Mechanisms of Wogonoside in the Treatment of Atherosclerosis Based on Network Pharmacology, Molecular Docking, and Experimental Validation

doi:10.21203/rs.3.rs-4461287/v1

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Research Article

Mechanisms of Wogonoside in the Treatment of Atherosclerosis Based on Network Pharmacology, Molecular Docking, and Experimental Validation

https://doi.org/10.21203/rs.3.rs-4461287/v1

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Background

Atherosclerosis serves as the fundamental pathology for a variety of cardiovascular disorders, with its pathogenesis being closely tied to the complex interplay among lipid metabolism, oxidative stress, and inflammation. Wogonoside, a natural flavonoid derived from Scutellaria baicalensis, exhibits various biological activities including anti-inflammatory, anti-cancer, and anti-angiogenesis properties. Despite these known effects, the specific role of wogonoside in the context of atherosclerosis remains to be elucidated.

Purpose

To validate the efficacy of wogonoside in the treatment of atherosclerosis and to investigate its possible therapeutic mechanisms.

Methods

Network pharmacology was used to obtain the core targets and signaling pathways that may be efficacious in the treatment of atherosclerosis with wogonoside, which were validated using molecular docking and molecular dynamics simulations. To further validate the core targets in the signaling pathway, we performed in vivo experiments using apolipoprotein E (ApoE)-/- mice. This included pathological morphology and lipid deposition analysis of mouse aorta, serum lipid level analysis, Elisa analysis, oxidative stress analysis, ROS fluorescence assay, immunohistochemical analysis and protein blot analysis.

Results

Predictions were obtained that wogonoside treatment of atherosclerosis has 31 core targets, which are mainly focused on pathways such as Toll-like receptor (TLR) signaling pathway and NF-kappa B signaling pathway. Molecular docking and molecular dynamics simulations showed that wogonoside has good binding properties to the core targets. In vivo experimental results showed that wogonoside significantly inhibited aortic inflammatory response and lipid deposition, significantly reduced the release levels of TC, TG, LDL-C, ox-LDL and FFA, and significantly inhibited the release of inflammatory factors TNF-α, IL-1β, IL-6 and oxidative stress in ApoE-/- mice. Further molecular mechanism studies showed that wogonoside significantly inhibited the activation of TLR4/NF-κB signaling pathway in ApoE-/- mice.

Conclusion

Wogonoside may be an effective drug monomer for the treatment of atherosclerosis, and its mechanism of action is closely related to the inhibition of the activation of the TLR4/NF-κB signaling pathway.

Atherosclerosis

Wogonoside

Inflammation

Network pharmacology

Atherosclerosis is the underlying pathology for numerous cardiovascular disorders. The pathogenesis of AS relates to the intricate connection between lipid metabolism, oxidative stress, and inflammation [1]. This ultimately results in serious cardiovascular ailments [2]. The introduction of pharmacologic interventions and surgical treatments has lowered the disability and mortality rates. However, intolerance to drugs such as statins, poor compliance, and incomplete hemodialysis for interventional and surgical treatments are still prevalent in AS patients [3–5]. Research efforts are increasingly directed toward treating the persistent inflammatory response in AS [6]. The development of natural drugs targeting the inflammatory pathways of AS is anticipated to introduce new treatment options for the disease [7–9].

Wogonoside, the principal flavonoid compound originating from the root of Scutellaria baicalensis, exhibits anti-inflammatory, anti-angiogenic, and chemotherapy-induced anticancer activities [10, 11]. Multiple studies have demonstrated that the complete flavonoid content of Scutellaria baicalensis has a marked therapeutic impact on cardiovascular disease (CVD) [12, 13]. It achieves this by blocking apoptosis, controlling oxidative stress responses, contributing to the inflammatory response, guarding against myocardial fibrosis, suppressing myocardial hypertrophy and regulating blood vessels. A recent study reported that wogonoside has a protective effect on non-alcoholic fatty liver disease in mice [14]. The anti-inflammatory and oxidative stress inhibiting properties of wogonoside may contribute to this effect. Furthermore, wogonoside was found to prevent ischemia/reperfusion-induced myocardial injury by suppressing apoptosis, inflammation, and fibrosis through modulating the Nrf2/HO-1 pathway, which confirms the therapeutic potential of wogonoside in treating CVD [15]. However, the impact of wogonoside on AS and its molecular mechanisms have not been fully explored.

The advent of network pharmacology has allowed us to find effective targets that are common to both drugs and diseases, improving the accuracy of signaling pathways for selecting drugs to treat diseases [16]. In this study, firstly, we screened and predicted the possible targets of the active ingredient of wogonoside in the treatment of AS, and further identified the possible signaling pathways. Then, through molecular docking and molecular dynamics simulations, we visualized the interactions between wogonoside and important therapeutic targets, further determining the binding stability and adaptability of the therapeutic targets. Finally, the therapeutic actions and mechanism of wogonoside anti-atherosclerosis were researched based on an atherosclerotic ApoE-deficient mice model induced by high-fat feed. This evidence is an indication that wogonoside has the potential to be a drug for the treatment of AS.

Network Pharmacology Analysis

First, we performed the acquisition of wogonoside-related targets. We obtained 4 targets using the TCM Systematic Pharmacology Study Platform (TCMSP) (http://www.tcmip.cn/TCMIP/index.php/Home/Index/index.html) [17], 16 targets using the Swiss target prediction (http://www.swisstargetprediction.ch/) [18], 7 targets using the Comparative Toxicogenomics Database(https://ctdbase.org/ ) [19] and finally 65 targets using the TargetNet database(http://targetnet.scbdd.com/) [20]. Then, to obtain relevant targets for the disease AS. We acquired 2529 related targets using AS as the keyword in GeneCards(https://www.genecards.org/) [21]. 3 targets using the Online Mendelian Inheritance in Man (OMIM) (https://www.omim.org) [22], 28 targets using the comparative toxicogenomics database (CTD)( https://ctdbase.org/) [19], and 35 targets using the therapeutic target database (TTD)( https://bidd.group/group/cjttd/ ) [23]. We plotted Venn diagrams of common targets with ggVenn (R package), and protein-protein interaction network (PPI) models were available using STRING 11.5 (https://cn.string-db.org/ ) and Cytoscape 3.9.0 software, where the species was set to "Homo sapiens", the minimum required interaction score set to "highest confidence (0.900)" [24]. We used the Database for Annotation, Visualization and Integrated Discovery (DAVID 6.8) (https://david.ncifcrf.gov/home.jsp) to analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway[25]. The date of access to the above databases is April 15, 2024.

Molecular docking

Molecular docking analysis was performed with the protein targets of the chosen ligands using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) platform[26, 27]. CB-Dock2 is a molecular docking tool based on AutoDock Vina analysis which can automatically identify and analyze the site where the ligand binds to the receptor. It enhances molecular docking accuracy while simplifying docking processes. Pymol software (free version) and Discovery Studio Visualizer (version 2021) software were utilized to further show us the ligand-receptor interaction in detail. The structure of wogonoside was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov), and was then charged with H atoms and subjected to free energy minimization. The three-dimensional structures of RELA (PDB ID: 2vge), TLR4 (PDB ID: 2Z62), and TNF (PDB ID: 5m2j) proteins were retrieved from RCSB PDB (https://www.rcsb.org/) [28]. Subsequently, hydrogen atoms were added and all water molecules were removed during processing. We made assured that the whole wogonoside was contained within the grid box. The Vina score was computed using CB-Dock2, which projected the optimal docking model with the least amount of energy. The "Cartoon" option was selected for the ligand and receptor. The terms "Element" and "Secondary structure" were used to present ligands and colored receptors, respectively. The date of access to the above databases is April 13, 2024.

Molecular dynamics simulation

To investigate the stability of AS target structures complexed with the wogonoside ligand, molecular dynamics (MD) simulations were conducted using GROMACS version 2020.6. The protein parameterization followed the Amber99SB force field, while the ligand topology was based on Sobtop (Tian Lu, Sobtop, Version 1.0(dev3.1), http://sobereva.com/soft/Sobtop ). The system, comprising the protein-ligand complex, was solvated in a dodecahedron box using a simple point charge (SPC) model, with periodic boundary conditions and a minimum solute-to-box distance of 1.5 nm. Ion addition was used to maintain charge neutrality. Energy minimization was carried out with the steepest descent method, followed by equilibration in both NPT and NVT ensembles at a constant temperature of 300 K and pressure of 1 bar using the Parrinello-Rahman barostat. The 100 ns molecular dynamics simulations, totaling 50,000,000 steps, were analyzed with GROMACS to assess structural stability via root mean square deviation (RMSD). Additionally, root-mean-square fluctuation (RMSF) of the protein and radius of gyration (Rg) were used to evaluate structural flexibility and dynamics, shedding light on conformational changes and folding.

Reagents

Wogonoside (purity ≥ 95%) was purchased from Sigma Chemicals (St. Louis, MO, USA). Atorvastatin (Lipitor; SFDA No. H20051408) was purchased from Pfizer Pharmaceutical Co., Ltd. The total cholesterol (TC), triglycerides (TG), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), oxidized low density (ox-LDL), free fatty acids (FFA), malondialdehyde (MDA) and superoxide Dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bioengineering Inc. (Nanjing, China). Toll-like receptor 4 (TLR4), anti-phosphor-NF-κB antibodies were obtained from Affintiy (Jiangsu, China).

Animals and Treatment

Thirty-two 6-8-week-old male ApoE-/- mice and eight 6-8-week-old male C57BL/6 mice were obtained from Beijing Huafukang Biotechnology Co., Ltd. The mice were housed in pathogen-free units at the Animal Center of Guangzhou University of Traditional Chinese Medicine. The housing conditions included a temperature of 22 ± 1°C, a relative humidity of 60–70%, and a 12-hour light/dark cycle. The ApoE-/- mice were fed a high-fat diet (HFD) for 12 weeks, consisting of 15% lard, 0.5% sodium cholate, 2% vitelline, 1.5% CHOL, and 81% basic feed[29]. Subsequently, they were randomly divided into four groups: model group, atorvastatin group (5 mg/kg/d), low dose wogonoside group (20 mg/kg/d), and high dose wogonoside group (40 mg/kg/d)[30]. The ApoE-/- mice received daily doses of atorvastatin and wogonoside for 8 weeks along with the HFD. Additionally, eight healthy C57BL/6 mice were included as the normal group and fed a regular diet without any interventions. All animal experiments were conducted in accordance with the Regulations on Animal Experiments established by the Animal Ethical Committee of The First Affiliated Hospital of Guangzhou University of Chinese Medicine (Ethical No. GZTCMF1-2022044).

Aortic root lesion analysis

The hearts of ApoE−/− mice were fixed in 4% PFA for 24 hours. To analyze atherosclerotic lesions, the hearts were embedded in OCT and frozen at − 80°C. The aortic root was then cut into 5 µm sections starting from the aortic valve. Three consecutive sections were stained with Oil Red O and Hematoxylin & Eosin for morphological examination. ImageJ was applied to analyze the staining results and calculate the ratio of plaque area/vessel area.

Serum lipid analysis

Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), oxidized LDL (ox-LDL), and free fatty acids (FFA) were assayed enzymatically.

Cytokine Expression Assessment

The concentrations of TNF-α, IL-1β, and IL-6 in both the serum and aorta were quantified using ELISA kits, in accordance with the provided instructions. The final concentrations were calculated by referencing the respective standard curves.

Oxidative stress analysis

The effect of wogonoside on the level of oxidative stress in ApoE-/- mice in vivo was assayed according to the instructions of the MDA and SOD assay kit.

Immunofluorescence assay

Reactive oxygen species (ROS) production in aortic tissue was assessed using the fluorescent probe dihydroethidium (DHE). Cryosections of the aortic root were treated with 10 µmol/L DHE in a dark environment at 37°C for 1 hour, followed by three rinses with 1X PBS. Fluorescence images were acquired with a fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

Immunohistochemistry assay

Immunohistochemistry was employed to visualize the expression of TLR4 and p-NF-κB in aortic arch tissues. Fixation of the aortic arch tissues was carried out using 4% paraformaldehyde, followed by embedding in paraffin for sectioning. After dewaxing, rehydration, and blocking with 3% bovine serum albumin (BSA), the sections were incubated with TLR4 and p-NF-κB antibodies overnight at 4 degrees. Subsequently, the sections were treated with secondary antibodies conjugated with HRP. The stained sections were examined under a microscope after DAB staining and hematoxylin re-staining. Quantification of TLR4 and p-NF-κB expression levels was performed using Image J software based on staining intensity scores.

Western blot analysis

Protein sampling of ApoE-/- mice tissue was performed using lysate. After protein sampling, electrophoresis was performed at 80 V for 20 minutes and then transferred to 120 V for 20 minutes. Proteins were then transferred to a PVDF membrane at 300 mA for 50 minutes and sealed in a sealing solution for 15 minutes. The gel is then incubated with primary antibodies at 4°C overnight. Exposure was performed on a Gel Imaging Analyzer System with ECL emitting solution. Independent experiments were repeated 3 times, and the grayscale values of each band were analyzed using ImageJ.

Statistical Analysis

The results are presented as mean ± SD and were analyzed using GraphPad Prism software (version 9.0). Statistical differences between groups were assessed using a one-way analysis of variance (ANOVA). p-values less than 0.05 were considered as indicating statistically significant differences.

Core target screening

The core target of wogonoside with AS co-action suggests that wogonoside has a potential target for the treatment of AS. We obtained 92 wogonoside targets and 2595 AS targets from three databases. By software calculation we obtained 31 co-acting targets as shown in Fig. 1A. Figure 1B shows the interactions of the 31 potential targets of action where wogonoside and AS work together, and from the size of the circle shading we can see the rank order of their correlation values. In Fig. 1C, the ten core targets (degree greater than 13) are listed in descending order of degree as TNF, STAT3, MAPK3, ESR1, PTGS2, TLR4, RELA, MAPK1, ESR2 and HNF4A.

GO Functional Annotation and KEGG Enrichment of Core Targets

Figures 2A-D present the results of GO enrichment analysis and KEGG pathway enrichment analysis for wogonoside and AS co-targets. A total of 155 biological processes (BPs), 44 molecular functions (MFs), 22 cellular components (CCs), and 107 KEGG pathways were annotated in our study. The GO analysis results include the top 10 enriched biological processes, cellular components, and molecular functions (Fig. 2A-C). Furthermore, Fig. 2D displays all 30 signaling pathways identified from the KEGG pathway enrichment analysis. The ten most upregulated BPs included inflammatory response, negative regulation of gene expression, positive regulation of ERK1 and ERK2 cascade, positive regulation of interleukin-8 production, positive regulation of interferon-gamma production, positive regulation of transcription from RNA polymerase II promoter, positive regulation of vascular endothelial growth factor production, positive regulation of interleukin-6 production, intracellular receptor signaling pathway, and lipopolysaccharide-mediated signaling pathway (Fig. 2A). The ten most upregulated CCs included processes such as the plasma membrane, cytoplasm, endoplasmic reticulum lumen, nucleoplasm, transcription factor complex, chromatin, caveola, endoplasmic reticulum, cytosol, and external side of apical plasma membrane (Fig. 2B). The ten most upregulated MFs included processes such as RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, transcription coactivator binding, protein homodimerization activity, enzyme binding, oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen, DNA binding, transcription factor activity, sequence-specific DNA binding, transcription regulatory region sequence-specific DNA binding, steroid hormone receptor activity, and protein binding (Fig. 2C). The first 15 core signaling pathways identified in the KEGG pathway enrichment analysis were Toll-like receptor signaling pathway, Prolactin signaling pathway, IL-17 signaling pathway, C-type lectin receptor signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, Sphingolipid signaling pathway, VEGF signaling pathway, Fc epsilon RI signaling pathway, B cell receptor signaling pathway, NOD-like receptor signaling pathway, Chemokine signaling pathway, NF-kappa B signaling pathway, cAMP signaling pathway, and Neurotrophin signaling pathway (Fig. 2D).

PPI view of the wogonoside-AS core target with KEGG focus pathway

Network pharmacology research indicates that the effective targets of wogonoside in treating AS may primarily be concentrated in the signaling pathways, with their correlation depicted in Fig. 3A. Through KEGG enrichment pathway analysis, it was revealed that 12 key targets, such as RELA, STAT3, TLR4, TLR9, TNF, ESR1, ESR2, MAPK1, MAPK3, PDE4D, PTGS2, and RAC1, were significantly enriched in the initial 15 signaling pathways. Figure 3A displays the core targets within the wogonoside-AS protein-protein interaction network were also enriched in the TLR4/NF-κB signaling pathway. Figure 3B displays an illustration of the NF-kappa B signaling pathway sourced from the official KEGG website. Notably, the core targets identified earlier were notably enriched in the TLR4/NF-κB signaling pathway, as highlighted in red. Consequently, the network pharmacology analysis suggests that the TLR4/NF-κB signaling pathway may play a crucial role in the mechanism of wogonoside in treating AS. Therefore, we hypothesize that the TLR4/NF-κB signaling pathway could serve as a significant mechanism of action for wogonoside in AS treatment.

Figure 3. (A) Relationship between common targets of wogonoside and AS therapy and the top 15 enriched targets of the KEGG signaling pathway. The light green Ellipse is the target. (B) NF-kappa B signaling pathway.

Molecular Docking

We screened 3 proteins, RELA (PDB ID: 2vge), TLR4 (PDB ID: 2Z62), and TNF (PDB ID: 5m2j), by network pharmacological prediction and further visualized molecular docking with wogonoside. The -CDOCKER energy values indicate binding affinity, with values below − 5.0 kJ/mol suggesting significant affinity between the ligand and receptor protein. The Vina scores for RELA (PDB ID: 2vge), TLR4 (PDB ID: 2Z62), and TNF (PDB ID: 5m2j) were calculated by CB-Dock2 to be -7.5 kcal/mol, -7.1 kcal/mol and − 6.6 kcal/mol, respectively (Fig. 4). The Vina score further validates that wogonoside is expected to modulate AS through TLR4/NF-κB signaling pathway.

Molecular dynamics simulation

To further investigate the interactions between protein complexes and small molecules, molecular dynamics simulations were performed on wogonoside complexed with RelA, TLR4, and TNF over a duration of 100 ns. RMSD was used to measure deviations from the initial conformation of each protein-ligand complex. Generally, higher RMSD values suggest greater instability, while a broad range of RMSD values indicates significant conformational changes within the molecule. The results, illustrated in Fig. 5A-C, showed that the average RMSD values for the complexes were consistently below 0.4 nm, with each complex quickly reaching a dynamic equilibrium within 5 ns. This rapid stabilization suggests a strong and effective binding of wogonoside to these targets. Notably, the wogonoside-RelA complex displayed some fluctuations during the simulation, yet these remained within a stable threshold of 0.1–0.4 nm. The RMSF analysis (Fig. 5D-F) indicated that areas related to ligand binding in all three molecules exhibited low fluctuations, with stable peaks, suggesting high structural stability throughout the simulation. In contrast, the regions with higher fluctuations, marked by larger peaks, were not involved in binding, likely relating to other biological processes within the molecules. Additionally, the Rg analysis of the complexes (Fig. 5G-I) showed smooth trajectories with fluctuations not exceeding 0.1 nm, indicating that the overall compactness and folding integrity of the protein-ligand complexes remain largely preserved after binding with wogonoside. This minimal impact on structural compactness confirms the suitability of wogonoside as a stable ligand for therapeutic targeting. Thus, the molecular dynamics simulation results further validated that wogonoside may treat AS through the TLR4/NF-κB signaling pathway.

Effect of wogonoside on body weight in the mice

The chemical structure of wogonoside is depicted in Fig. 6A. Over time, the weight of all groups increased, and by week 8, there was a noticeable difference in weight between the normal group and the other groups. The mouse weight in the atorvastatin and wogonoside groups demonstrated a distinct effect in slowing down weight gain, as illustrated in Fig. 6B.

Effect of wogonoside on pathological morphology and lipid deposition in the aorta of mice

As demonstrated in Fig. 6C-E, the plaque area in the model group showed significant thickening, formation of foam cells, accumulation of inflammatory cells, and a higher number of lipid droplets compared to the normal group. The aortic plaque area, intima thickness, foam infiltration, and lipid droplet count in the plaques were all significantly reduced in the atorvastatin and wogonoside groups compared to the model group. It was found that wogonoside significantly improved atherosclerotic plaque thickness, reduced inflammatory infiltration and lipid deposition.

Effect of wogonoside on serum lipid level

In the assessment of the serum lipid profile, the levels of TC, TG, LDL-C, ox-LDL and FFA in the model group were markedly higher than those in the normal group. However, no such changes were observed in HDL-C. Atorvastatin and wogonoside decreased TC, TG, LDL-C, ox-LDL and FFA levels (Fig. 7).

Effect of wogonoside on the expression of cytokines and oxidative stress

Owing to wogonoside's potent anti-inflammatory and antioxidant characteristics, we subsequently assessed how wogonoside affected the inflammatory response and oxidative stress in ApoE−/− mice. Figure 8 illustrates that the model group's serum and aorta had considerably higher levels of TNF-α, IL-1β, and IL-6 expression when compared to the normal group. On the other hand, the administration of wogonoside had an inhibitory effect on the inflammatory cytokine production.

Figure 8. Effect of wogonoside on the expression of cytokines in the mice. (A) TNF-α, (B) IL-1β and (C) IL-6 in serum of mice. (D) TNF-α, (E) IL-1β and (F) IL-6 in the aorta of mice. Data are represented as means ± SD for n = 6 per group. * p < 0.05 and ** p < 0.01 vs. model group.

Oxidative stress was observed in the model group as evidenced by the increase in MDA and decrease in SOD levels. Wogonoside treatment subsequently mitigated these modifications to some extent (Fig. 9A-B). We also looked at how wogonoside affected the expression of ROS in the aorta, and the findings revealed that ROS expression dramatically rose in the model group and dramatically decreased following wogonoside intervention (Fig. 9C). These results indicate that in ApoE-/-mice, wogonoside reverses the production of inflammatory cytokines and oxidative stress.

Effect of wogonoside on the TLR4/NF-κB signaling pathway

To investigate the molecular mechanism of wogonoside in treating AS, we examined its impact on the TLR4/NF-κB signaling pathway by analyzing the expression of p-TLR4 and p-NF-κB in mouse aorta through immunohistochemical methods. Our results showed an up-regulation of p-TLR4 expression and activation of p-NF-κB in the aorta of the model group (Fig. 10A-C). Conversely, the wogonoside group displayed a significant decrease in p-TLR4 expression and inhibition of activated p-NF-κB compared to the model group.

To further investigate the mechanism of action of wogonoside in the treatment of AS by regulating the TLR4/NF-κB signaling pathway, we performed Western Blot analysis of the protein expression levels of TLR4 and NF-κB and their phosphorylation in mouse aorta. The results showed that the ratio of p-NF-κB/NF-κB decreased significantly after treatment with wogonoside, as did the ratio of p-TLR4/TLR4 (Fig. 11A-B).

Figure 11. Effect of wogonoside on TLR4/NF-κB signaling pathway in the mice. (A) Reduced p-NF-κB/NF-κB expression in aorta was detected by Western blotting. (B) Reduced p-TLR4/TLR4 expression in aorta detected by Western blotting. The above results reached *p < 0.05 and **p < 0.01 compared to the model group.

Atherosclerosis is the most common underlying pathology of coronary artery disease, peripheral artery disease, and cerebrovascular disease. The most common complications, myocardial infarction and stroke, are caused by spontaneous thrombotic vessel occlusion and represent the most common cause of death worldwide[31, 32]. Lipid accumulation, inflammation and oxidative stress are the main mechanisms leading to AS, and the three are causal and influence each other[33]. One of the key processes in the development of AS is the buildup of lipids, and the creation of foam cells is caused by the accumulation of intracellular modified lipids, particularly ox-LDL. Every stage of AS involves inflammation, all AS risk factors trigger an inflammatory response, and inflammatory cytokines cause lipids to build up inside cells, ultimately rupturing them[34, 35]. Oxidative stress and apoptosis occur in arterial wall cells due to an increase in pro-inflammatory cytokines. In summary, various pathological factors such as lipoprotein particle aggregation, endothelial cell inflammation, leukocyte recruitment, foam cell formation, inflammation, and oxidative stress play a crucial role in the initiation, continuation, and eventual resolution of the atherogenic process[36, 37].

Natural remedies and medicinal herbs have gained prominence in recent years as one of the most potent anti-atherosclerotic treatments and preventative measures for AS[38, 39]. Scutellaria baicalensis is the source of the flavonoid known as wogonoside, which has a variety of biological and pharmacological uses[40]. In traditional Chinese medicine, Scutellaria baicalensis is a popular plant that has been used for a long time to treat a variety of AS symptoms. Scutellaria baicalensis regulates inflammatory pathways, lowers cholesterol, reduces free radicals, and lowers vascular resistance to avoid AS and its detrimental consequences. It also possesses potential antioxidant, antiatherogenic, and antithrombotic activities[41, 42]. Wogonoside's putative qualities have garnered significant attention, indicating a need for further investigation.

One of the key pathophysiological processes for the start and development of AS has been identified as chronic inflammation. Numerous investigations have demonstrated that wogonoside possesses a broad spectrum of anti-inflammatory properties and may efficiently impede the production of inflammatory mediators, including TNF-α and IL-1β[43, 44]. One of the factors contributing to the pathophysiology of AS is oxidative stress, which is defined as the body's increased production of ROS above its capacity for scavenging, resulting in tissue damage[45]. In mice with nonalcoholic fatty liver disease, wogonoside can also reduce oxidative stress by controlling the expression of NDA, SOD, ROS, and other related molecules[14]. This reduces lipid deposition and the inflammatory response. Recent studies have shown that wogonoside has a protective effect against myocardial ischemia/reperfusion and acute myocardial ischemia. It has been found that wogonoside regulates various metabolic indicators, targets, and pathways, which ultimately provide cardio-protective effects on myocardial ischemia and myocardial ischemia/reperfusion injury after blood supply restoration[15, 35]. In our study, we demonstrated that wogonoside effectively down-regulated the levels of TC, TG, LDL-C, ox-LDL, and FFA, while also significantly reducing aortic inflammatory response and lipid deposition in ApoE-/- mice. Further research has shown that wogonoside inhibited the release of inflammatory factors TNF-α, IL-1β, and IL-6, as well as oxidative stress in ApoE-/- mice, thereby contributing to the treatment of AS.

In order to further elucidate the mechanism of wogonoside in the treatment of AS, we applied network pharmacology and molecular docking methods to screen the signaling pathways in which wogonoside might interfere with AS. We found that the core targets in the wogonoside-AS PPI network were enriched in the TLR/NF-κB signaling pathway. Molecular docking and molecular dynamics simulation experiments further demonstrated the good binding of the ligand components to the receptor proteins, which validated that wogonoside is expected to regulate ankylosing spondylitis through the TLR4/NF-κB signaling pathway.

The TLRs signaling pathway plays a crucial role in the development of inflammatory conditions, such as AS. TLR4 is expressed in lipid-rich and atherosclerotic plaques[46]. Overexpression of TLR4 in macrophages occurs at different stages of AS in both human and mouse atherosclerotic lesions[47]. Additionally, TLR4 activation leads to the upregulation of various cytokines and chemokines involved in inflammatory cell recruitment and proliferation. This, in turn, initiates the inflammatory response and promotes the accumulation of intimal foam cells[48]. NF-κB, a downstream nuclear transcription factor of the TLR4 signaling pathway, is highly activated and often causes the expression of cytokines and oxidative stress, resulting in a local inflammatory response in AS[49, 50]. In our study, we have demonstrated that wogonoside inhibits the TLR4/NF-κB signaling pathway, providing insights into the molecular mechanism of wogonoside in AS treatment.

This comprehensive study aimed to investigate the role and mechanism of wogonoside in ameliorating AS using network pharmacology, molecular docking, molecular dynamics simulation and in vivo experiments. The results showed that wogonoside has significant anti-atherosclerotic effects by improving lipid accumulation, inhibiting inflammation and reducing oxidative stress. It was also shown that wogonoside may treat AS by inhibiting the TLR4/NF-κB signaling pathway. These findings provide valuable insights into the molecular mechanisms underlying the potential of wogonoside to treat AS.

Data accessibility declaration

Upon request, the corresponding author will provide the data used to support the study's conclusions.

Ethics Statement

The Ethics Committee of The First Affiliated Hospital of Guangzhou University of Chinese Medicine granted exemption from review for available data from public databases involving in our research. The animal experiments conducted in this study were approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (Ethics No. GZTCMF1-2022044).

Author Contributions

All authors have made significant contributions to the work reported, including conception, study design, execution, data collection, analysis and interpretation, drafting or writing, substantive revision, and critical review of the article. It was agreed by all authors that the article would be submitted to this journal and the review process was completed. Furthermore, all authors have agreed to take responsibility for the content of the article.

Funding

The work is supported by GuangDong Basic and Applied Basic Research Foundation (No. 2022A1515110789 and No. 2022A1515220138), Guangzhou City Science and Technology Bureau city school (college) joint Foundation (No. 202201020257), National Natural Science Foundation of China (No. 82105047), Guangzhou Basic and Applied Basic Research Foundation (No. 2023A04J1928).

Disclosure

The authors report no conflicts of interest in this work.

Wojtasińska A, Frąk W, Lisińska W, Sapeda N, Młynarska E, Rysz J, Franczyk B: Novel Insights into the Molecular Mechanisms of Atherosclerosis. Int J Mol Sci 2023, 24(17).
Song P, Fang Z, Wang H, Cai Y, Rahimi K, Zhu Y, Fowkes FGR, Fowkes FJI, Rudan I: Global and regional prevalence, burden, and risk factors for carotid atherosclerosis: a systematic review, meta-analysis, and modelling study. The Lancet Global Health 2020, 8(5):e721-e729.
Rached F, Santos RD: The Role of Statins in Current Guidelines. Curr Atheroscler Rep 2020, 22(9):50.
Ward NC, Watts GF, Eckel RH: Statin Toxicity. Circ Res 2019, 124(2):328-350.
Shamaki GR, Markson F, Soji-Ayoade D, Agwuegbo CC, Bamgbose MO, Tamunoinemi BM: Peripheral Artery Disease: A Comprehensive Updated Review. Curr Probl Cardiol 2022, 47(11):101082.
Tuñón J, Badimón L, Bochaton-Piallat ML, Cariou B, Daemen MJ, Egido J, Evans PC, Hoefer IE, Ketelhuth DFJ, Lutgens E et al: Identifying the anti-inflammatory response to lipid lowering therapy: a position paper from the working group on atherosclerosis and vascular biology of the European Society of Cardiology. Cardiovasc Res 2019, 115(1):10-19.
Singh L, Sharma S, Xu S, Tewari D, Fang J: Curcumin as a Natural Remedy for Atherosclerosis: A Pharmacological Review. Molecules 2021, 26(13).
Sobenin IA, Myasoedova VA, Iltchuk MI, Zhang DW, Orekhov AN: Therapeutic effects of garlic in cardiovascular atherosclerotic disease. Chin J Nat Med 2019, 17(10):721-728.
Zhang S, Li L, Chen W, Xu S, Feng X, Zhang L: Natural products: The role and mechanism in low-density lipoprotein oxidation and atherosclerosis. Phytother Res 2021, 35(6):2945-2967.
Liao H, Ye J, Gao L, Liu Y: The main bioactive compounds of Scutellaria baicalensis Georgi. for alleviation of inflammatory cytokines: A comprehensive review. Biomed Pharmacother 2021, 133:110917.
Zhao T, Tang H, Xie L, Zheng Y, Ma Z, Sun Q, Li X: Scutellaria baicalensis Georgi. (Lamiaceae): a review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. J Pharm Pharmacol 2019, 71(9):1353-1369.
Chen Q, Rahman K, Wang SJ, Zhou S, Zhang H: Scutellaria barbata: A Review on Chemical Constituents, Pharmacological Activities and Clinical Applications. Curr Pharm Des 2020, 26(1):160-175.
Shi X, Zhang B, Chu Z, Han B, Zhang X, Huang P, Han J: Wogonin Inhibits Cardiac Hypertrophy by Activating Nrf-2-Mediated Antioxidant Responses. Cardiovasc Ther 2021, 2021:9995342.
Jiang G, Chen D, Li W, Liu C, Liu J, Guo Y: Effects of wogonoside on the inflammatory response and oxidative stress in mice with nonalcoholic fatty liver disease. Pharm Biol 2020, 58(1):1177-1183.
Zhang B, Xu D: Wogonoside preserves against ischemia/reperfusion-induced myocardial injury by suppression of apoptosis, inflammation, and fibrosis via modulating Nrf2/HO-1 pathway. Immunopharmacol Immunotoxicol 2022, 44(6):877-885.
Sun T, Quan W, Peng S, Yang D, Liu J, He C, Chen Y, Hu B, Tuo Q: Network Pharmacology-Based Strategy Combined with Molecular Docking and in vitro Validation Study to Explore the Underlying Mechanism of Huo Luo Xiao Ling Dan in Treating Atherosclerosis. Drug Des Devel Ther 2022, 16:1621-1645.
Xu HY, Zhang YQ, Liu ZM, Chen T, Lv CY, Tang SH, Zhang XB, Zhang W, Li ZY, Zhou RR et al: ETCM: an encyclopaedia of traditional Chinese medicine. Nucleic Acids Res 2019, 47(D1):D976-D982.
Daina A, Michielin O, Zoete V: SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res 2019, 47(W1):W357-W364.
Davis AP, Wiegers TC, Johnson RJ, Sciaky D, Wiegers J, Mattingly CJ: Comparative Toxicogenomics Database (CTD): update 2023. Nucleic Acids Res 2023, 51(D1):D1257-d1262.
Yao Z-J, Dong J, Che Y-J, Zhu M-F, Wen M, Wang N-N, Wang S, Lu A-P, Cao D-S: TargetNet: a web service for predicting potential drug–target interaction profiling via multi-target SAR models. Journal of Computer-Aided Molecular Design 2016, 30(5):413-424.
Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, Stein TI, Nudel R, Lieder I, Mazor Y et al: The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics 2016, 54:1.30.31-31.30.33.
Amberger JS, Bocchini CA, Schiettecatte F, Scott AF, Hamosh A: OMIM.org: Online Mendelian Inheritance in Man (OMIM®), an online catalog of human genes and genetic disorders. Nucleic Acids Res 2015, 43(Database issue):D789-798.
Zhou Y, Zhang Y, Lian X, Li F, Wang C, Zhu F, Qiu Y, Chen Y: Therapeutic target database update 2022: facilitating drug discovery with enriched comparative data of targeted agents. Nucleic Acids Res 2022, 50(D1):D1398-d1407.
Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, Doncheva NT, Legeay M, Fang T, Bork P et al: The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res 2021, 49(D1):D605-d612.
Sherman BT, Hao M, Qiu J, Jiao X, Baseler MW, Lane HC, Imamichi T, Chang W: DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res 2022, 50(W1):W216-w221.
Liu Y, Yang X, Gan J, Chen S, Xiao Z-X, Cao Y: CB-Dock2: improved protein–ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Research 2022, 50(W1):W159-W164.
Yang X, Liu Y, Gan J, Xiao Z-X, Cao Y: FitDock: protein–ligand docking by template fitting. Briefings in Bioinformatics 2022, 23(3).
Consortium TU: UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Research 2022, 51(D1):D523-D531.
Xuan Y, Cai Y, Wang XX, Shi Q, Qiu LX, Luan QX: [Effect of Porphyromonas gingivalis infection on atherosclerosis in apolipoprotein-E knockout mice]. Beijing Da Xue Xue Bao Yi Xue Ban 2020, 52(4):743-749.
Cai Y, Wen J, Ma S, Mai Z, Zhan Q, Wang Y, Zhang Y, Chen H, Li H, Wu W et al: Huang-Lian-Jie-Du Decoction Attenuates Atherosclerosis and Increases Plaque Stability in High-Fat Diet-Induced ApoE(-/-) Mice by Inhibiting M1 Macrophage Polarization and Promoting M2 Macrophage Polarization. Front Physiol 2021, 12:666449.
Wolf D, Ley K: Immunity and Inflammation in Atherosclerosis. Circ Res 2019, 124(2):315-327.
Bäck M, Yurdagul A, Jr., Tabas I, Öörni K, Kovanen PT: Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities. Nat Rev Cardiol 2019, 16(7):389-406.
Malekmohammad K, Bezsonov EE, Rafieian-Kopaei M: Role of Lipid Accumulation and Inflammation in Atherosclerosis: Focus on Molecular and Cellular Mechanisms. Front Cardiovasc Med 2021, 8:707529.
Zhong S, Li L, Shen X, Li Q, Xu W, Wang X, Tao Y, Yin H: An update on lipid oxidation and inflammation in cardiovascular diseases. Free Radic Biol Med 2019, 144:266-278.
Bhattacharya P, Kanagasooriyan R, Subramanian M: Tackling inflammation in atherosclerosis: Are we there yet and what lies beyond? Curr Opin Pharmacol 2022, 66:102283.
Chen PY, Qin L, Li G, Wang Z, Dahlman JE, Malagon-Lopez J, Gujja S, Cilfone NA, Kauffman KJ, Sun L et al: Endothelial TGF-β signalling drives vascular inflammation and atherosclerosis. Nat Metab 2019, 1(9):912-926.
Raggi P, Genest J, Giles JT, Rayner KJ, Dwivedi G, Beanlands RS, Gupta M: Role of inflammation in the pathogenesis of atherosclerosis and therapeutic interventions. Atherosclerosis 2018, 276:98-108.
Meng T, Li X, Li C, Liu J, Chang H, Jiang N, Li J, Zhou Y, Liu Z: Natural products of traditional Chinese medicine treat atherosclerosis by regulating inflammatory and oxidative stress pathways. Front Pharmacol 2022, 13:997598.
Wang D, Yang Y, Lei Y, Tzvetkov NT, Liu X, Yeung AWK, Xu S, Atanasov AG: Targeting Foam Cell Formation in Atherosclerosis: Therapeutic Potential of Natural Products. Pharmacol Rev 2019, 71(4):596-670.
Gu Y, Zheng Q, Fan G, Liu R: Advances in Anti-Cancer Activities of Flavonoids in Scutellariae radix: Perspectives on Mechanism. Int J Mol Sci 2022, 23(19).
Baradaran Rahimi V, Askari VR, Hosseinzadeh H: Promising influences of Scutellaria baicalensis and its two active constituents, baicalin, and baicalein, against metabolic syndrome: A review. Phytother Res 2021, 35(7):3558-3574.
Xu Y, Li Y, Chen G, Fan M, Hu G, Guo M: Screening and identification of potential hypoglycemic and hypolipidemic compounds from aqueous extract of Scutellaria baicalensis Georgi root combing affinity ultrafiltration with multiple drug targets and in silico analysis. Phytochem Anal 2023.
Huang S, Fu Y, Xu B, Liu C, Wang Q, Luo S, Nong F, Wang X, Huang S, Chen J et al: Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway. Phytomedicine 2020, 68:153179.
Xu X, Xia J, Zhao S, Wang Q, Ge G, Xu F, Liu X, Zhang W, Yang Y: Qing-Fei-Pai-Du decoction and wogonoside exert anti-inflammatory action through down-regulating USP14 to promote the degradation of activating transcription factor 2. Faseb j 2021, 35(9):e21870.
Batty M, Bennett MR, Yu E: The Role of Oxidative Stress in Atherosclerosis. Cells 2022, 11(23).
Choi SH, Sviridov D, Miller YI: Oxidized cholesteryl esters and inflammation. Biochim Biophys Acta Mol Cell Biol Lipids 2017, 1862(4):393-397.
Li S, Navia-Pelaez JM, Choi SH, Miller YI: Macrophage inflammarafts in atherosclerosis. Curr Opin Lipidol 2023, 34(5):189-195.
Zeng F, Zheng J, Shen L, Herrera-Balandrano DD, Huang W, Sui Z: Physiological mechanisms of TLR4 in glucolipid metabolism regulation: Potential use in metabolic syndrome prevention. Nutr Metab Cardiovasc Dis 2023, 33(1):38-46.
Zeng X, Guo R, Dong M, Zheng J, Lin H, Lu H: Contribution of TLR4 signaling in intermittent hypoxia-mediated atherosclerosis progression. J Transl Med 2018, 16(1):106.
Kong P, Cui ZY, Huang XF, Zhang DD, Guo RJ, Han M: Inflammation and atherosclerosis: signaling pathways and therapeutic intervention. Signal Transduct Target Ther 2022, 7(1):131.

No competing interests reported.

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Mechanisms of Wogonoside in the Treatment of Atherosclerosis Based on Network Pharmacology, Molecular Docking, and Experimental Validation

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Abstract

Background

Purpose

Methods

Results

Conclusion

Figures

Introduction

Material and methods

Network Pharmacology Analysis

Molecular docking

Molecular dynamics simulation

Reagents

Animals and Treatment

Aortic root lesion analysis

Serum lipid analysis

Cytokine Expression Assessment

Oxidative stress analysis

Immunofluorescence assay

Immunohistochemistry assay

Western blot analysis

Statistical Analysis

Results

Core target screening

GO Functional Annotation and KEGG Enrichment of Core Targets

PPI view of the wogonoside-AS core target with KEGG focus pathway

Molecular Docking

Molecular dynamics simulation

Effect of wogonoside on body weight in the mice

Effect of wogonoside on pathological morphology and lipid deposition in the aorta of mice

Effect of wogonoside on serum lipid level

Effect of wogonoside on the expression of cytokines and oxidative stress

Effect of wogonoside on the TLR4/NF-κB signaling pathway

Discussion

Conclusion

Declarations

Data accessibility declaration

Ethics Statement

Author Contributions

Funding

Disclosure

References

Additional Declarations

Supplementary Files

Status:

Version 1