ABSTRACT
The Huashi Baidu Formula (HSBDF), a key Chinese medical drug, has a remarkable clinical efficacy in treating acute lung injury (ALI), and it has been officially approved by the National Medical Products Administration of China for drug clinical trials. Nevertheless, the regulated mechanisms of HSBDF and its active compounds in plasma against ALI were rarely studied. Based on these considerations, the key anti-inflammatory compounds of HSBDF were screened by molecular docking and binding free energy. The key compounds were further identified in plasma by LC/MS. Network pharmacology was employed to identify the potential regulatory mechanism of the key compounds in plasma. Next, the network pharmacological prediction was validated by a series of experimental assays, including CCK-8, EdU staining, test of TNF-α, IL-6, MDA, and T-SOD, and flow cytometry, to identify active compounds. Molecular dynamic simulation and binding interaction patterns were used to evaluate the stability and affinity between active compounds and target. Finally, the active compounds were subjected to predict pharmacokinetic properties. Molecular docking revealed that HSBDF had potential effects of inhibiting inflammation by acting on IL-6R and TNF-α. Piceatannol, emodin, aloe-emodin, rhein, physcion, luteolin, and quercetin were key compounds that may ameliorate ALI, and among which, there were five compounds (emodin, aloe-emodin, rhein, luteolin, and quercetin) in plasma. Network pharmacology results suggested that five key compounds in plasma likely inhibited ALI by regulating inflammation and oxidative damage. Test performed in vitro suggested that HSBDF (0.03125 mg/ml), quercetin (1.5625 µM), emodin (3.125 µM), and rhein (1.5625 µM) have anti-inflammatory function against oxidative damage and decrease apoptosis in an inflammatory environment by LPS-stimulation. In addition, active compounds (quercetin, emodin, and rhein) had good development prospects, fine affinity, and stable conformations with the target protein. In summary, this study suggested that HSBDF and its key active components in plasma (quercetin, emodin, and rhein) can decrease levels of pro-inflammatory factors (IL-6 and TNF-α), decrease expression of MDA, increase expression of T-SOD, and decrease cell apoptosis in an inflammatory environment. These data suggest that HSBDF has significant effect on anti-inflammation and anti-oxidative stress and also can decrease cell apoptosis in treating ALI. These findings provided an important strategy for developing new agents and facilitated clinical use of HSBDF against ALI.
ABSTRACT
Angelicae Sinensis Radix excels in activating blood, but the scientific mechanism has not been systematically analyzed, thus limiting the development of the medicinal. This study employed the computer-aided drug design methods, such as structural similarity-based target reverse prediction, complex network analysis, molecular docking, binding free energy calculation, cluster analysis, and ADMET(absorption, distribution, metabolism, excretion, toxicity) calculation, and enzyme activity assay in vitro, to explore the components and mechanism of Angelicae Sinensis Radix in activating blood. Target reverse prediction and complex network analysis yielded 40 potential anticoagulant targets of the medicinal. Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis indicated that the targets mainly acted on the complement and coagulation cascade signaling pathway to exert the anticoagulant function. Among them, the key enzymes thrombin(THR) and coagulation factor Xa(FXa) in coagulation cascade and thrombosis were the drug targets for thromboembolic diseases. At the same time, molecular docking and cluster analysis showed that the medicinal had high selectivity for FXa. According to binding free energy score, 8 potential active components were selected for enzyme activity assay in vitro. The results demonstrated that 8 components inhibited THR and FXa, and the inhibition was stronger on FXa than on THR. The pharmacophore model of 8 active compounds was constructed, which suggested that the components had the common pharmacophore AAHH. The ADMET calculation result indicated that they had good pharmacokinetic properties and were safe. Based on target reverse prediction, complex network analysis, molecular docking and binding free energy calculation, anticoagulant activity in vitro, spatial binding conformation of molecules and targets, pharmacophore model construction, and ADMET calculation, this study preliminarily clarified the material basis and molecular mechanism of Angelicae Sinensis Radix in activating blood from the perspective of big data, and calculated the pharmacology and toxicology parameters of the active components. Our study, for the first time, revealed that the medicinal had obvious selectivity and pertinence for different coagulation proteins, reflecting the unique effect of different Chinese medicinals and the biological basis. Therefore, this study can provide clues for precision application of Angelicae Sinensis Radix and the development of the blood-activating components with modern technology.