Elucidating the chemical interaction effects of herb pair Danshen-Chuanxiong and its anti-ischemic stroke activities evaluation

Salvia miltiorrhiza Bunge (Danshen) and Ligusticum chuanxiong Hort. (Chuanxiong) is the core herb pair in traditional Chinese medicines (TCMs) formulae for treating ischemic stroke. However, the synergistic effect of Danshen-Chuanxiong against anti-ischemic stroke and its compatibility mechanism remains unclear. This study aimed to uncover the compatibility mechanism of Danshen-Chuanxiong against ischemic stroke through chemical profiling, pharmacodynamics evaluation, network pharmacology and experimental validation. Ultra-high performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS) and UHPLC connected with tandem triple quadrupole mass spectrometry (QQQ-MS) were utilized to conduct the chemical interaction analysis. Then the synergistic effects of Danshen-Chuanxiong against ischemic stroke were comprehensively evaluated by the middle cerebral artery occlusion reperfusion (MCAO/R) mice model, zebrafish ischemic stroke model and glutamic acid-induced PC12 cells injury model. Afterwards, network pharmacology and molecular docking were applied to dissect the significant active compounds and potential mechanisms. Finally, the key target proteins were experimentally validated by Western blot. 83 compounds were characterized in Danshen-Chuanxiong by UHPLC-QTOF-MS analysis, and 4 compounds were tentatively identified for the first time. The quantification results (24 accurately identified compounds) in 13 proportions of Danshen-Chuanxiong revealed that Danshen significantly increased the dissolution of most phthalides (from Chuanxiong), while Chuanxiong facilitated the dissolution of most phenolic acids (from Danshen) in solution. The anti-ischemic stroke effects of Danshen-Chuanxiong were significantly better than Danshen or Chuanxiong in attenuating infarct size, reducing brain edema and neurological scores in MCAO/R mice. Also, compared with single herbs, this herb pair exerted better effects of suppressing the incidence of cerebral thrombosis in zebrafish, and increasing the cell viability of glutamic acid-induced PC12 cells. In network pharmacology, 7 effective compounds (rosmarinic acid, chlorogenic acid, salvianolic acid B, (Z)-ligustilide, ferulic acid, caffeic acid, tanshinone IIA) and 5 hub targets (AKT, TNF−α, IL-1β, CASP3 and BCL2) as well as 4 key pathways were predicted. Western blot results showed that Danshen-Chuanxiong exert therapeutic effects mainly through decreasing the protein expressions of TNF-α, IL-1β and Cleaved-CASP3, elevating the levels of p-AKT and BCL2. This work provided an integration strategy for uncovering the synergistic effects and compatibility mechanism of Danshen-Chuanxiong herb pair for treating ischemic stroke, and laid foundation for the further development and utilization of this herb pair.