed to verify key targets of IPF. Haematoxylin-eosin and Masson's trichrome staining were used to observe lung tissue pathology. Cytokine levels in the bronchoalveolar lavage fluid (BALF) were measured using ELISA kits. Gene and protein expression levels were determined using PCR and western blotting, respectively. The contents of Q-marker candidates in different batches of SSPF were then determined for traceability research, and the quality consistency of SSPF was objectively evaluated using principal component analysis (PCA). Finally, pharmacokinetic research was performed, and candidates with desirable metabolite and bioavailability parameters were confirmed as Q-markers of SSPF.
The compound-target network included 56 compounds and 14 therapeutic targets. Animal experiments showed that SSPF attenuates lung fibrosis. SSPF decreased CC motif chemokine 2 (CCL2) and CC chemokine receptor type 2 (CCR2) levels in the BALF and downregulated the gene and protein expression of IPF therapy-related molecules, such as 5-hydroxytryptamine receptor 2A (HTR2A), CCL2, and CCR2, in the lungs. Cell experiments showed that nine Q-marker candidates in SSPF regulated the expression of CCL2 and CCR2, as predicted. Phytochemical analysis and PCA indicated that the qualities of SSPF in the nine batches were relatively stable. Pharmacokinetic studies demonstrated that mangiferin, salvianolic acid B, tanshinone IIA, naringin, and glycyrrhizic acid could be effectively absorbed into rat plasma, which ensured desirable bioavailability and confirmed their roles as Q-markers to represent anti-pulmonary fibrotic activity.
Our study is an integrated strategy, based on network pharmacology with experimental verification and phytochemical and pharmacokinetic analyses that provides a novel approach for Q-marker selection and validation of SSPF for IPF treatment.
双参平肺方（SSPF）是一种经典的中药衍生方剂，已被证明对特发性肺纤维化（IPF）具有治疗作用。然而，SSPF的质量控制化合物仍不明确。
基于网络药理学、细胞生物学、动物药效学以及植物化学和药代动力学分析，筛选并确认SSPF的Q-标志物。
根据先前研究构建化合物-靶点网络。此外，选择SSPF的高连接度化合物作为潜在的Q-标志物候选物。进行动物和细胞学实验以验证IPF的关键靶点。采用苏木精-伊红染色和Masson三色染色观察肺组织病理学变化。使用ELISA试剂盒检测支气管肺泡灌洗液（BALF）中的细胞因子水平。分别使用PCR和蛋白质印迹法测定基因和蛋白质表达水平。然后测定不同批次SSPF中Q-标志物候选物的含量进行溯源研究，并使用主成分分析（PCA）客观评价SSPF的质量一致性。最后进行药代动力学研究，将具有理想代谢物和生物利用度参数的候选物确认为SSPF的Q-标志物。
化合物-靶点网络包含56种化合物和14个治疗靶点。动物实验表明，SSPF可减轻肺纤维化。SSPF降低了BALF中CC基序趋化因子2（CCL2）和CC趋化因子受体2型（CCR2）的水平，并下调了肺中与IPF治疗相关分子如5-羟色胺受体2A（HTR2A）、CCL2和CCR2的基因和蛋白质表达。细胞实验表明，SSPF中的9种Q-标志物候选物如预期那样调节了CCL2和CCR2的表达。植物化学分析和PCA表明，9个批次的SSPF质量相对稳定。药代动力学研究表明，芒果苷、丹酚酸B、丹参酮IIA、柚皮苷和甘草酸可有效吸收进入大鼠血浆，这确保了理想的生物利用度，并证实了它们作为代表抗肺纤维化活性的Q-标志物的作用。
我们的研究是一种基于网络药理学并结合实验验证以及植物化学和药代动力学分析的综合策略，为IPF治疗中SSPF的Q-标志物选择和验证提供了一种新方法。
