Sirtuin 1 (SIRT1) has emerged as a promising therapeutic target for many diseases such as neurodegenerative disorders. Resveratrol, a plant polyphenol, is known to induce SIRT1 expression and activity. To excavate active compounds that can increase SIRT1 transcription from FDA-approved drugs, a cell-based SIRT1 up-regulator screening assay was developed. 293A cells were transfected with a SIRT1-promoter-luciferase reporter gene construct, and the stably transfected cell line was selected to establish a specific SIRT1 expression assay in 96-well microplate format using resveratrol as a positive control. The evaluating parameter Z' value of 0.67 supported the stability and reliability of this cell-based high-throughput screening (HTS) assay. Screening of 1523 drugs identified 17 compounds up-regulating SIRT1 transcriptional activity by over 200 %. The 17 compounds could elevate SIRT1 transcription in a dose-dependent character, and their EC50 values were lower than that of resveratrol. The top three active compounds including belinostat, panobinostat and vorinostat belonging to pan-histone deacetylase inhibitor (HDACi) were furtherly observed to enhance the SIRT1 mRNA level and the H3 acetylated protein level in 293A cells, and interact with SIRT1 revealed by molecular docking. Moreover, belinostat, a potential candidate for the treatment of the severe neurodegenerative disease multiple sclerosis (MS), could also up-regulate the SIRT1 mRNA and protein levels in the spinal cords of experimental autoimmune encephalomyelitis (EAE) mice. These findings suggest that SIRT1 up-regulators originated from this HTS assay might become new drug candidates for the treatment of neurodegenerative disease, which provides an approach to repositioning approved drugs through HTS assay.

Introduction: Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Recanalization of blocked blood vessels and restoring blood supply to ischemic brain tissue are crucial for post-stroke rehabilitation. The decoction Naodesheng (NDS) composed of five Chinese botanical drugs, including Panax notoginseng (Burk.) F. H. Chen, Ligusticum chuanxiong Hort., Carthamus tinctorius L., Pueraria lobata (Willd.) Ohwi, and Crataegus pinnatifida Bge., is a blood-activating and stasis-removing herbal medicine commonly used for the clinical treatment of cerebrovascular diseases in China. However, the material basis of NDS on the effects of blood circulation improvement and vascular tone regulation remains unclear.Methods: A database comprising 777 chemical metabolites of NDS was constructed. Then, the interactions between various herbal metabolites of NDS and five vascular tone modulation G-protein-coupled receptors (GPCRs), including 5-HT1AR, 5-HT1BR, β2-AR, AT1R, and ETBR, were assessed by molecular docking. Using network analysis and vasomotor experiment of the cerebral basilar artery, the potential material basis underlying the vascular regulatory effects of NDS was further explored.Results: The Naodesheng Effective Component Group (NECG) was found to induce relaxation of rat basilar artery rings precontracted using Endothelin-1 (ET-1) and KCl in vitro in a dose-dependent manner. Several metabolites of NDS, including C. tinctorius, C. pinnatifida, and P. notoginseng, were found to be the main plant resources of metabolites with high docking scores. Furthermore, several metabolites in NDS, including formononetin-7-glucoside, hydroxybenzoyl-coumaric anhydride, methoxymecambridine, puerarol, and pyrethrin II, were found to target multiple vascular GPCRs. Metabolites with moderate-to-high binding energy were verified to have good rat basilar artery-relaxing effects, and the maximum artery relaxation effects of all three metabolites, namely, isorhamnetin, kaempferol, and daidzein, were found to exceed 90%. Moreover, metabolites of NDS were found to exert a synergistic effect by interacting with vascular GPCR targets, and these metabolites may contribute to the cerebrovascular regulatory function of NDS.Discussion: The study reports that various metabolites of NDS contribute to its vascular tone regulating effects and demonstrates the multi-component and multi-target characteristics of NDS. Among them, metabolites with moderate-to-high binding scores in NDS may play an important role in regulating vascular function.

Xiaoxuming decoction (XXMD) is a traditional Chinese herbal medicine (CHM) that is used for the treatment of stroke in China. Stroke injury damages the cerebral vasculature and disrupts the autoregulation of vasoconstriction and vasodilatation, which is crucial for maintaining constant cerebral blood flow (CBF). It has been reported that XXMD exerts a positive effect on cerebral circulation in animal models of stroke. However, the mechanisms underlying the regulatory effect of XXMD on vascular tone, and the interactions among the multiple components of XXMD, remain unclear. In this study, XXMD was found to induce relaxation of the basilar artery rings of rats precontracted by 5-hydroxytryptamine (5-HT) in vitro, in a dose-dependent manner. The modulation of vascular tone and the process of cerebral ischemia are mediated via the interactions between G protein-coupled receptors (GPCRs) and their ligands, including 5-HT, angiotensin II (Ang II), and urotensin II (UII). Thus, the potential synergistic effects of the different components of XXMD on the regulation of vasoconstriction and vasodilation were further investigated by molecular docking based on network pharmacology. We constructed and analyzed a database comprising 963 compounds of XXMD and studied the interactions between five vascular GPCRs (5-HT1A receptor (5-HT1AR), 5-HT1B receptor (5-HT1BR), Ang II type 1 receptor (AT1R), beta 2-adrenergic receptor (β2-AR), and UII receptor (UTR)) and the various herbal constituents of XXMD using molecular docking. By constructing and analyzing the compound-target networks of XXMD, we found that Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba were the three major herbs that contained a large number of compounds with high docking scores. We additionally observed that several constituents of XXMD, including gallotannin, liquiritin apioside, nariutin, 1,2,3,4,6-pentagalloylglucose, folic acid, and ginsenoside Rb1, targeted multiple vascular GPCRs. Moreover, the interactions between the components of XXMD and the targets related to vascular tone constituted the comprehensive cerebrovascular regulatory function of XXMD and provided a material basis of the vasoregulatory function of XXMD. The study reports the contributions of various components of XXMD to the regulatory effects on vascular tone and provides scientific evidence for the multicomponent and multitargeting characteristics of XXMD.

Xiaoxuming decoction (XXMD), a classic traditional Chinese medicine (TCM) prescription, has been used as a therapeutic in the treatment of stroke in clinical practice for over 1200 years. However, the pharmacological mechanisms of XXMD have not yet been elucidated. The purpose of this study was to develop neuroprotective models for identifying neuroprotective compounds in XXMD against hypoxia-induced and H2O2-induced brain cell damage. In this study, a phenotype-based classification method was designed by machine learning to identify neuroprotective compounds and to clarify the compatibility of XXMD components. Four different single classifiers (AB, kNN, CT, and RF) and molecular fingerprint descriptors were used to construct stacked naïve Bayesian models. Among them, the RF algorithm had a better performance with an average MCC value of 0.725±0.014 and 0.774±0.042 from 5-fold cross-validation and test set, respectively. The probability values calculated by four models were then integrated into a stacked Bayesian model. In total, two optimal models, s-NB-1-LPFP6 and s-NB-2-LPFP6, were obtained. The two validated optimal models revealed Matthews correlation coefficients (MCC) of 0.968 and 0.993 for 5-fold cross-validation and of 0.874 and 0.959 for the test set, respectively. Furthermore, the two models were used for virtual screening experiments to identify neuroprotective compounds in XXMD. Ten representative compounds with potential therapeutic effects against the two phenotypes were selected for further cell-based assays. Among the selected compounds, two compounds significantly inhibited H2O2-induced and Na2S2O4-induced neurotoxicity simultaneously. Together, our findings suggested that machine learning algorithms such as combination Bayesian models were feasible to predict neuroprotective compounds and to preliminarily demonstrate the pharmacological mechanisms of TCM.

In this study, bioinformatics methods such as molecular docking and network pharmacology were adopted to establish Xiaoxuming Decoction (XXMD) "compound-vasodilatory and vasoconstrictory related G protein-coupled receptors (GPCR) targets" network, then the vascular function regulatory effective components and the potential targets of XXMD were analyzed. Based on the XXMD herb sources, the chemical structures of the compounds were retrieved from the national scientific data sharing platform for population and health pharmaceutical information center, TCMSP database and the latest research literatures. The chemical molecular library was established after class prediction and screening for medicinal and metabolic properties. Then, five kinds of vasodilatory and vasoconstrictory related GPCR crystal structure including 5-HT receptors (5-HT1AR, 5-HT1BR), AT1R, β2-AR, hUTR and ETB were retrieved from RCSB Protein Data Bank database or constructed by homology modeling of Discovery Studio 4.1 built-in modeling tools. After virtual screening by Libdock molecular docking, the highest rated 50 compounds of each target were collected and analyzed. The collected data were further used to construct and analyze the network by Cytoscape 3.4.0. The results showed that most of the chemical composition effects were associated with different vasodilatory and vasoconstrictory related GPCR targets, while a few effective components could be applied to multiple GPCR targets at the same time, therefore forming synergies and vasorelaxant effects of XXMD.

BACKGROUND DL0805 (5-nitro-1H-indazole-3-carbonitrile) is a new Rho kinase inhibitor discovered by high-throughput screening (HTS) in our previous work, and it was found to have a vasorelaxant effect in rat aortic rings contacted by KCL or angiotensin II. However, whether it has the vasorelaxant effects on isolated rat basilar artery rings and the underlying mechanisms are still not known. METHODS Endothelium-intact and -denuded basilar artery rings were prepared from SD rats, and then stimulated with KCL, 5-hydroxytryptamine (5-HT) and endothelin-1(ET-1). The tension of the basilar artery rings was measured through an isometric force transducer. RESULTS DL0805 exerted vasorelaxation at a dose-dependent manner in endothelium-intact rings contracted by KCL (60 mM), 5-HT (10E-6M) and ET-1 (10E-8M). Preincubation with DL0805 attenuated contractile responses to KCl (10-60 mM), 5-HT (10E-9-10E-5 M) and ET-1 (10E-11-10E-7.5 M) in basilar artery rings. Accordingly, in human brain vascular smooth muscle cells (HBVSMCs), DL0805 was observed to inhibit the ET-1-induced cytoskeletal remodeling. The molecular docking results showed that DL0805 barely interacted with both 5-HT1B receptor (5-HT1BR) and ET1B receptor (ET1BR). Moreover, pre-incubation with pinocembrin dramatically suppressed the high K+-induced extracellular Ca2+ influx and ET-1-induced intracellular Ca2+ release in endothelium-denuded rings. CONCLUSIONS These results demonstrated that DL0805 has a vasorelaxant effect on isolated rat basilar artery rings. It may exert effects by blockade of Ca2+ channels via blocking ET1BR.

Objective: Pinocembrin (5,7-dihydroxyflavanone) is one of the primary flavonoids in propolis with various biological activities. We found that pinocembrin increased cerebral blood flow and improved the outcome after focal or global cerebral ischemia/reperfusion injury in rats. In the present study, the vasorelaxant effect of pinocembrin on the rat basilar artery and its potential mechanisms were studied. Methods: Endothelium-intact and -denuded basilar artery rings were prepared from SD rats, and then stimulated with KCl, 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1). The tension of the basilar artery rings was measured through an isometric force transducer. Results: Pinocembrin induced vasorelaxation in a dose-dependent manner in endothelium-intact rings contracted by KCl, 5-HT, and ET-1. However, partial loss of the vasorelaxation induced by pinocembrin was observed in endothelium-denuded rings contracted by KCl. The vasorelaxant effect of pinocembrin was significantly reduced by pre-incubation with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME), the guanylate cyclase inhibitor ODQ and the cyclooxygenase inhibitor indomethacin. In addition, the pinocembrin-induced relaxations were remarkably attenuated by the voltage-dependent K + channel blocker 4-aminopyridine, the ATP-sensitive K + channel blocker glibenclamide and the Ca 2+ -activated K + channel blocker tetraethylammonium. Moreover, pre-incubation with pinocembrin dramatically suppressed the high K + -induced extracellular Ca 2+ influx and ET-1-induced intracellular Ca 2+ release in endothelium-denuded rings. Conclusion: These results demonstrated that pinocembrin has a vasorelaxant effect in isolated rat basilar artery rings. It may exert its effects by an endothelium-dependent pathway involving NO-cGMP, and also through an endothelium-independent pathway, opening K + channels and blockade of Ca 2+ channels.