Exploring Active Compounds and Mechanisms of Angong Niuhuang Wan on Ischemic Stroke Based on Network Pharmacology and Molecular Docking

Methods The chemical ingredients of ANW were retrieved from TCMSP, TCMID, and literature. We predicted the potential targets of active ingredients by PubChem, Swiss Target Prediction, and STITCH databases. The targets related to ischemic stroke were retrieved using GeneCards, DisGeNET, DrugBank, TTD, and GEO databases. Subsequently, Venn diagrams were used to identify common targets of active ingredients and ischemic stroke. Protein-protein interaction (PPI) network was structured with STRING platform and Cytoscape 3.8.2. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of key targets were performed in the Metascape database. Finally, molecular docking was conducted by AutoDock Tools and PyMOL software. Results A total of 2391 targets were identified for 230 active ingredients of ANW, and 1386 of them overlapped with ischemic stroke targets. The key active ingredients were mainly quercetin, β-estradiol, berberine, wogonin, and β-sitosterol, and the key targets were also identified, including IL-6, AKT1, MAPK3, PIK3CA, and TNF. The biological process (BP) results indicated that ANW may have therapeutic effects through response oxidative stress, inflammatory response, cellular response to lipid, and response to nutrient levels. Furthermore, the ingredients of ANW were predicted to have therapeutic effects on ischemic stroke via the HIF-1 signaling pathway, FoxO signaling pathway, chemokine signaling pathway, fluid shear stress and atherosclerosis, and neurotrophin signaling pathway. The molecular docking results all showed that the core ingredients were strong binding activity with the core targets. Conclusion In conclusion, the bioinformatics and pharmacological results reveal that counteracting oxidative stress, suppressing inflammation, inhibiting the development of AS, and even protecting neurological function are critical pathways for ANW in the treatment of ischemic stroke. These results may help to elucidate the mechanism of ANW on ischemic stroke for experimental studies and clinical applications.


Introduction
Ischemic stroke is a common cerebrovascular event due to an abrupt cerebral artery occlusion, resulting in insufficient perfusion, which then causes edema, inflammation, and necrosis of the affected tissue and severely damages to neurological function. e World Health Organization reports that ischemic stroke is the main cause of death and long-term disability in the world, which causes a tremendous psychological and financial burden on patients [1]. However, the pathological process of ischemic stroke involves multiple aspects, including energy metabolism disorder, oxidative stress, inflammation, and neuronal damage, and there is no sovereign remedy [2,3]. erefore, it is significantly important to explore drugs or active ingredients with multiple targets for the treatment of cerebral ischemia.
Notably, many of the Chinese herbs have been proven to produce therapeutic effects on ischemic stroke in clinical research [4]. As a famous Chinese herbal formula, Angong Niuhuang Wan (ANW) is widely used in clinical practice for the treatment of ischemic stroke, which contains 11 herbs, including Moschus, Realgar, Curcumae Radix, Borneolum, Scutellariae Radix, Coptidis Rhizoma, Gardeniae Fructus, Bovis Calculus, Bubali Cornu, Margarita, and Cinnabaris. Studies indicated that ANW had effect on reducing infarct size, protecting the integrity of the blood-brain barrier (BBB), improving antioxidant capacity, and inhibiting inflammation injury to produce neuroprotection; furthermore, it may improve the development of early atherosclerosis (AS) by suppressing inflammation [5][6][7]. However, the pharmacological effects of ANW on ischemic stroke have still not been elucidated.
In this study, we aim to elucidate the possible mechanism of ANW on ischemic stroke and reveal the interaction between ANW, target, and ischemic stroke from a holistic perspective through a network pharmacology approach. e workflow diagram of the study is presented in Figure 1.

Screening of Active Ingredients in ANW.
e effective ingredients of ANW were searched through TCMSP (https://tcmspw.com/tcmsp.php) [8], TCMID (http://www. megabionet.org/tcmia/), and literature. e active compounds were screened for oral bioavailability (OB), druglikeness (DL), and blood-brain barrier permeability (BBB) prediction. e selection of OB, DL, and BBB referred to the recommendations of the TCMSP database. erefore, we finally screened the compounds with OB ≥ 0.2, DL ≥ 0.1, and BBB ≥ -0.3, which were considered as parameters for selecting potentially pharmacological ingredients [9][10][11], in addition, the ingredients with high content or pharmacological effects searched from literature and TCMID that did not contain the above parameters, which were also included in the further analysis. Besides, the threshold values were considered based on the following points: firstly, extracting more useful information from fewer compounds; secondly, maintaining concordance with the proven pharmacological data.

Protein-Protein Interaction Network Construction and
Analysis. Protein-protein interaction (PPI) network was constructed through the STRING database (https://stringdb.org/) [15] with a confidence score >0.7. And topology analysis was performed by Cytoscape software.
e key targets were sorted and screened according to the value of degree, betweenness centrality, and closeness centrality of the topological analysis results [16]. In addition, we screened important functional modules in PPI networks with the Cytoscape plugin MCODE.

Functional Enrichment and Pathways Analysis.
e Gene ontology (GO) including biological process (BP), molecular function (MF), and cellular component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database (https://metascape.org) [17]. e statistical significance threshold was set at the cutoff values of P < 0.01. In addition, the bioinformatics platform (http:// www.bioinformatics.com.cn/) was used to visualize GO and KEGG enrichment analysis with the bubble charts.

Construction of Active Ingredients-Targets-Pathway
Network. An ingredients-targets-network was constructed by Cytoscape software. e key active ingredients of ANW were sorted and screened according to the value of degree, betweenness centrality, and closeness centrality based on topological analysis.

Molecular Docking.
e 3D structures of candidate ingredients were obtained from PubChem, which were transformed by Open Babel Toolkit (version 2.4.1) into a mol2 file format. e 3D structures of the core target were downloaded from the PDB database (http://www.rcsb.org/). e AutoDockTool 1.5.6 was used to add hydrogen and optimize protein structure for molecular docking after removing water and original ligands.  Evidence-Based Complementary and Alternative Medicine

Protein-Protein Interaction Network Analysis.
A total of 4963 potential targets were obtained of ischemic stroke, and 1386 common targets were obtained after intersecting with 2391 potential targets of the active ingredients ( Figure 2). e topological results of 1386 targets were obtained 130 significant targets according to the degree, betweenness centrality, and closeness centrality. e PPI network included 130 nodes and 2946 edges, among which 25 genes were more relevant to the ischemic stroke according to the MalaCards database (https://www.malacards.org/) [18], so they were identified as key targets ( Figure 3, Table 2). MCODE has screened 5 functional modules according to the 130 targets ( Figure 4). e biological functions of the subnetwork are shown in Table 3. e BP analysis revealed

Construction of Active Ingredients-Targets Network.
As shown in Figure 5, we constructed a network of active ingredients-targets using Cytoscape software (version 3.8.0). e active ingredients-targets network contained 310 nodes (including 180 ingredients and 130 genes) and 2110 edges. e top 20 active ingredients were screened by topology analysis (Table 4).

GO Enrichment
Analysis. GO enrichment results include 296 BP terms, 99 MF terms, and 92 CC terms. e key items of BP mainly included response to oxidative stress, inflammatory response, cellular response to lipid, and response to nutrient levels. e main results of MF included oxidoreductase activity, cytokine receptor binding, lipid binding, and neurotransmitter receptor activity, and CC mainly included neuronal cell body, dendritic tree, axon, and postsynapse. We individually selected top 20 remarkably enriched terms in BP, MF, and CC classification as presented in Figure 6.

KEGG Pathway Enrichment Analysis and Ingredients-
Targets Pathway Network Construction. KEGG pathway enrichment analysis may elaborate the mechanism of ANW on ischemic stroke. 139 signal pathways were obtained based on the 130 core targets. After removing pathways associated with cancer and unrelated to disease, the main results of KEGG pathways included the HIF-1 signaling pathway, FoxO signaling pathway, chemokine signaling pathway, fluid shear stress and atherosclerosis, and neurotrophin signaling pathway. 20 significantly enriched pathways were selected as shown in Figure 7. An ingredients-targets pathway network was built involving pathways, targets, and corresponding ingredients to further elucidate the molecular    3.6. Docking Results Analysis. We selected the core targets, including IL-6, AKT1, MAPK3, PIK3CA, and TNF for molecular docking with the quercetin, β-estradiol, berberine, wogonin, and β-sitosterol. e results suggested that the 5 key ingredients all had a strong affinity with IL-6, AKT1, MAPK3, PIK3CA, and TNF, and the results of the docking were visualized by PyMOL software (Table 5, Figure 9).

Discussion
Stroke is classified as ischemic or hemorrhagic. Cerebral hemorrhage and cerebral ischemia have the possibility to cause serious inflammatory response, cerebral edema, and neurological deficits [19,20]. e studies found that ANW reduced brain edema and intracranial pressure in cerebral ischemia and cerebral hemorrhage by regulating the expression of MMP-9 and AQP4 which were closely related to the formation of brain edema and the disruption of the BBB [21,22]; in addition, it was able to exert neuroprotective function by reducing the inflammatory response and  Evidence-Based Complementary and Alternative Medicine inhibiting oxidative stress and neurotoxicity in brain tissue of cerebral ischemia and cerebral hemorrhage [23,24]. At present, the incidence of cerebral ischemia is far higher than cerebral hemorrhage; therefore, the paper is focused on the mechanism of Angong Niuhuang Wan in the treatment of cerebral ischemia. Network analysis increases the understanding of multiple mechanisms of drug action. Systems pharmacology may provide new avenues for drug discovery    strong binding activity to the IL-6, AKT1, MAPK3, PIK3CA of the core targets. IL-6 is a pleiotropic cytokine that plays a crucial role in host defense [25]. However, trans-signaling of IL-6 induces vascular endothelial cells to express and release the pro-inflammatory chemokine MCP-1, which is mediated through the JAK/STAT3 and PI3K/AKT pathways [26]. e studies found that administration of β-estradiol from Moschus reversed neuronal damage by regulating the JAK-STAT3 pathway and protected neurons from acidosis-mediated neurotoxicity and ischemic cerebral injury, thus promoting remodel and repair after brain injury [27,28]. Liao et al. [29] demonstrated that β-sitosterol inhibited the secretion of inflammatory factors such as TNF-α, IL-1β, IL-6 to suppress the inflammatory response. TNF is a versatile pro-inflammatory cytokine involved in all stages of ischemic stroke. e study confirmed that quercetin from Coptidis Rhizoma and Gardeniae Fructus attenuated TNF-induced inflammation by suppressing the NF-κB pathway [30].  MAPK is involved in inflammatory and apoptotic processes in cerebral ischemia-reperfusion injury. Studies had shown that quercetin inhibited inflammation and regulated JNK and ERK signaling pathways to produce antiapoptosis, thereby improving ischemic brain injury [31,32]. AKT1, as a threonine protein kinases, is an important regulator of the AKT-mTOR signaling pathway that controls the tempo of newborn neurons during adult neurogenesis. PIK3CA is involved in the cell signaling of various growth factors. Yan et al. [33] demonstrated that activation of the PI3K/Akt/mTOR pathway inhibited oxidative stress-related neuronal autophagy and exerted neuroprotective functions. e research showed that berberine can reduce the apoptosis of striatum and mitochondrial through regulating PI3K/Akt signaling pathway and reducing intracellular ROS levels to exert neuroprotective effects [34,35].
According to the results of KEGG enrichment analysis, ANW is considered to affect important pathways that are closely related to the pathogenesis of ischemic stroke, including HIF-1 signaling pathway, FoxO signaling pathway, chemokine signaling pathway, fluid shear stress and atherosclerosis, and neurotrophin signaling pathway. e results of GO enrichment were also closely related to response to oxidative stress, inflammatory response, cellular response to lipid, and response to nutrient levels. Furthermore, the BP analysis revealed that the subnetworks were mainly associated with inflammatory response, response to lipid, neuroapoptosis, and development.
HIF-1α is a primary modulator of cellular and systemic homeostatic reactions to hypoxia. Evidence showed that HIF-1 facilitated the transcription of various prosurvival proteins engaged in energy metabolism, angiogenesis, and neurogenesis, exerting a neuroprotective effect against ischemic stroke in ischemic conditions [36]. Research showed that estradiol facilitated neurogenesis in rats after stroke, possibly via increasing HIF-1α and VEGF protein expression [37]. e FoxO family of transcription factors is a critical regulator of cellular stress responses and facilitated the antioxidant defense of cells. Akt and p38MAPK are known stress-responsive kinases targeted to FoxO and are involved in the regulation of FoxO activity [38]. Zhang et al. [39] found that regulation of PI3K/Akt/FoxO-3a signaling pathway facilitated the proliferation of neural stem/progenitor cells and reduced ischemia-reperfusion injury. e inflammatory immune reaction needs leukocytes to be recruited to the site of inflammation. Chemokines are critical in protecting the host response by providing directional cues for cellular transport. Research confirmed that ANW downregulated the expression of chemokine receptors CCR2, CXCR3, and cell adhesion molecules in the arterial vasculature and alleviated the development of atherosclerosis by suppressing inflammation [7].
Atherosclerosis is a major cause of stroke onset or recurrence, and blood flow-induced shear stress has become an essential characteristic of atherosclerosis. e fluid resistance exerted on the vessel wall is mechanically translated into biochemical signals that lead to alter vascular behavior. erefore, the maintenance of physiological laminar shear stress is essential for normal vascular function [40].
Quercetin alleviates vascular calcification by suppressing oxidative stress and mitochondrial division [41]. Moreover, Fan et al. [42] stated that ANW suppressed the development of atherosclerosis by regulating immune homeostasis and suppressing chronic inflammation. Neurotrophins have been proved to control survival, development, and function of neurons in the central nervous system. Studies asserted that quercetin and berberine alleviated neuronal apoptosis of ischemic stroke in the rat by activating the BDNF-TrkB-PI3K/Akt signaling pathway to increase the expression of BDNF [34,43].
is suggests a potential application of neurotrophins in the therapy of ischemic stroke.
is research based on a pharmacological network explored the potential mechanisms of ANW for the treatment of ischemic stroke. e findings highlighted the improvement of the inflammatory response, immune defense, and neuroprotection of ANW against ischemic stroke. Our results were consistent with published studies that upregulation of HIF-1 signaling pathway, FoxO signaling pathway, and neurotrophin signaling pathway and downregulation of chemokine signaling pathway had positive effects on cerebral ischemia [42,[44][45][46][47]. In addition, we also provided some potential targets for treating ischemic stroke, which would contribute to the exploitation of new therapeutic strategies.

Conclusion
In conclusion, the bioinformatics and pharmacological results reveal that counteracting oxidative stress, suppressing inflammation, inhibiting the development of AS, and even protecting neurological function are critical pathways for ANW in the treatment of ischemic stroke. ese results may help to elucidate the mechanism of ANW on ischemic stroke for experimental studies and clinical applications.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Yasu Zhang and Xiaomin Liu conceived and designed the research; Junzi Long, Xue Cheng, and Xinyu Wang analyzed the data. Xiaodong Feng reviewed the paper for intellectual content. All authors read and approved the final version of the manuscript. Yasu Zhang and Xiaomin Liu contributed equally to this work.