Naodesheng decoction regulating vascular function via G-protein-coupled receptors: network analysis and experimental investigations.

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, ß2-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.

Quercetin attenuates ischemia reperfusion injury by protecting the blood-brain barrier through Sirt1 in MCAO rats.

The purpose of the present study was to examine the protective action and mechanisms of quercetin on the blood-brain barrier (BBB) in rats subjected to transient middle cerebral artery occlusion (tMCAO) and reperfusion. Quercetin (10, 30, 50 mg/kg) was intraperitoneally administered at the onset of reperfusion. The results showed that quercetin significantly reduced cerebral infarct volume, neurological deficit, BBB permeability and ROS generation via Sirt1/Nrf2/HO-1 signaling pathway. Moreover, EX527, a selective inhibitor of Sirt1, reversed these neuroprotective effects. Our findings indicate that quercetin has neuroprotective effects against cerebral ischemia-reperfusion injury by protecting BBB through Sirt1 signaling pathway in MCAO rats.

The histone deacetylase inhibitor belinostat ameliorates experimental autoimmune encephalomyelitis in mice by inhibiting TLR2/MyD88 and HDAC3/ NF-κB p65-mediated neuroinflammation.

Multiple sclerosis (MS) is a Th cell-mediated inflammatory demyelinating autoimmune disease. MS cannot be cured, and long-term drug treatment is still needed for MS patients. In this study, we examined the effect of belinostat, a pan-histone deacetylase inhibitor (HDACi), on experimental autoimmune encephalomyelitis (EAE) and elucidated its mechanism of action. We found that belinostat alleviates the clinical symptoms, histopathological central nervous system (CNS) inflammation and demyelination outcomes in EAE mice. Compared to the MS oral drug dimethyl fumarate (DMF) (100 mg/kg), belinostat (30 mg/kg) treatment exhibited better efficacy in improving the clinical symptoms of EAE mice. Belinostat treatment significantly suppressed the activation of M1 microglia and the proinflammatory cytokine expression; but it had no effects on the M2 microglial polarization. Belinostat also decreased both NO and iNOS levels in LPS-stimulated BV2 microglia. Accordingly, belinostat treatment of EAE mice significantly inhibited activation of the TLR2/MyD88 signaling pathway and downregulated the expression of HDAC3 while upregulating the acetylated NF-κB p65 levels. Taken together, these data demonstrate for the first time that belinostat ameliorates EAE in mice through inhibiting neuroinflammation via suppressing M1 microglial polarization, and implicating belinostat as a potential candidate for the treatment of multiple sclerosis.

Xiaoxuming Decoction Regulates Vascular Function by Modulating G Protein-Coupled Receptors: A Molecular Docking Study.

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 (ß2-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.

Network Pharmacology-Based Analysis of Xiao-Xu-Ming Decoction on the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) has become a worldwide disease that is harmful to human health and brings a heavy economic burden to healthcare system. Xiao-Xu-Ming Decoction (XXMD) has been widely used to treat stroke and other neurological diseases for more than 1000 years in China. However, the synergistic mechanism of the constituents in XXMD for the potential treatment of AD is still unclear. Therefore, the present study aimed to predict the potential targets and uncover the material basis of XXMD for the potential treatment of AD. A network pharmacology-based method, which combined data collection, drug-likeness filtering and absorption, distribution, metabolism, excretion and toxicity (ADME/T) properties filtering, target prediction and network analysis, was used to decipher the effect and potential targets of XXMD for the treatment of AD. Then, the acetylcholinesterase (AChE) inhibitory assay was used to screen the potential active constituents in XXMD for the treatment of AD, and the molecular docking was furtherly used to identify the binding ability of active constituents with AD-related target of AChE. Finally, three in vitro cell models were applied to evaluate the neuroprotective effects of potential lead compounds in XXMD. Through the China Natural Products Database, Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database, Traditional Chinese Medicine (TCM)-Database @Taiwan and literature, a total of 1481 compounds in XXMD were finally collected. After ADME/T properties filtering, 908 compounds were used for the further study. Based on the prediction data, the constituents in XXMD formula could interact with 41 AD-related targets. Among them, cyclooxygenase-2 (COX-2), estrogen receptor α (ERα) and AChE were the major targets. The constituents in XXMD were found to have the potential to treat AD through multiple AD-related targets. 62 constituents in it were found to interact with more than or equal to 10 AD-related targets. The prediction results were further validated by in vitro biology experiment, resulting in several potential anti-AD multitarget-directed ligands (MTDLs), including two AChE inhibitors with the IC50 values ranging from 4.83 to 10.22 µM. Moreover, fanchinoline was furtherly found to prevent SH-SY5Y cells from the cytotoxicities induced by sodium nitroprusside, sodium dithionate and potassium chloride. In conclusion, XXMD was found to have the potential to treat AD by targeting multiple AD-related targets and canonical pathways. Fangchinoline and dauricine might be the potential lead compounds in XXMD for the treatment of AD.

Activation of Transient Receptor Potential Channel Vanilloid 4 by DPP-4 (Dipeptidyl Peptidase-4) Inhibitor Vildagliptin Protects Against Diabetic Endothelial Dysfunction.

Endothelial dysfunction is an early step to the progression of cardiovascular diseases in diabetes. Apart from their anti-diabetic action, DPP-4 (dipeptidyl peptidase-4) inhibitors also reduce cardiovascular events in diabetic patients. However, the underlying mechanism of the beneficial effect of DPP-4 inhibitor on endothelial function is still obscure. In this study, we intervened type 1 or 2 diabetic model mice with vildagliptin for 4 weeks and measured the vascular reactivity. We found that vildagliptin improved endothelium-dependent vasodilation in diabetic mice independent of GLP-1 (glucagonlike peptide-1), but this effect was blocked by a SIRT1 (Sirtuin 1) inhibitor, Ex527. Mechanistically, vildagliptin-activated Transient Receptor Potential Channel Vanilloid 4 (TRPV4) to promote extracellular calcium uptake in endothelial cells, which activated AMPK (AMP-activated protein kinase)/SIRT1 pathway to counteract hyperglycemia-induced endothelial reactive oxygen species generation and senescence. Vildagliptin directly binds to TRPV4 by forming a hydrogen bond, which is critical to vildagliptin-evoked endothelial calcium intake. Knockout or inhibition of TRPV4 erased the beneficial role of vildagliptin. In addition, activation of SIRT1 by SRT1720 improved endothelial function independent of TRPV4 and reduced TRPV4 transcription to maintain an appropriate calcium level. In summary, our findings prove that vildagliptin protects against hyperglycemia-induced endothelial dysfunction by activating TRPV4-meditaed Ca2+ uptake, which helps to re-understand the mechanism of DPP-4 inhibitors and expand the therapeutic scope.

Evaluation and Identification of the Neuroprotective Compounds of Xiaoxuming Decoction by Machine Learning: A Novel Mode to Explore the Combination Rules in Traditional Chinese Medicine Prescription.

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.

[Network pharmacology study of Xiaoxuming Decoction based on vasodilatory and vasoconstrictory related GPCR targets].

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, ß2-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.