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 (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.

Stroke is a type of acute cerebral vascular disease, which is the leading cause of adult disability and the second most common cause of death worldwide [11]. Stroke occurs when the blood supply to a part of the brain is interrupted, and the brain cells die due to oxygen or nutrient insufficiency. There are two main categories of stroke, namely, ischemic stroke, in which the flow of blood is restricted, and hemorrhagic stroke, which is characterized by bleeding. Ischemia induces cerebral damage, which impairs the vasculature and adversely affects cerebral blood flow (CBF) [12]. As insufficient blood supply is presently considered to be a major pathology of stroke, drugs that induce recovery of the cerebral blood supply are of enormous promise. The drugs that modulate the vascular tone and dilate the cerebral arteries can restore blood supply to the ischemic area and thus provide a material basis for the subsequent antireperfusion injury and neuronal protection.
Network pharmacology is based on the theory of systems biology and is used to perform network analysis of biological systems. In network pharmacology, a specific signal node (nodes) can be selected to perform novel analyses of a multitarget drug for molecular design. Network pharmacology can serve as a robust method for studying "drugs-target-disease" networks, which may be closer to elucidating the complex nature of the disease being investigated [25]. To date, network-based methods have been used as a novel strategy for discovering the constituents of traditional Chinese medicine (TCM), identifying bioactive compounds, and elucidating the mechanisms underlying the action of herbal formulae [26]. Molecular docking is a computer-assisted approach that is primarily used to study the interactions between two or several molecules using both geometric complementarity and energy matches. Molecular docking has been widely used in drug discovery and serves as an important tool for the investigation and evaluation of herbal medicines [27,28]. The combined use of molecular docking and network pharmacology can provide valuable information and improve the accuracy of the prediction.
Although some progress has been made in the pharmacological investigation of XXMD over the past few years [1][2][3][4][5][6][7][8][9][10]29], the interactions among the multiple components of XXMD remain to be elucidated. The investigation of TCM using network pharmacology can aid in the discovery of multiple therapeutic targets for the various active ingredients to formulate effective interactions and therapeutic targets. This approach can be used to identify the targets, the group of active constituents that play a role in the interactions, and the material basis of the active herbal components of XXMD.
In this study, we found that XXMD could induce relaxation of the basilar artery rings of rats precontracted by 5-HT in a dose-dependent manner. However, the mechanism  BioMed Research International underlying the modulation of vascular tone and the material basis of XXMD remain unclear. We performed molecular docking for investigating the potential synergistic effects of the constituents of different herbs in XXMD on the regulation of vascular tone. The interactions between the five GPCRs linked to vasoconstriction (5-HT1AR, 5-HT1BR, AT1R, β2-AR, and UTR) and the various herbal constituents of XXMD were assessed. The compound-target networks for XXMD were subsequently constructed and analyzed. The rats were euthanized by cervical dislocation, following which the basilar artery was rapidly isolated and cut into 2 mm long segments in ice-cold physiological saline solution (PSS) having the following composition (mmol/L): NaCl 130, KCl 4.7, MgSO 4 1.17, KH 2 PO 4 1.18, NaHCO 3 14.9, glucose 5.5, ethylene diamine tetraacetic acid 0.026, and CaCl 2 1.6. For intact tissue preparation, extreme care was taken to avoid endothelial cell damage. The segments were then mounted on 40 μm stainless steel wires in a Multi Myograph System (Danish Myograph Technology, Aarhus, Denmark), and the contractile responses were determined. The organ chamber was filled with 5 mL of PSS, through which 95% O 2 /5% CO 2 was continually bubbled, and the temperature was maintained at 37°C. The segments of the basilar arteries were normalized to the optimal initial tension, according to the instructions provided with the Multi Myograph System. The segments were then allowed to equilibrate for 60 min at the resting tension. The PSS was changed every 20 min during equilibration. After equilibration, the arterial rings were constricted twice with a high K + (60 mmol/L) PSS (K-PSS)    3 14.9, glucose 5.5, ethylene diamine tetraacetic acid 0.026, and CaCl 2 1.6. The integrity of a functional endothelium was verified by the ability of acetylcholine (Ach) (20 μmol/L) to induce more than 60% relaxation in the arterial rings precontracted with 60 mmol/L K-PSS [30].

Preparation of Ligands and Target
Proteins. Five vascular GPCRs (5-HT1AR, 5-HT1BR, AT1R, β2-AR, and UTR) were considered for the molecular docking study. The crystal structures of 5-HT1BR, AT1R, and β2-AR were retrieved from the RCSB Protein Data Bank (PDB) (http://www.pdb .org/) [31], and the PDB codes were 4IAQ, 4YAY, and 4LDE, respectively. The structures of the GPCRs for which crystal structures were unavailable, namely, 5-HT1AR and UTR, were constructed by homology modeling using Discovery Studio 4.1. After sequence comparison, the sequences of β2-AR (PDB ID: 2RH1) and 5-HT1BR (PDB ID: 4IAQ) were selected as the templates for modeling UTR and 5-HT1AR, respectively. The Build Homology Model module in Discovery Studio was used for homology modeling, and the model with optimal reliability was selected for molecular docking.
A ligand library, comprising the different conformations of each ligand, was constructed using the Prepare Ligands tool. The existing ligands and water of crystallization in the target proteins were removed using the Prepare Protein tool. The ligand-receptor binding sites were determined using the Define and Edit Binding Site tool, based on the information obtained from the binding sites of the cocrystallized ligands in the protein structures. For 5-HT1BR and AT1R, the binding sites of the cocrystallized ligands, dihydroergotamine and 5,7-diethyl-1-{[2′-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl}-3,4-dihydro-1,6-naphthyridin-2(1H)-one, respectively, were defined as the active ligand-binding pocket. For β2-AR, the binding site of BI167107 was defined as the active ligandbinding pocket.
2.6. Molecular Docking. The LibDock module of Dock Ligands in Discovery Studio 4.1 was used to perform molecular docking. The conformation method was set to fast to ensure docking accuracy and velocity, and the other parameters were set to default. In this semiflexible docking approach, 100 or less conformations of each ligand were generated to ensure computational speed and comprehensiveness. The output index and LibDock scores were subsequently obtained. The scores indicate the binding affinity between the receptors and the ligand conformations. The higher the score of a small molecule, the stronger the protein binding affinity. For each target, the 50 top-scoring compounds were selected, and their scores were considered to be the highest among all the conformations for each ligand. The LibDock scores of the agonists and antagonists, retrieved from the IUPHAR database, were also determined for each target, for assessing the potential activity of the herbal compounds in XXMD. The following agonists and antagonists were used for molecular docking: 5-HT1AR (agonists: U92016A, vilazodone, and vortioxetine; antagonists: (S)-UH 301 and WAY-100635); 5-HT1BR (agonists: CP94253, eletriptan, and L-694,247; antagonists: GR-55562 and SB236057); AT1R (agonists: L-162,313; antagonists: candesartan, irbesartan, and valsartan), β2-AR (agonists: formoterol, salmeterol, and zinterol; antagonists: carvedilol, propranolol, and timolol; and UTR (agonists: AC-7954 and FL104; antagonists: palosuran and SB-706375).

Compound-Target Network Construction Using Cytoscape.
The information pertaining to the chemical composition and related compound-target network interactions were analyzed using Cytoscape version 3.7.1. The compound-target network was established using Cytoscape, and the interactions were subsequently visualized.       Table 1 and Figure 3. The results demonstrated that the five top-ranking chemical categories in the prescription were essential oils (32%), flavonoids (24%), alkaloids (13%), saponins (7%), phenols, and organic acids (5%). However, it should be noted that the results reflect the number of different types of compounds rather than their abundance or relative content in the prescription. In order to identify the potentially effective constituents of XXMD, the compounds derived from the different herbs of XXMD were retrieved and analyzed. The names, plant sources, and chemical categories of forty compounds are enlisted in Table 2. Of the compounds that were derived from multiple plant sources, the majority were essential oils, flavonoids, phenols, or organic acids. This result was consistent with the data pertaining to the major chemical constituents of XXMD.
For each GPCR target, the compounds with the highest scores were selected to generate a scatter diagram, as depicted in Figure 4. The LibDock scores of the agonists and antagonists of the GPCR targets are provided in Figure 4. Most of the chemical constituents of XXMD could be successfully docked to the target GPCRs. For each GPCR, some of the compounds exhibited higher LibDock scores than the agonists and antagonists, indicating their strong binding affinity to the GPCR targets. Nevertheless, some of the compounds failed to dock to the target GPCRs owing to their structural characteristics.
In order to analyze the overall results of ligand-receptor docking, we determined the number of compounds that were successfully docked and the percentage of compounds with LibDock scores higher than the average scores of the agonists and antagonists. As demonstrated in Table 3, 76.52%, 84.46%, 84.89%, 76.96%, and 78.70% of the compounds in XXMD successfully docked to 5-HT1AR, 5-HT1BR, AT1R, β2-AR, and UTR, respectively. Additionally, more than 10% of the compounds of XXMD had higher LibDock scores than the average score of the GPCR agonists and antagonists. Therefore, these results suggested that most of the compounds of XXMD have the potential to bind and interact with the five vascular GPCRs considered herein. It subsequently follows that the vascular tone can be comprehensively modulated by XXMD via the interactions between the multiple chemical constituents of XXMD and the different vascular GPCR targets in the compound-target network.

Construction and Analysis of Compound-Target
Interaction Network of XXMD. In order to elucidate the material basis of the modulation of vascular tone by XXMD, the 50 top-scoring compounds that targeted the vascular GPCRs were selected, and the compound-target network of      Demethyldelavaine A Aconiti Lateralis Radix Praeparata 2 9 BioMed Research International XXMD was constructed using Cytoscape version 3.7.1. As depicted in Figure 5, the blue and yellow nodes represent the chemical components and targets, respectively, while the edges represent the interactions. The network analyzer plugin of Cytoscape was used to analyze the network by calculating the number of nodes, network density, and network heterogeneity, among other factors. The compound-target network comprised 249 edges, 135 nodes, 130 compounds (after removal of duplicates), and 5 targets. Network analysis revealed that multiple compounds targeted the same GPCR, while some of the compounds had multiple targets, which demonstrated that the perspective of CHM is very different from the philosophy of WCM, which considers that one compound interacts with one specific target.
In order to explore the relationships between the chemical constituents of the twelve herbs of XXMD and the five vascular GPCR targets, the 50 top-scoring compounds with the highest docking scores were selected for each of the targets and categorized according to their herbal sources ( Figure 6). The three major herbs were Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba.

Discussion
To date, there are no definitive treatment strategies for ischemic stroke. With the development of recent research on multi-component-multi-target drugs, the functions of TCM

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BioMed Research International in the treatment of complex diseases have become increasingly important. Owing to the potent compound group formulation used in TCM prescriptions, the efficacy of TCM is attributed to the interactions between multiple active ingredients and multiple disease targets. Several animal experiments and clinical studies have demonstrated that XXMD, a CHM with relaxant effects on cerebral blood vessels, can improve the treatment outcomes in both acute and chronic ischemic brain injury [4,5,10]. By relaxing the cerebral blood vessels, XXMD can restore the supply of blood and nutrients to the ischemic area, providing a material basis of the prevention of reperfusion injury and neuronal protection. In this study, the vasorelaxant effects of XXMD on rat basilar arteries contracted by 5-HT were determined. The modulation of vascular tone and the process of cerebral ischemia are mediated via the interactions between GPCRs and their ligands, including 5-HT, Ang II, and UII. Therefore, molecular docking was performed to determine the potential synergistic effects of the components of XXMD on the regulation of vascular tone via modulation of the activity of vascular GPCRs. First, a database of 963 compounds of XXMD, with different chemical structures, was constructed and analyzed. Molecular docking was used to investigate the interactions between the chemical constituents of XXMD and the five vascular GPCRs, namely, 5-HT1AR, 5-HT1BR, AT1R, β2-AR, and UTR. The 50 top-scoring compounds with high docking scores to each of the GPCR targets were selected and sorted. The herbal sources of these compounds were determined for identifying the roles of the twelve herbs of XXMD in regulating the vascular tone via modulation of GPCR activity.
The results of our study suggested that several overlapping compounds and their analogues are widely distributed in the different medicinal herbs of XXMD. The contents of these ingredients are relatively high in the XXMD prescription, compared to those of the other compounds. Furthermore, previous studies have demonstrated that most of these compounds have similar physiological activities, including antitumor, anti-inflammatory, antioxidative, neuroprotective, blood lipid-regulating, and vasorelaxant properties. These compounds, derived from a variety of different plants in XXMD, have different chemical structures and can produce synergistic therapeutic effects under the same pathological conditions. Therefore, they are likely to be the key components that play a crucial role in the therapeutic effect of XXMD.
Apart from their pharmacological activities, some of the chemical ingredients, including essential oils, promote the absorption of other drugs. It is speculated that ginger is indispensable in the treatment of cerebral apoplexy by XXMD, and the maximum dosage should be five Liang [32]. Furthermore, it has been demonstrated that ginger extract improves memory deficits and neuronal density in rats with focal cerebral ischemia [33]. It can therefore be concluded that the essential oils, which are the main chemical components of ginger, are likely to enhance the absorption of other constituents in several CHMs.
Classification of the herbal sources of the 50 top-scoring compounds revealed that the three major herbs were     [34]. Glycyrrhizin (GL) [35] and 18β-glycyrrhetinic acid (GA) [36], isolated from the roots of licorice, have also been reported to have neuroprotective effects in focal cerebral ischemia/reperfusion brain injury, which are mediated via the anti-inflammatory, antioxidant, and antiapoptotic effects of GL, or the strong antioxidant and radical scavenging properties of GA in global cerebral ischemia/reperfusion brain injury. Isoliquiritigenin (ISL), a flavonoid present in the roots of licorice, is known to have vasorelaxant effects and protects against injury due to cerebral ischemia by ameliorating cerebral energy metabolism and via its antioxidant properties [37]. Ginsenoside Rd, belonging to the class of Ginseng Radix et Rhizoma compounds in herbal medicine, has been shown to attenuate damage due to ischemic stroke via suppression of oxidative stress and inflammation [38]. The active metabolite of ginsenosides in the gut, 20(S)-protopanaxadiol, induces vasorelaxation in isolated rat thoracic aorta via an endothelium-independent pathway [39]. A combination of Paeoniae Radix Alba and Rhizoma Ligustici Chuanxiong was reported to ameliorate focal cerebral ischemia in rats with MCAO by attenuating the endoplasmic reticulum stress-dependent apoptotic signaling pathway [40]. Therefore, the results of this study indicate that Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Paeoniae Radix Alba play a key role in the modulatory effect of XXMD on vascular tone and its effects on suppressing ischemic stroke via regulation of the compound-target interaction network in XXMD and necessitate further investigation.
It has been reported that TCM influences disease progression by modulating GPCR activity. The Chinese herbal formula, Sini Tang (SNT), comprises Glycyrrhizae Radix et Rhizoma, Aconiti Lateralis Radix Praeparata, and Zingiberis Rhizoma Recens. SNT improves cardiac function by inhibiting the excessive activation of the renin-angiotensinaldosterone system, which involves a reduction in the levels of plasma angiotensin II and downregulation of the protein and gene levels of AT1R in heart failure after myocardial infarction in rats [41]. ISL has also been reported to exhibit in vitro 5-HT2B receptor antagonistic activity [42]. Ginsenoside Rb1 has been reported to possess antidepressant-like activity, which is mediated via the modulation of 5-HT 2A receptors [43]. The results of our study demonstrated that XXMD inhibited the constriction of rat basil artery induced by 5-HT. However, the effects of XXMD on vasoconstriction induced by other GPCR ligands, and the underlying mechanism involving the modulation of GPCRs and their signaling pathways, necessitate further investigation.
By reviewing the relevant literature, we found that most of the components of XXMD that target multiple vascular GPCRs possess vasorelaxant effects and suppress ischemic stroke. For instance, 1,2,3,4,6-pentagalloylglucose, a major component of Paeoniae Radix Alba, binds to 4 vascular GPCR targets and has been reported to induce vasorelaxation and suppress the vascular inflammatory process by activating the NO/cGMP signaling pathway [44]. Folic acid, a member of the vitamin B complex, and isolated from Ginseng Radix et Rhizoma and Chuanxiong Rhizoma, was found to bind to 4 GPCR targets. Interestingly, folic acid has been reported to improve endothelial function [45] and alleviate various microglia-mediated neuroinflammation via the Notch1/nuclear factor kappa B p65 pathway in the hippocampus

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BioMed Research International following brain I/R injury in rats [46]. Ginsenoside Rb1, an active component extracted from Ginseng Radix et Rhizoma, was found to bind to three GPCR targets and has been reported to promote poststroke axonal regeneration by activating the cAMP/PKA/CREB signaling pathway [47]. Few studies have investigated the vasorelaxant and anti-ischemic stroke effects of the three compounds of XXMD, namely, gallotannin, liquiritin apioside, and narirutin, which bound to all the five vascular GPCR targets. It is therefore necessary to study the effects of these three compounds in further detail. The results of our study demonstrate that the compounds that targeted the vascular GPCRs could mediate the modulatory effect of XXMD on vascular tone and provide a material basis of the treatment of ischemic stroke with XXMD. However, the results of this study need to be verified using experiments and data validation.
In conclusion, the current study clearly demonstrated that XXMD was able to relax the cerebral artery rings of rats. XXMD comprises numerous compounds with different chemical structures and is derived from multiple plant sources. Therefore, XXMD might regulate cerebrovascular function via the network of interactions between the constituents of XXMD and the GPCRs involved in vasoconstriction. Our results demonstrated that the various herbal constituents of XXMD contribute to the regulation of vascular tone via the modulation of the target GPCRs. By investigating and analyzing the monomeric component of XXMD from the perspective of chemical composition and the relationships between the chemical constituents and targets, we were able to provide evidence for understanding the mechanisms underlying the effect of XXMD in the treatment of stroke.

Data Availability
The data used to support the findings of this study are included within the article. Other data are available from the corresponding author upon request.