Network Pharmacology and Molecular Docking Analysis on Molecular Targets and Mechanisms of Buyang Huanwu Decoction in the Treatment of Ischemic Stroke

Methods The bioactive components and potential targets of BHD were screened by TCMSP, BATMAN-TCM, ETCM, and SymMap databases. Besides, compounds that failed to find the targets from the above databases were predicted through STITCH, SwissTargetPrediction, and SEA. Moreover, six databases were searched to mine targets of IS. The intersection targets were obtained and analyzed by GO and KEGG enrichment. Furthermore, BHD-IS PPI network, compound-target network, and herb-target-pathway network were constructed by Cytoscape 3.6.0. Finally, AutoDock was used for molecular docking verification. Results A total of 235 putative targets were obtained from 59 active compounds in BHD. Among them, 62 targets were related to IS. PPI network showed that the top ten key targets were IL6, TNF, VEGFA, AKT1, etc. The enrichment analysis demonstrated candidate BHD targets were more frequently involved in TNF, PI3K-Akt, and NF-kappa B signaling pathway. Network topology analysis showed that Radix Astragali was the main herb in BHD, and the key components were quercetin, beta-sitosterol, kaempferol, stigmasterol, etc. The results of molecular docking showed the active components in BHD had a good binding ability with the key targets. Conclusions Our study demonstrated that BHD exerted the effect of treating IS by regulating multitargets and multichannels with multicomponents through the method of network pharmacology and molecular docking.


Introduction
e incidence of ischemic stroke (IS) is particularly high, and survivors have more or less neurological function deficits, which brings about a large burden on society and patients' families. With high mortality and morbidity rate, stroke has been the third most common cause of death following by coronary heart disease and cancer in the world [1]. IS accounts for 70%-80% of all stroke, which is the most common type of stroke in clinic [2]. At present, thrombolysis has been considered as the fastest and most effective treatment for IS, but the clinical effect of thrombolysis therapy is limited due to strict indications, a short time window, high risk of bleeding, and reperfusion injury [3]. A large number of experimental studies and clinical observations have confirmed that Chinese medicine has unique advantages in treating IS. Various empirical prescriptions, single drugs, and active ingredient extracts have shown clear neuroprotective effects on IS [4][5][6].
Buyang Huanwu decoction (BHD) is a classical prescription for the treatment of IS. is prescription is mainly composed of Huangqi (Radix Astragali), Honghua (Carthami flos), Taoren (Persicae Semen), Chishao (Radix Paeoniae Rubra), Danggui (Angelicae sinensis Radix), Chuangxiong (Chuanxiong Rhizoma), and Dilong (Pheretima). Among them, Radix Astragali is the most widely used in the original prescription, which is the monarch drug in BHD. A systematic review and meta-analysis of nineteen RCTs with 1580 individuals showed that BHD could significantly improve the neurological deficit score and the ability of self-care of patients with IS [7]. BHD could promote angiogenesis, attenuate infiltration of natural killer cells, and facilitate neurorehabilitation through an improvement of synaptic plasticity after cerebral ischemia/ reperfusion injury. It could significantly decrease cerebral edema and rat neurological function scores and reduce brain infarct volume [8][9][10][11][12]. Pharmacological researches have shown that Radix Astragali has the function of dilating blood vessels and improving microcirculation, which can significantly increase the brain's ability to withstand periods of severe hypoxia and/or ischemia [13,14]. Carthami flos, Persicae Semen, Radix Paeoniae Rubra, Angelicae sinensis Radix, and Chuanxiong Rhizoma can effectively improve the microcirculation of the body, significantly inhibit the proliferation of fibrous tissue, and reduce the inflammatory response [15][16][17].
However, the molecular mechanism of BHD has not been certainly clear. e clinical effect of the decoction is a comprehensive result of the complex biological processes in human body. Network pharmacology, based on system biology and multidirectional pharmacology, integrates the contents of computer biology and network analysis [18]. It explains the integrity and systematization of drug-target-disease interaction from the perspective of multicomponents, multitargets, and multichannels, which is consistent with the holistic view of Chinese medicine [19]. erefore, it provides a method for the study of multicomponents' mechanisms of Chinese medicine [20,21]. In this paper, network pharmacology was used to explore the molecular mechanism of BHD against IS. e detailed workflow of the study is shown in Figure 1.

Protein-Protein Interaction
Data. String 11.0 (https:// string-db.org/) [32] is a database for storing known and predicted protein interactions, including direct and indirect protein interactions. It scores each protein interaction. A higher score means a higher confidence of protein interaction. e selected intersection targets were imported into String for protein interaction analysis, and the protein interaction network was obtained with the species limited to "Homo sapiens" and a confidence score >0.7. e protein interaction data were imported into Cytoscape 3.6.0 (https:// cytoscape.org/) to construct the PPI network.

Gene Ontology (GO) and Pathway Enrichment.
DAVID (https://david.ncifcrf.gov/) [33] database integrates various types of database resources and uses the improved Fisher precision test algorithm to analyze the enrichment of gene sets. A cutoff P value and false discovery rate (FDR) < 0.05 were used to indicate statistical significance. GO annotation and KEGG PATHWAY analysis were carried out for the intersection genes. Finally, we could get the pathway maps from KEGG PATHWAY Database (https://www.kegg.jp/) [34].
All networks can be constructed via utilizing the network visualization software Cytoscape, which displays network graphically. Cytoscape makes it possible for data integration, analysis, and visualization of complicated networks. In the network diagram, "node" represents the active component and target in BHD, and "edge" represents the relationship between the active component and target.
e "degree" parameter, presenting the number of connections between the nodes in the network, was used to evaluate important targets [30]. Evidence-Based Complementary and Alternative Medicine

Cluster of BHD-IS PPI Network.
e closely related regions in protein-protein interaction networks are defined as topological modules or clusters. ese clusters or functional modules can put nodes of similar or related function together in the same network. By the help of MCODE, a plug-in of Cytoscape, we can get clusters.

Molecular Docking Verification.
To validate the compound-target associations, the AutoDock software (version 4.2) was used to perform the molecular docking program [35]. RCSB PDB (http://www.rcsb.org/) [36] was used to retrieve and download the 3D structure files of key target proteins. 3D structure files of compounds were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) [37]. Finally, the AutoDock platform was used for molecular docking verification [38]. e binding energy was calculated to evaluate binding interactions between the compounds and their targets. e binding energy less than "−5" indicates a good binding interaction between the compound and target [39].

Targets of BHD and IS.
As 19 compounds of BHD had no targets in TCMSP, BATMAN-TCM, ETCM, and SymMap, Canonical SMILES of these compounds were found in PubChem. Based on chemical structural similarity, we used databases like STITCH, SwissTargetPrediction, and SEA, to predict their targets. ese compounds were excluded because of the targets score less than 50% eventually. In brief, 235 targets were adopted in this research.
By means of the six available resources, namely, Dis-GeNET, OMIM, TTD, DrugBank, PharmGKB and Mala-Cards databases, we obtained 460 IS-related targets.
Based on targets of the candidate ingredients and IS, intersection targets were got by R software. 62 intersection genes were found eventually, shown in Figure 2(a). e details of intersection targets are described in Table 2. Evidence-Based Complementary and Alternative Medicine

Pathway Enrichment.
rough comprehensive analysis, we obtained an integrated IS pathway based on our current knowledge of IS pathogenesis to illuminate the integral role of BHD in treating IS. TOP 10 KEGG signaling pathways of BHD were obtained and constructed based on P value as shown in Figure 3(d).

BHD-IS PPI Network Analysis
3.4.1. BHD-IS PPI Network. 62 intersection targets were imported into the String database, and TSV text showing the interaction relationship was obtained, as shown in Figure 2(b). en, the network topology analysis was applied by the software of Cytoscape 3.6.0. Importing the TSV text into the Cytoscape software, we could get BHD-IS network, as shown in Figure 2(c). is network contained 59 nodes and 664 edges. In this network, the rose red nodes had higher degrees. e number of those nodes' edges was 36 in IL6, 31 in TNF, 29 in VEGFA, 28 in AKT1, 27 in MMP-9, 26 in IL1B, 23 in MAPK1, 22 in ICAM1, 22 in PTGS2, and 20 in IL10, respectively. is suggested that these genes might be the key or central genes in IS development. Bar graph of all protein nodes degree related to the targets is shown in Figure 2(d).
e target proteins in the PPI network were modularized and analyzed by using the plug-in of cluster maker of the software Cytoscape 3.6.0. e results showed that 62 targets were divided into four modules, including 24 in module one, 18 in module two, 11 in module three, and 9 in module four, as shown in Figure 2(e). Functional annotation of the 4 modules is shown in Table 3.

Compound-Target Network
Analysis. e active components and their targets were constructed to establish the compound-target network using Cytoscape.
is network was composed of 294 nodes (235 compound-target nodes and 59 compound nodes) and 1192 edges. In this network, we could find that one target could be hit by several compounds (central nodes, such as IL6, MMP-9, TNF, AKT1, ICAM1, IL1B, PTGS2, IL-10, VEGFA, and MAPK1), but some were modulated by only one compound in this network. Furthermore, one potential active compound could correspond to multiple targets. Top twelve compounds with high degree were quercetin, beta-sitosterol, kaempferol, stigmasterol, baicalein, luteolin, hederagenin, 7-o-methylis omicron ulatol, formononetin, isorhamnetin, dimethoxy, and myricanone, shown in Table 4. It could be seen that the neuroprotective mechanism of BHD had the characteristics of multicomponents, multitargets, and multi mechanisms. e compound-target network is shown in Figure 4.

Herb-Target-Pathway
Network. By importing 62 targets which overlapped with IS disease genes into DAVID, we could get 20 IS-related pathways. Radix Astragali and Carthami flos had the highest degree, which means that the two herbs might be the main herbs in treating IS. Meanwhile, TNF signaling pathway showed the highest degree, followed by PI3K-Akt signaling pathway, MAPK signaling pathway,   Evidence-Based Complementary and Alternative Medicine      Figure 5.

Target Path Analysis.
e pathway map of BHD in treating IS was obtained from KEGG PATHWAY Database, as shown in Figure 7. e related pathways were marked in red, and the targets of BHD in treating IS were marked in rose red. e results showed that the main pathways of BHD in treating IS included TNF signaling pathway, MAPK signaling pathway, NF-κB signaling pathway, and PI3K/ AKT signaling pathway.

Discussion
In our study, we found the molecular mechanism of BHD's neuroprotection effect against IS using network pharmacology strategy. e network pharmacology strategy is helpful to clarify the mechanism of TCM's function from a systematic viewpoint [40,41]. Furthermore, this method provides a multidimensional research strategy for various complicated herbal decoctions. At present, the application of network pharmacology to study the mechanism of Chinese medicine has become a research hotspot. In this study, we found that 59 active components of BHD could act on 62 targets related to IS. Further analysis showed that BHD could act on many biological processes of IS and had an influence on the outcome of stroke through TNF, PI3K-Akt, MAPK, and NF-kappa B signaling pathway. It further confirmed that BHD had the characteristics of multicomponents, multichannels, and multitargets.
Core ingredients with the highest degree in compoundtarget network were considered to be responsible for neuroprotection, including quercetin, beta-Sitosterol, kaempferol, stigmasterol, baicalein, luteolin, hederagenin, 7-O-methylisomucronulatol, formononetin, isorhamnetin, Dimethoxy, and myricanone. Six of these components belong to Radix Astragali and Carthami flos. e results of network topology analysis showed that the degrees of Radix Astragali and Carthami flos were the highest in BHD. As the core herb in BHD, the dosage of Radix Astragali is the highest, indicating that the results of network pharmacology are consistent with the clinical application of Chinese medicine. Quercetin and kaempferol are common components of Radix Astragali and Carthami flos. It was found that quercetin could pass through the blood-brain barrier with the highest passage rate [42]. A research showed that quercetin had effects of antioxidant stress and promoting autophagy, which was helpful for the prevention and treatment of stroke [43,44]. In addition, quercetin could also regulate protein phosphatase 2A subunit B (PP2A) to produce significant neuroprotective effects on rats with cerebral ischemia-reperfusion injury and HT22 cell model of glutamate injury [45]. Lu et al. found that quercetin could inhibit the expression and release of many inflammatory factors such as TNF-α, IL-1 β, and IL6 by reducing the production of NF-κB in elderly mice [46]. Kaempferol, a common flavonoid compound, has been widely concerned because of its anti-inflammatory, antioxidant, antibacterial, and antiviral effects. e neuroprotective effect of kaempferol has been confirmed in the Evidence-Based Complementary and Alternative Medicine acute phase of cerebral infarction [47,48]. One study confirmed that kaempferol inhibited oxygen-glucose deprivation (OGD) induced cell viability decline, oxidative stress, mitochondrial dysfunction, and apoptosis [49]. ese findings suggested that kaempferol might be a promising choice for the intervention of IS. Baicalein is a common component of Carthami flos and Radix Paeoniae Rubra. As an important flavonoid compound, baicalein has many pharmacological effects, such as antioxidant stress, anti-inflammatory, antiexcitatory toxicity, antiapoptosis, stimulating neurogenesis, and promoting the expression of neuroprotective factors [50][51][52]. Liu et al. found that baicalein had protective effect on transient middle cerebral artery occlusion model rats and could significantly reduce the apoptosis of ischemic penumbra cells around the ischemic infarct of middle cerebral artery occlusion (MCAO) model rats [53]. As an ingredient of Carthami flos, luteolin could downregulate the expression of TLR4, TLR5, NF-κ B, and P-P38MAPK, upregulate the expression of p-ERK, and protect cerebral ischemia in rats [54]. Experiments performed in vivo also demonstrated that luteolin reduced the infarct volume. It was suggested that luteolin had a potential in the treatment of IS through inhibiting MMP-9 and activating PI3K/Akt signaling pathway [55]. Beta-sitosterol and stigmasterol are the common components of Carthami flos, Persicae Semen, Radix Paeoniae Rubra, and Angelicae sinensis Radix. ey are both sterol compounds, mainly having the functions of reducing blood fat, antioxidation, and anti-inflammation [56].
PPI analysis showed that IL6, TNF, VEGFA, AKT1, MMP-9, IL1B, MAPK1, ICAM1, PTGS2, and IL10 were the top ten targets with high degrees. Followed by cluster of the PPI network, the network could be divided into four modules, which were mainly related to angiogenesis, inflammation, coagulation, and blood-brain barrier. Inflammation plays a critical role in the pathological process of stroke [57]. IL1B, IL10, TNF, IL6, and ICAM1 are closely related to the inflammatory response after stroke, among which IL-10 is an important anti-inflammatory factor, while L1B, TNF, and IL6 are proinflammatory factors. ICAM1 is       Figure 7: Pathway map of BHD against IS. e key targets of BHD in the treatment of IS are shown as rose red in the TNF signal pathway. an important adhesion molecule mediating the adhesion reaction, which plays an important role in stabilizing the interaction between cells and promoting the migration of leukocytes and endothelial cells. Ischemic cascade reaction leads to microglial activation, which will promote the release of proinflammatory cytokines (TNF-α, IL1B, and IL6) and anti-inflammatory cytokines (IL10 and TGF-β) [58]. MMP-9 is a kind of matrix metalloproteinases (MMPs) closely related to the development of IS, which promotes embryo development, inflammation, atherosclerosis, and other biological functions. Under the stimulation of cerebral ischemia and hypoxia, microglia and astrocytes produce part of MMP-9 under the guidance of inflammatory factors. By hydrolyzing the tight junction protein on the basement membrane of cerebrovascular, the integrity of blood-brain barrier is destroyed [59]. VEGF is a double-edged sword in the development of cerebral infarction. In the hyper acute stage of cerebral infarction, the increase of VEGF concentration increases the permeability of blood-brain barrier, leading to brain edema and aggravate clinical symptoms. In the postinfarction recovery stage, the high content of VEGF is conducive to the establishment of collateral circulation of ischemic focus and penumbra and the damage and repair of neurons [60]. AKT1 is one of serine/threonine-protein kinases (AKT1, AKT2, and AKT3), and it regulates many processes including metabolism, proliferation, cell survival, growth, and angiogenesis. AKT1 gene deletion induces dysfunction of vascular endothelial cells, migration, and survival of vascular smooth muscle cells [61].
Pathway enrichment analysis results showed that TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and NF-kappa B signaling pathway were the main pathways. TNF signaling pathway is an important inflammatory pathway. As an important cytokine, TNF can induce apoptosis, cell survival, inflammation, immunity, and other intracellular signaling pathways. TNFR1 signal transduction can induce the activation of many genes, which are mainly controlled by NF-kappa B and MAPK cascade. In this present study, TNF, IL-1B, MYC, and TGFB1 are potential targets of BHD, suggesting that BHD plays a neuroprotective role against ischemia-reperfusion injury through TNF signaling pathway. PI3K/Akt signaling pathway is one of the important pathways of cerebral ischemia and neuronal apoptosis. A study found that activating PI3K/ Akt signal pathway could inhibit the apoptosis of nerve and reduce the occurrence of blood-borne brain edema. A series of studies have shown that many Chinese herbal extracts play a protective role in IS through this pathway [62,63]. Another study found that baicalein also decreased the LC3-II/LC3-I ratio and promoted phosphorylation of the PI3K/ Akt/mTOR signaling pathway which implied inhibition of autophagy. e reduction of phosphorylation Akt and glycogen synthase kinase-3beta (GSK3beta) induced by OGD was restored by Baicalein, which was associated with preserved levels of phosphorylation of PTEN [53,64]. It was reported that baicalein could activate PI3K/AKT pathway, inhibit caspase activation, and reduce cerebral infarct volume in MCAO rats [65]. Besides, formononetin mediated neuroprotection against cerebral ischemia/reperfusion in rats via downregulation of the Bax/Bcl-2 ratio and upregulation PI3K/Akt signaling pathway [66]. e MAPK signaling pathway may be a therapeutic pathway for stroke [67]. Researches showed that suppressing the NF-κB and MAPK signaling pathways would downregulate the expression of proinflammatory factors. e MAPK signaling pathway could be a promising candidate for future applications in CNS injury treatment [68]. BHD alleviated pressure overload induced cardiac remodeling by suppressing TGF-β/Smads and MAPKs signaling activated fibrosis [69].
However, our research also has some limitations. For example, the accuracy and integrity of existing databases need further verification. Higher quality databases of traditional Chinese medicine and more accurate background network databases are needed. Moreover, the results of network pharmacology need experimental support. e application of network pharmacology in the study of Chinese medicine is just in its start-up step. We need to promote the interdisciplinary researches integrating network science, bioinformatics, computer science, mathematics, and pharmacology in the future.

Conclusion
In this study, we explored and discussed the characteristic of "multicomponents, multitargets, and multichannels" of BHD-mediated IS treatment through the method of network pharmacology and molecular docking. In the future, we should provide experimental evidence for the neuroprotective effect of BHD against IS according to the results of network pharmacology research.

Data Availability
All data obtained or analyzed during this study are included within the article.

Conflicts of Interest
All authors declare no conflicts of interest.

Authors' Contributions
Qiang Gao contributed to conceptualization and writing original draft. Danfeng Tian contributed to software and data curation. Zhenyun Han contributed to supervision, review, and editing. Jingfeng Lin and Ze Chang contributed to methodology and software. Dandan Zhang contributed to supervision. Dayong Ma contributed to supervision, funding acquisition, and review and editing. Qiang Gao and Danfeng Tian contributed equally to this work.