School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China.
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
J Ethnopharmacol. 2020 Oct 5;260:113051. doi: 10.1016/j.jep.2020.113051. Epub 2020 Jun 4.
Ermiao fang (EMF) is a traditional Chinese medicinal herbal formula from ancient times and recorded in the pharmacopeia of the People's Republic of China. It is composed of two typical Chinese herbal medicines, Cortex Phellodendri (Huangbai), the bark of Phellodendron chinensis Schneid. (Rutaceae), and Rhizoma Atractylodis (Cangzhu), the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae). EMF has been clinically used for the treatment of endometritis for many years in China.
This study was aimed to identify the active ingredients, potential targets, and mechanism of action of EMF for the treatment of endometritis.
In this research, the pharmacological effects of EMF on endometritis were first evaluated by establishing a rat model of endometritis. A network pharmacology-based analytical strategy was then used to predict its targets and signaling pathways. An endometritis-related protein target and compound database was built for EMF. The compounds in EMF and those absorbed into the blood were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). High-throughput virtual screening and molecule docking methods were used to predict the protein targets of EMF. The surface plasmon resonance analysis (SPR) method was used to validate the affinity between the compound and proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to predict the related pathways. Western blotting analysis was used to evaluate the expression of key proteins in the related pathways.
The animal study showed that EMF could reduce uterine inflammation in rats with endometritis. Then, an ingredient database including 187 compounds and a protein target database including 836 proteins were constructed. Twenty-four compounds in EMF were identified by UHPLC-Q-TOF/MS, among which eight compounds were present in rat plasma after an oral administration of EMF. Afterward, 39 potential target proteins were predicted by the high-throughput screening method, and 20 of them were selected after further screening using molecular docking. Subsequently, an ingredient-target network was constructed, and the target proteins were classified into the NF-κB and MAPK signal pathways by KEGG pathway enrichment analysis. Finally, the affinity between the active ingredients and the target proteins was verified by SPR. The Western blotting analysis showed that EMF significantly inhibited the elevated NF-κB and MAPK pathway proteins in rats with endometritis.
EMF exhibited a significant pharmacological effect on rats with endometritis. Network pharmacology analysis revealed that eight compounds were absorbed into the blood after oral administration and interacted with 20 targets. Western blotting analysis indicated that EMF exerted anti-inflammatory effects by inhibiting the NF-κB and MAPK signaling pathway proteins in the treatment of endometritis.
二妙方（EMF）是一种传统的中药方剂，来源于古代，记录在中国药典中。它由两种典型的中草药组成，黄柏（Huangbai），即黄皮树（Phellodendron chinensis Schneid.）的树皮（芸香科），和苍术（Cangzhu），即白术（Atractylodes lancea (Thunb.) DC.）的根茎（菊科）。EMF 在中国临床上已用于治疗子宫内膜炎多年。
本研究旨在确定 EMF 治疗子宫内膜炎的活性成分、潜在靶点和作用机制。
在这项研究中，首先通过建立子宫内膜炎大鼠模型来评估 EMF 对子宫内膜炎的药理作用。然后，采用基于网络药理学的分析策略来预测其靶点和信号通路。为 EMF 建立了一个子宫内膜炎相关蛋白靶标和化合物数据库。通过超高效液相色谱-四级杆飞行时间质谱联用（UHPLC-Q-TOF/MS）鉴定 EMF 中的化合物和被吸收到血液中的化合物。采用高通量虚拟筛选和分子对接方法预测 EMF 的蛋白靶标。表面等离子体共振分析（SPR）方法用于验证化合物与蛋白之间的亲和力。京都基因与基因组百科全书（KEGG）通路富集分析用于预测相关通路。Western blot 分析用于评估相关通路中关键蛋白的表达。
动物研究表明，EMF 可减轻子宫内膜炎大鼠的子宫炎症。然后，构建了一个包含 187 种化合物的成分数据库和一个包含 836 种蛋白质的蛋白质靶标数据库。通过 UHPLC-Q-TOF/MS 鉴定出 EMF 中的 24 种化合物，其中 8 种化合物在大鼠口服 EMF 后存在于血浆中。随后，通过高通量筛选方法预测出 39 个潜在的靶标蛋白，进一步通过分子对接筛选后选择了 20 个。随后，构建了一个成分-靶标网络，通过 KEGG 通路富集分析将靶标蛋白分类为 NF-κB 和 MAPK 信号通路。最后，通过 SPR 验证了活性成分与靶蛋白之间的亲和力。Western blot 分析表明，EMF 显著抑制了子宫内膜炎大鼠中升高的 NF-κB 和 MAPK 通路蛋白。
EMF 对子宫内膜炎大鼠具有显著的药理作用。网络药理学分析表明，口服给药后有 8 种化合物被吸收到血液中，并与 20 个靶标相互作用。Western blot 分析表明，EMF 通过抑制 NF-κB 和 MAPK 信号通路蛋白在治疗子宫内膜炎中发挥抗炎作用。
