Department of Natural Medicinal Chemistry, College of Pharmacy, Inner Mongolia Medical University, Jinshan Development Zone, Hohhot 010110, PR China; Engineering Technology Research Center of Pharmacodynamic Substance and Quality Control of Mongolian Medicine in Inner Mongolia, Hohhot 010110, PR China.
Inner Mongolian International Mongolian Hospital, University East Street, Hohhot 010065, PR China.
Phytomedicine. 2022 Oct;105:154382. doi: 10.1016/j.phymed.2022.154382. Epub 2022 Aug 3.
Nutmeg-5, an ancient and classic formula in traditional Mongolian medicine comprising five kinds of traditional Chinese medicine, is widely used in the treatment of myocardial infarction (MI, called heart "Heyi" disease in Mongolian medicine). Cardiac fibrosis plays a critical role in the development and progression of heart failure after MI. However, the material basis and pharmacological mechanisms of the effect of Nutmeg-5 on cardiac fibrosis after MI remain unclear.
The aim of this study was to first explore the potential material basis and molecular mechanism of action of Nutmeg-5 in improving cardiac fibrosis after MI via a multiomics approach.
The constituents in Nutmeg-5 were identified by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). High-performance liquid chromatography (HPLC) and gas chromatography (GC)-based fingerprints of Nutmeg-5 were analysed, and characteristic peaks were identified by comparison to standard samples. A rat MI model was created by permanent ligation of the left anterior descending artery. The protective effect of Nutmeg-5 on cardiac fibrosis after MI was evaluated by tissue histology and measurement of the serum biomarkers of myocardial injury. Cardiac fibrosis levels were evaluated by Sirius red staining. Differentially expressed proteins in the myocardium and metabolites in the serum were explored by proteomic and untargeted metabolome analyses, respectively. Pearson correlation analysis was performed to explore the association between serum metabolites and myocardial proteins.
A total of 67 constituents were identified in Nutmeg-5 by UPLC-MS/MS. Sixteen components were identified in the fingerprint of Nutmeg-5 by comparison with a standard sample. Six lactones were isolated from Nutmeg-5 and quantified by HPLC and GC. MI was significantly alleviated in Nutmeg-5-treated rats compared to MI rats, as demonstrated by their decreased mortality, improved cardiac function, and attenuated cardiac fibrosis and myocardial injury. A total of 252 significant differential metabolites were identified in plasma between model and Nutmeg-5-treated rats by untargeted metabolome analysis. Among these, 36 critical metabolites were associated with Nutmeg-5 activity. Proteomic analysis identified 338 differentially expressed proteins in the rat myocardium between MI and Nutmeg-5-treated rats, including 204 upregulated and 134 downregulated proteins. Protein set enrichment analysis revealed that Nutmeg-5 treatment significantly inhibited the extracellular matrix (ECM)-receptor interaction pathway, which was activated in the myocardium of MI rats. A significant decrease in collagen and alpha smooth muscle actin expression levels was found in the myocardium of Nutmeg-5-treated rats compared to MI rats. These results illustrated that Nutmeg-5 had a significant protective effect on cardiac fibrosis after MI. A significant correlation was found between the ECM-receptor interaction pathway in the myocardium and critical metabolites in the serum. In addition, there were positive correlations between the levels of critical metabolites and the expression levels of transforming growth factor (TGF)-β1 and Smad2 in the rat myocardium.
Nutmeg-5 alleviated cardiac fibrosis after MI in rats by inhibiting the myocardial ECM-receptor interaction pathway and TGF-β1/Smad2 signalling, which was achieved by regulating plasma metabolites.
荜茇-5 是蒙医传统医学中的一种古老而经典的配方，由五种中药组成，广泛用于治疗心肌梗死（在蒙医中称为“心脏 Heyi”疾病）。心肌纤维化在心肌梗死后心力衰竭的发展和进展中起着关键作用。然而，荜茇-5 改善心肌梗死后心肌纤维化的物质基础和药理机制仍不清楚。
本研究旨在通过多组学方法首先探讨荜茇-5 改善心肌梗死后心肌纤维化的潜在物质基础和作用机制。
采用超高效液相色谱-串联质谱联用技术（UPLC-MS/MS）鉴定荜茇-5 中的成分。分析荜茇-5 的高效液相色谱（HPLC）和气相色谱（GC）指纹图谱，并通过与标准样品比较鉴定特征峰。通过永久性结扎左前降支动脉建立大鼠心肌梗死模型。通过组织病理学和心肌损伤血清标志物测量评估荜茇-5 对心肌梗死后心肌纤维化的保护作用。采用天狼猩红染色评估心肌纤维化程度。通过蛋白质组学和非靶向代谢组学分析分别探讨心肌中的差异表达蛋白和血清中的差异代谢物。采用 Pearson 相关性分析探讨血清代谢物与心肌蛋白之间的关联。
通过 UPLC-MS/MS 共鉴定出荜茇-5 中的 67 种成分。通过与标准样品比较，在荜茇-5 的指纹图谱中鉴定出 16 种成分。从荜茇-5 中分离出 6 种内酯，并通过 HPLC 和 GC 进行定量。与心肌梗死大鼠相比，荜茇-5 治疗的大鼠死亡率降低、心功能改善、心肌纤维化和心肌损伤减轻，表明心肌梗死后大鼠的心肌梗死明显缓解。通过非靶向代谢组学分析，在模型和荜茇-5 治疗的大鼠血浆中共鉴定出 252 种显著差异代谢物。其中，36 种关键代谢物与荜茇-5 的活性有关。蛋白质组学分析在心肌梗死和荜茇-5 治疗的大鼠之间鉴定出 338 个差异表达蛋白，包括 204 个上调蛋白和 134 个下调蛋白。蛋白质集富集分析表明，荜茇-5 治疗显著抑制了心肌中细胞外基质（ECM）-受体相互作用途径，该途径在心肌梗死大鼠中被激活。与心肌梗死大鼠相比，荜茇-5 治疗的大鼠心肌中胶原蛋白和α平滑肌肌动蛋白的表达水平显著降低。这些结果表明荜茇-5 对心肌梗死后心肌纤维化具有显著的保护作用。在心肌中，ECM-受体相互作用途径与血清中的关键代谢物之间存在显著相关性。此外，关键代谢物的水平与大鼠心肌中转化生长因子（TGF）-β1 和 Smad2 的表达水平呈正相关。
荜茇-5 通过抑制心肌 ECM-受体相互作用途径和 TGF-β1/Smad2 信号通路，调节血浆代谢物，减轻大鼠心肌梗死后的心肌纤维化，从而发挥治疗作用。
