A Network Pharmacology Approach to Predict the Proangiogenesis Mechanism of Huangqi-Honghua Herb Pair after Cerebral Ischemia

Objective Huangqi-Honghua herb pair is known for its medicinal value to treat Qi deficiency and blood stasis syndrome with a long history in clinical practice. To understand its possible mechanism in a systematic study, a network pharmacological method was addressed. Methods Detailed information on the HH compounds was obtained from two public databases, and oral bioavailability (OB) and drug-like (DL) of the compounds were evaluated. A correlation between HH compounds, its potential targets, and known targets was extrapolated, and the herb-compound-target-disease (H-C-T-D) network was established. Next, the pathway enrichment and essential genes were analyzed. Then, three key genes (VEGFA, VEGFR2, and eNOS), highly associated with angiogenesis, were screened and verified through western blot assay. Results Out of 276 compounds, 21 HH compounds and 78 target genes regulating the major pathways associated with CI in the network are analyzed. The bioactive compounds in HH were active in various signal transduction pathways such as the toll-like receptor signaling pathway, VEGF signaling pathway, TNF signaling pathway, and HIF-1 signaling pathway are important pathways that may regulate anti-inflammatory, antiapoptotic, immune correlation, and antioxidative effects. The core genes are PTGS2, TNF, NOS2, IL6, BCL2, IL1B, SOD2, NOS3, SOD1, MMP9, and VEGFA. The in vitro results suggested that HH treatment could significantly elevate the expression of proangiogenic genes such as VEGFA, VEGFR2, and eNOS compared with OGD groups. Conclusions Our results predict that HH may regulate the expression of VEGFA, VEGFR2, and eNOS via the VEGF and HIF-1 signaling pathway to promote angiogenesis and alleviate cerebral ischemia injury.


Introduction
Ischemic stroke (cerebral ischemia, CI) has become a major public health concern with morbidity, mortality, and health care costs [1]. Irrespective of the current standard of care options (e.g., mechanical or pharmacological (tissue plasminogen activator, tPA) reperfusion) available in the clinical practice, there is an unmet need for ischemic stroke because of the narrow treatment time window [2]. us, there is an unmet need for developing regenerative medicine to address CI and its secondary effects. One of the devastating consequences of CI is decreased blood supply to the injured brain, and it leads to the functional impairment of the brain tissue in the affected area. Developing an effective strategy to increase the blood supply to the ischemic area may potentially rescue the brain tissue from death and may improve the recovery rate of the patients [3]. Previous studies have indicated that the higher the vascular density of ischemic penumbra, the longer the survival time of ischemic stroke patients [4,5]. erefore, proangiogenesis may be a valid antistroke strategy to address current clinical hurdles associated with a stroke patient, which has the potential to repair and rewire the injured nerve connections.
Traditional Chinese medicine (TCM) has been shown to be beneficial in promoting developmental angiogenesis, which highlights "reinforcing qi to enrich the blood, dispelling stasis to promote regeneration" theory by associating with angiogenesis together. Radix Astragali (Huangqi) is the dry root of Astragalus membranaceus (Fisch.) Bge, which has been commonly used in patients with stroke or chronic weakness in China because it can enhance Qi and dispel blood stasis, with antioxidation, anti-inflammatory, immunomodulation, and anticancer effects in modern medical research [6,7]. Carthamus tinctorius L. (Honghua), a dried flower, often used to promote blood circulation, dispel blood stasis, and relieve pain [8,9]. Huangqi-Honghua (HH) herb pair has been used widely in clinical practice for treating Qi deficiency and blood stasis syndrome, especially cerebrovascular and cardiovascular diseases, with a long history, such as Buyang Huanwu decoction, a wellknown TCM formula [10][11][12]. In our previous study, by using a rat model of middle cerebral artery occlusion (MCAO), we have validated the synergetic effect of hydroxysafflor yellow A and astragaloside IV (main constituents of HH) in removing blood stasis (QDBS) syndrome [13].
Network pharmacology follows the dogma of "drugtargets-gene-disease," to predict drug targets and improve the efficiency of drug discovery effort. Network pharmacology maintains integrity and systematic characteristic, similar to the principle of traditional Chinese medicine that involves multiple components and multiple targets [14]. Network pharmacology has become a novel and efficient way to interpret the complex pharmacological mechanism of TCMs scientifically, particularly, formulae orchestration [15].
Despite a few studies that have proved the protective effect of HH in the cerebral hemisphere, pharmacologybased prediction of complete profiling of bioactive components and the network of their target pathways has not fully been elucidated. us, in the present study, we have explored the proangiogenesis mechanisms of HH in the etiology of CI. In this study, we have established the herbcompound-target-disease (H-C-T-D) networks by utilizing the network pharmacology approach. Furthermore, to demonstrate angiogenesis, one key pathway of HH in cerebral protection, we have identified three key targets, which is highly associated with angiogenesis, through immunoblot assay. e workflow is shown in Figure 1.

Oral Bioavailability and Drug-Likeness of HH
Components. All compounds in HH were evaluated for their oral bioavailability (OB) and drug-likeness (DL). OB is one of the most common pharmacokinetic parameters in the drug screening process, which explains the percentage of an oral drug that remains unchanged after entering into the systemic circulation. In addition, OB provides a clue for the fusion of the ADME process. DL measures the extent of the "drug-like" property of a compound and determines whether the compound influences absorption, distribution, metabolism, and excretion (ADME) in the human body like an approved drug. In this study, chemicals only with an OB ≥30 and DL ≥0.18 (recommended by TCMSP database) were considered for further valuation [16].

Collection Target Genes Relevant to the Screened
Compound. UniProt (http://www.uniprot.org/) platform, by setting the filter to "Homo sapiens," was used to identify the target genes, closely relevant to chemicals. Meanwhile, information on target genes related to the CI was obtained from the therapeutic target database (TTD; http://www. bidd.Nus.edu.sg/group/cjttd/TTD%20HOME.asp) and DisGeNETv6.0 (http://www.disgenet.org/web/DisGeNET/ menu/home). Finally, we assigned the gene names and ID by constructing herbs-ingredients-targets relationship dataset for HH.

Network Construction and Pathway Analyses.
e herbcompound-target-disease (H-C-T-D) networks were established using Cytoscape 3.3.0, JAVA software. Functional pathways annotation related to CI and enrichment evaluation was evaluated using the Kyoto Encyclopedia of Genes (KEGG), Genomes (GO), and the Database for Annotation, Visualization, and Integrated Discovery (DA-VID) version 6.8 (http://david.ncifcrf.gov/).

Protein-Protein Interaction (PPI)
Data. PPI data were collected from the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/, 11.0 version), and possible protein-protein interactions were predicted. We chose and showed the top 20 target genes with confidence score >0.9 as the core genes for CI.  30 min. e whole cell lysates were separated by 10% sepharose gel, and PVDF membranes (Millipore, USA) were used to transfer protein. e membranes were incubated with 5% bovine serum albumin (BSA) and overnight with primary antibodies against VEGFA (1 : 1000, rabbit), VEGFR2 (1 : 1000, rabbit), and eNOS (1 : 1000, rabbit) at 4°C. After that, membranes were incubated with secondary antibody at a 1 : 5000 dilution at 37°C for 1 h. After ECL-Plus reagent (Santa Cruz, USA) treatment, the blots were analyzed with Quantity One System image analysis software (Bio-Rad, USA).

Statistical Analysis.
e results were expressed as mean ± standard deviation (S.D.) and analyzed with oneway analysis of variance (ANOVA), followed by a Tukey's post hoc test was used for analyzing differences between groups. p < 0.05 presented statistical significance.

Identification of Active Compounds in HH.
HH consists of a total of 276 compounds, 87 in Astragalus (Huangqi) and 189 in Safflower (Honghua). All 276 compounds were screened with OB and DL, out of which, only 42 compounds were recommended for the final screening. e remaining compounds were discarded. e therapeutic effect of a few selected compounds on CI was confirmed through the related literature search. erefore, preselected active ingredients were added manually, including hydroxysafflor yellow A and astragaloside IV [17][18][19]. After that, the repeated ingredients and the ingredients with the unclear target are deleted. e selected 27 compounds from HH is shown in Table 1 and Figure 2(a).

Compounds-Targets Analysis.
e active ingredients of HH obtained from the screening assay were predicted using TCMSP and CTD database (https://ctdbase.org/). e repeated targets were deleted, and the unclear targets were eliminated. A total of 459 predicted target genes were obtained related to the 27 identified compounds in HH (Figures 2(a) and 2(c)). e compound-target gene network includes 683 nodes and 1146 edges as shown in Figure 2  Evidence-Based Complementary and Alternative Medicine 3 HH are listed in Table 2. Kaempferol is the compound of maximum interactions with target genes.

Pathway and GO Term Enrichment Analysis.
To explore the signaling pathway and functions of the identified target genes, 78 candidate targets were analyzed by GO and enriched by the KEGG pathway. e top 6 enriched conditions were found to be involved in the biological process (BP), cell component (CC), and molecular function (MF) (Figure 4(a)). According to the results of GO enrichment analysis, the positive regulation of transcription from RNA polymerase II promoter, inflammatory response, response to lipopolysaccharide, aging, response to drug, signal transduction etc., dominates the biological process. Forty-two targets, the largest proportion, account for cell components. Screening of p < 0.05 signal pathway by KEGG enrichment analysis eliminates signaling pathways that are not associated with CI and get the top 18 signaling pathways (Figure 4(b) and Table 4). erefore, we speculate that the TNF signaling pathway, toll-like receptor signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, NFkappa B signaling pathway, and VEGF signaling pathway may be the important pathways to exert their synergistic effects against cerebral ischemia injury.

Experimental Validation.
To confirm the results from the network and to verify that angiogenesis is indeed one of the key pathways of HH in cerebral protection, we selected three targets related to angiogenesis (VEGFA, VEGFR2, and eNOS) for pharmacological validation ( Figure 6). Western blot analysis indicated that treatment with HH could significantly increase the expression of VEGFA, VEGFR2, and eNOS, compared with the OGD group ( * p < 0.05, * * p < 0.01 compared with control group, and #p < 0.05 compared with OGD group).

Discussion
Multicompound TCMs, with multiple biological targets, are the prime focus in Chinese clinical practice for more than thousand years. Surprisingly, TCMs are known to target multiple biological pathways to defend against diseases. However, the traditional method of usage of TCMs barely provides insights into the biological complexity of compounds, its biological targets, and associated disease, which limits the development and reformulation of TCMs. Lately, network pharmacology approach of analyzing TCMs gains more popularity that dissects the pharmacological mechanism of action [20][21][22].
In the current study, a network pharmacology analysis of HH identified 2 herbs, 21 compounds, as well as 78 target gene-regulated major pathways associated with CI. rough the pathway enrichment analysis, we found that the targets of active ingredients in HH against cerebral ischemia injury     Figure 5(b)), such as TNF, IL6, MAPK3, VEGFA, IL10, and MMP9. In addition, three key targets (VEGFA, VEGFR2, and eNOS), predicted in the network, highly related to angiogenesis were verified by using western blot assay.
According to the TCM theory, Qi and blood dysfunction is one key pathogenesis of CI. In the clinic, Qi-tonifying     drugs, blood-activating drugs, and reinforcing Qi and activating blood drugs team were usually treated for CI with significant efficacy. A fewer side effects [23,24] of these drugs could promote postischemic angiogenesis to recovery the cerebral blood flow and tissue perfusion, supporting the survival of neurons and neural progenitor cells and promoting long-term functional recovery [25,26]. Huangqi-Honghua (HH), a common reinforcing Qi and activating blood drugs team, has been used widely in clinical practice for treating Qi deficiency and blood stasis syndrome, especially, CI with Qi deficiency and blood stasis [7,27]. Based on the available data, there are 276 compounds found in HH and categorized into flavonoids, lignans, terpene, sterols, triterpenoids, and their glycosides. After screening with OB and DL, we selected 21 compounds that were correlated with 78 human protein targets for CI. e top 10 key compounds related to more than five genes were found to be astragaloside IV, calycosin, formononetin, bifendate, isorhamnetin, hydroxysafflor yellow A, quercetin, luteolin, kaempferol, and beta-sitosterol. Uniformly, hydroxysafflor yellow A and astragaloside IV are in the list. Our previous study has indicated that HH could significantly ameliorate cerebral ischemia injury, and the mainly effective compounds are hydroxysafflor yellow A and astragaloside IV [13]. Moreover, in a recent study, astragaloside IV also has also been shown to possess anti-inflammatory and antioxidant activation potential [28]. rough the network analysis results, kaempferol has the maximum interactions with target genes, especially can attenuate neuroinflammation and blood brain barrier dysfunction to improve neurological deficits in  Evidence-Based Complementary and Alternative Medicine cerebral I/R rats by regulating the NK-κB pathway [29]. However, further studies need to understand if kaempferol is the most important pharmacodynamics substance. Calycosin, which strikingly downregulates HUVEC TGF-betal, ICAM1, and RAGE expression [30], has antitumor, neuroprotective, anti-inflammatory, and proangiogenesis effects [31]. In vivo study also revealed quercetin to have antiinflammatory effects by inhibiting oxidative stress and cytokine production [32]. Formononetin exert its therapeutic effects by regulating these biological processes such as hormone metabolism, apoptosis, cell communication, and signal transduction [33]. ese findings were consistent with the results from the active component target and indicate that the main components of HH are effective for treating target disease(s). Angiogenesis is similar to "reinforcing qi to enrich blood, Dispelling stasis to promote regeneration" theory and regulated by angiogenesis inducers and inhibitors. Angiogenesis involves basilar membrane degradation, chemotactic migration, and proliferation of EC (endothelial cell) and EPC (endothelial progenitor cell). In the PPI system analysis, top 20 center genes were recognized, and among these genes, VEGFA and eNOS are highly correlated with angiogenesis. Over the few decades, vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been regarded as the principal drivers of angiogenesis and the development and maintenance of vascular system [34]. VEGFA (commonly known as VEGF) is the prototype member of the VEGF family of proteins, which stimulate angiogenesis in blood vascular endothelial cells via receptor-binding with VEGFR2 [35,36]. Previous report suggests that overexpressed IncRNA ANRIL upregulates VEGF and promotes angiogenesis by activating NF-κB pathway in CI rats [37]. Endothelial nitric oxide synthase (eNOS/NOS3) provides continuous local production of nitric oxide (NO), a crucial angiogenesis mediator and effector [38]. e increase in NO production via the upregulation of NOS3 by VEGF indicates that the angiogenic effect of VEGF seems to be mediated by NO [39]. erefore, we verified the function of three essential genes (VEGFA, VEGFR2, and eNOS) using western blot assay, and the results show that VEGFA, VEGFR2, and eNOS were upregulated in BMECs with oxygen-glucose deprivation treatment with HH, which is beneficial for angiogenesis. According to the KEGG pathway analysis, these three target genes interact mainly with HIF-1 signaling pathway and VEGF signaling pathway. Low oxygenation concentrations in tissues (hypoxia) often trigger angiogenesis [40], which can be initiated independently of VEGF-related pathways, as well as lead to expression of multiple growth factors such as VEGF and NOS, via the HIF pathway [41]. HIF-regulated proangiogenic factors can increase vascular permeability, endothelial cell proliferation, migration, adhesion, and tube formation [42]. erefore, we speculate that HH can regulate VEGFA, VEGFR2, and eNOS via the pathways associated with promote angiogenesis and alleviate cerebral ischemia injury. However, our current study still have some limitations, the compounds in the database may be incomplete, and it is incomprehensive that the vitro experiments only be used to confirm the results from the network. In the future, further studies require to design and implement a range of pharmacological experiment to investigate the possible pathway that controls proangiogenesis.
In summary, network pharmacology analysis of HH identified 2 herbs, 21 compounds, and 78 target gene-regulated major pathways associated with CI. e bioactive compounds in HH mainly participate in numerous signal transduction pathways such as TNF signaling pathway, tolllike receptor signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, and VEGF signaling pathway, and these important pathways may regulate anti-inflammatory, antiapoptotic, immune correlation, and antioxidative effects. Furthermore, through the pharmacological experiment, we predict that HH can regulate VEGFA, VEGFR2, and eNOS via the HIF-1 signaling pathway and VEGF signaling pathway to promote angiogenesis and alleviate cerebral ischemia injury.

Data Availability
e 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.