Explore the mechanism of Citri Reticulatae Pericarpium (Chenpi) in atherosclerosis Based on Network Pharmacology, Molecular Docking and Experimental Evidence

Explore the mechanism of Citri Reticulatae Pericarpium (Chenpi) in atherosclerosis Based on Network Pharmacology, Molecular Docking and Experimental Evidence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Explore the mechanism of Citri Reticulatae Pericarpium (Chenpi) in atherosclerosis Based on Network Pharmacology, Molecular Docking and Experimental Evidence Yumeng Pan, Ping Weng, Yilin Wen, Liming Yang, Yueyue Li, Chengju Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4241694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Citri Reticulatae Pericarpium (CRP), a traditional Chinese medicine, is extensively used to prevent and treat cardiovascular diseases. However, the exact target and pharmacological mechanism of CRP remain unclear. This study aims to investigate the potential mechanism of CRP in treating atherosclerosis (AS) using network pharmacology, molecular docking, and experimental verification. Methods: The chemical constituents and targets of CRP were retrieved, collected, and screened in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database. Potential AS targets were obtained from GeneCards and OMIM databases. Subsequently, the STRING database was used to establish a protein-protein interaction network, and Cytoscape was employed to construct the CRP-AS-potential target gene network to identify core targets. After GO and KEGG enrichment analysis, naringenin and core targets were selected for molecular docking simulation. Finally, the anti-AS mechanism of naringenin was validated through cell experiments. Results: Five potential active components of CRP were identified, and 54 common targets of the disease and drugs, including 15 core targets (such as MAPK3 and MMP9), were obtained. Lipid and atherosclerosis were found to be the most prominent pathways of action. Molecular docking demonstrated the strong binding of naringenin with MMP9 and MAPK3. In vitro experiments, it was revealed that naringenin might inhibit lipid accumulation in smooth muscle cells and slow down the occurrence of atherosclerosis by decreasing the expression of MAPK3. Conclusions: Through network pharmacological analysis, molecular docking, and experimental verification, this study found that naringenin, the core active ingredient of CRP, may inhibit the occurrence of smooth muscle cell foam by reducing the expression of MAKP3 in vascular smooth muscle cells (VSMCs)and play an anti-AS role, providing a new idea for further research on CRP and naringenin in the prevention and treatment of AS. Atherosclerosis Citri Reticulatae Pericarpium Network pharmacology Chinese traditional medicine Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Background Atherosclerosis (AS) is a chronic progressive disease characterized by the deposition of lipids and infiltration of inflammatory cells. It presents significant clinical manifestations, including ischemic heart disease, ischemic stroke, and peripheral artery disease [ 1 ]. AS has emerged as the primary cause of cardiovascular illnesses and is increasingly prevalent among younger individuals, now serving as the leading cause of global mortality [ 2 , 3 ]. Consequently, effective control of AS occurrence and development is paramount in reducing cardiovascular disease incidents. Dyslipidemia, particularly elevated levels of plasma LDL-cholesterol, marks the beginning of AS [ 4 ]. Lipid-lowering drugs, such as statins, are widely used in the clinical treatment of AS for primary and secondary prevention of atherosclerotic cardiovascular diseases. They aim to improve vascular risk factors and exert anti-inflammatory effects [ 5 ]. However, the clinical utilization of statins is limited due to adverse effects, including Statin-associated muscle symptoms (SAMS) and liver damage, and may even lead to increased cardiovascular adverse outcomes [ 6 – 8 ]. Hence, it is imperative to conduct further research to identify more effective and safe lipid-regulating drugs. The theory of ancient Traditional Chinese Medicine (TCM) suggests that the dysfunction of body organs leads to the stagnation of blood, which in turn results in the formation of AS [ 9 ]. TCM has a long history of application due to its positive therapeutic effects, including minimal side effects and superior curative outcomes for AS [ 10 ]. One particular component of TCM, CRP, commonly known as Chenpi in Chinese, belongs to the citrus genus and Rutaceae family. It is a dried and ripe orange peel [ 11 ]. CRP consists of various active components that possess multiple functions, such as reducing blood lipids, anti-inflammatory properties, anti-thrombotic effects, and inhibition of oxidative stress. As a result, CRP has been widely used as a health food and dietary supplement for the prevention and treatment of cardiovascular diseases [ 11 , 12 ]. However, the target and specific pharmacological mechanism of CRP in preventing and treating AS have not been fully established, considering the complex formation process of AS, the intricate composition of traditional Chinese medicine components, and the ambiguity surrounding the target of action [ 13 ]. Network pharmacology is a powerful technology used to investigate the practical components and mechanisms of traditional Chinese medicine in treating various diseases. This approach involves extensive database mining and analyzes the intricate relationship between drug components, action targets, action pathways, and diseases. By constructing a visual network, it systematically and comprehensively reveals the action mechanism of multi-component drug plants [ 14 , 15 ]. In this study, the network pharmacology approach, combined with molecular docking, was adopted to create a network of "drug composition - target of action - pathway of action." In vitro experiments were then conducted to confirm and predict the probable target of CRP for AS and its mode of action. These findings serve as the foundation for further basic research and offer a more comprehensive outlook on the clinical application of CRP. The workflow to study the molecular mechanism of Chenpi in AS was exhibited in Fig. 1 . 2. Methods 2.1. Data Preparation The components of CRP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database (TCMSP, https://tcmspw.com/index.php ) [ 16 ]. This retrieval and collection process aimed to acquire the oral bioavailability (OB) and drug-likeness (DL) values of the compounds. Screening conditions were set based on the ADME principle, with OB ≥ 30% and DL ≥ 0.18 serving as criteria for identifying active ingredients [ 17 ]. The target proteins were named using the UniProt database ( https://www.uniprot.org/ ). The GeneCards database ( https://www.genecards.org/ ) and Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/ ) were utilized to search for and collect information related to the keyword "atherosclerosis." The obtained data were then summarized and collated to intersect with the targets of CRP components. The VennDiagram package of R 4.2.1 software was employed to process the data and generate a Venn diagram, which allowed for the identification of common disease targets and potential drugs. These findings suggest that CRP has the potential to be a treatment for atherosclerosis. 2.2. Network Construction The STRING database ( https://string-db.org/ ) was chosen as a resource to obtain protein interactions. The process involved introducing the disease-drug interaction protein, selecting "Homo sapiens" as the organism, setting the minimum required interaction score to be greater than 0.4, and hiding disconnected nodes in the network. This resulted in the generation of a protein-protein interaction (PPI) network. The protein interaction network file was then imported into Cytoscape 3.8.0 software for visual analysis, allowing for the construction of a network diagram representing the "drug component-target-pathway" interactions and identifying potential core targets. Additionally, the connection results were enriched using R 4.2.1 software, which facilitated the creation of visual bar plots. 2.3. Enrichment Analysis The R 4.2.1 software was utilized to convert the gene symbols of CRP and AS into Entrez ID. Subsequently, various R packages were employed to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, resulting in the generation of bar and bubble charts. The Perl computer program language was used to screen the GO enrichment results and extract the functions of the corresponding genes. For further analysis, only the GO enrichment results with a p-value < 0.05 were considered statistically significant and retained. 2.4. Molecular Docking Proteins were obtained from the RCSB database, and protein receptor pretreatment was carried out using AutoDockTools 1.5.6. The pretreatment involved dehydrating, hydrogenating, removing the primary ligand, and setting the proteins as receptors. Small molecules were acquired from the PubChem database. A slight energy minimization was performed on the small molecules using OpenBabel 2.4.1 with the MMFF94 force field. The small molecule ligands were then processed using AutoDockTools 1.5.6, hydrogenated, and the number of rotatable bonds was set. These prepared ligands were used in molecular docking conducted with the software Autodock Vina 1.1.2. The docking process involved generating ten docking conformations, and the conformation with the most favorable absolute value of binding energy was selected from the docking results. Finally, the selected conformation was analyzed and mapped using Discovery Studio 2019 Client. 2.5. Experimental verification 2.5.1. Cytotoxic assay Mouse aortic vascular smooth muscle cells (MOVAS) were seeded in 96-well plates one day prior to the experiment. After a 12-hour serum starvation treatment, the control group was supplemented with DMEM/12F medium containing 10% FBS, while the treatment group received naringenin at concentrations of 10 µM, 25 µM, 50 µM, and 100 µM, respectively. The cells were then incubated in a CO2 incubator at 37°C for 24 hours. Following incubation, the medium was removed, and the cells were washed twice with PBS to remove any residual substances. Next, a mixture of CCK-8 medium was added at a 10% concentration, and the cells were further incubated for 1 hour in the incubator. The optical density (OD) at 450 nm was measured using an enzymometer. 2.5.2. Nile Red staining The cells were seeded in 96-well plates one day prior to the experiment. After a 12-hour serum starvation treatment, the control group was supplemented with DMEM/12F medium containing 1% FBS. Meanwhile, the treatment group received 100 µg/ml Ox-LDL + DMSO and 100 µg/ml Ox-LDL + 10 µM naringenin, respectively. The cells were then incubated for an additional 24 hours. Following incubation, the medium was discarded, and the cells were washed twice with PBS. They were then fixed with 4% paraformaldehyde for 15 minutes, followed by three washes with PBS. Subsequently, the cells were stained with 1 µM Nile Red/PBS for 30 minutes, followed by three more washes with PBS. Additionally, the cells were stained with DAPI for 10 minutes, followed by three more washes with PBS. Finally, fluorescence quenching was performed using an anti-fluorescence quencher under a Leica inverted microscope. 2.5.3. Western Blot Analysis For cell culture and grouping, please refer to step 2.5.2. Following removing the medium, the cells were rinsed twice with PBS and then treated with RIPA lysate containing 1% PMSF for cell lysis. This lysate was collected into a 1.5ml EP tube and heated in a metal bath at 100℃ for 5 minutes. The subsequent Western blotting procedure was conducted: 30 µg of total protein lysate was loaded and separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in TBST for 2 hours at room temperature, the membranes were incubated overnight at 4℃ with various primary antibodies, including anti-MAPK3. Secondary antibodies were added to the membrane at room temperature for 2 hours. β-actin was used as both the loading control and for normalization purposes. The expression of the MAPK3 antibody (Proteintech, 11257-1-AP) was finally detected. 2.5.4. Thermal Stability Test For details, see [ 18 ]. 3. Results 3.1.Data Preparation The components of CRP were obtained by retrieving and collecting data from TCMSP database. Active components and their corresponding targets were screened, and the names and serial numbers of the identified drug targets were recorded. A total of 60 drug targets were identified. Using OB ≥ 30% and DL ≥ 0.18 as screening criteria, five active ingredients were obtained: sitosterol, naringenin, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one, Citromitin, and nobiletin (Table 1 ). Genes obtained from the GeneCards and OMIM databases were excluded, resulting in a final list of 6015 AS-related genes. The R 4.2.1 software was employed to create a Venn diagram (Fig. 2 ), revealing 54 common targets shared by both the disease and the drug. These targets represent potential candidates for CRP treatment of AS, serving as a basis for further research. Table 1 The ID, OB, and DL of five active compounds in CRP. NO Molecule ID Molecule name Molecule weight OB(%) DL 1 MOL000359 sitosterol 414.79 36.91 0.75 2 MOL004328 naringenin 272.27 59.29 0.21 3 MOL005100 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one 302.30 47.74 0.27 4 MOL005815 Citromitin 404.45 86.90 0.51 5 MOL005828 nobiletin 402.43 61.67 0.52 3.2. Network Construction The protein-protein interaction (PPI) networks (Fig. 3 ) were constructed by importing 54 target genes into the STRING database. The PPI map of the 54 overlapping genes associated with CRP and AS consists of 54 nodes and 421 edges. Each node represents a gene, and the size of the node is proportional to the degree of interaction of that gene with other genes in the network. The connectivity results were enriched using R 4.2.1 software, and a bar chart (Fig. 4 ) was generated to display the top twenty proteins based on their degrees. The bar chart reveals that the targets with higher degree values include AKT1, TP53, CASP3, PPARG, PPARA, PTGS2, ESR1, MAPK3, MMP9, HSP90AA1, CAT, CREB1, MAPK8, MAPK1, and ADIPOQ. Subsequently, this data was imported into Cytoscape 3.8.0 software for visual analysis and the construction of the CRP-AS-potential target gene network (Fig. 5 ). 113 connections were identified, enabling the examination of the target genes and essential chemicals. It is worth noting that a node's core density increases with its size. The image demonstrates the significance of naringenin, the primary constituent of CRP (MOL004328), in combating illnesses and influencing its targets. In the network, the size of each node represents its degree value, indicating the number of connections it has with other nodes. The gray connecting lines indicate the interconnectedness between the nodes. 3.3. Enrichment Analysis 3.3.1. GO enrichment analysis The data of disease drug targets was transformed using the R software package BiocManager to display their gene IDs, facilitating later data enrichment analysis. The GO enrichment analysis encompassed three aspects: biological process (BP), cellular component (CC), and molecular function (MF). The function of the GO gene was enriched using R 4.2.1 software, resulting in the identification of 1685 significant GO items (P < 0.05). To determine the roles of the corresponding genes, the enrichment results were screened using the Perl computer programming language. The gene GO ontology entries were sorted based on their p-values, and the top 10 scatterplots and histograms for BP, CC, and MF were selected for plotting (Fig. 6 ). The top twenty GO entries for BP, CC, and MF were displayed in the plot. Regarding BP, the entries were primarily enriched in response to oxidative stress and nutrient levels. For CC, the entries were primarily enriched in the endoplasmic reticulum lumen. The MF entries specifically included DNA-binding transcription factor binding. These results highlight the crucial role of CRP targets in treating AS across BP, CC, and MF domains. The plot's x-axis represents the gene ratio, and the y-axis displays enriched GO entries. The size of each dot corresponds to the number of genes associated with that entry. The dot color indicates the degree of enrichment, ranging from red to green, corresponding to decreasing Q values. 3.3.2. KEGG pathway analysis The potential target of CRP in treating AS was analyzed through KEGG pathway analysis using R software, which enabled the creation of a signal pathway map for a deeper understanding of CRP's mechanism of action. The analysis identified and screened 152 significant signal pathways (P < 0.05). To provide a visual representation, a chart (Fig. 7 ) was generated to display the top 20 crucial signaling pathways. By examining the path annotation in the KEGG database, it was found that the lipid and atherosclerosis pathway is the most well-known pathway, involving 17 genes. Among the 15 targets with the most significant potential effects of CRP (as suggested in Fig. 4 ), eight targets, namely AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8, are part of this central pathway. These findings suggest that these targets may play a key role in the therapeutic effects of CRP. 3.4. Molecular docking The molecular docking technique was employed to assess the strength of naringenin's binding ability to the eight targets mentioned earlier: AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8. The results obtained from this analysis helped identify the most likely targets for naringenin (Fig. 8 , supplementary materials). Typically, a higher absolute value of binding energy signifies a stronger binding affinity. A binding energy of -4.6 kcal/moL suggests primary binding, while values below − 5 kcal/moL indicate excellent binding, and values below − 7 kcal/moL indicate a good binding effect. In the visualization provided (Fig. 8 of the supplementary materials), small molecular ligands are represented by green structures, and binding bonds are represented by dotted lines. The alpha-number type notes represent residue names for proteins, while pure number notes represent bond lengths. Upon observing the figure, it becomes evident that naringenin forms multiple binding bonds with the two target proteins, MMP9 and MAPK3, which exhibit the best binding affinity. This suggests that naringenin and these two target proteins are closely bound to each other. 3.5. Experimental verification Next, we conducted experimental verification using MOVAS. Initially, we used CCK-8 to determine the safe concentration range of naringenin (Figure. 9A). Based on these results, we selected a concentration of 10 µM for further treatment. Subsequently, thermal stability experiments demonstrated that naringenin could affect the stability of MAPK3 (Figure. 9B), suggesting a direct interaction between the two. Furthermore, naringenin exhibited a significant reduction in the expression of MAPK3 induced by Ox-LDL (Figure. 9C). We then investigated the lipid-lowering effect of naringenin, which was assessed using Nile Red staining. Our findings revealed a notable reduction in lipid accumulation in the naringenin group compared to the Ox-LDL group (Figure. 9D-E). Additionally, KEGG analysis predicted the lipid-lowering effect of naringenin, suggesting that it may inhibit lipid accumulation in smooth muscle cells and impede the occurrence of atherosclerosis by suppressing the expression of MAPK3 during smooth muscle cell foam formation. 4. Discussion Atherosclerosis is a complex disease characterized by lipid aggregation, vascular endothelial cell damage, and the activation of inflammatory factors. Within the pathophysiology of AS, the accumulation of lipids in the arterial wall, particularly in foam cells, is a hallmark lesion. Using TCMSP database, we found 5 active ingredients of CRP. By intersecting these compounds with AS-related targets, we identified 54 potential action targets. Subsequently, a protein-protein interaction (PPI) analysis was performed, revealing naringenin as the primary core component and 15 core targets (AKT1, TP53, CASP3, PPARG, PPARA, PTGS2, ESR1, MAPK3, MMP9, HSP90AA1, CAT, CREB1, MAPK8, MAPK1, and ADIPOQ). Further examination of KEGG data indicated that CRP influences the progression of AS through various pathways. Notably, the "Lipid and atherosclerosis (hsa05417) pathway" emerged as a significant pathway, suggesting that CRP likely plays a crucial role in AS by affecting lipid metabolism. The "Lipid and atherosclerosis (hsa05417) pathway" overlapped with eight of the top 15 PPI targets, validating the accuracy of the network pharmacological analysis. Consequently, eight fundamental regulatory targets—AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8—were identified. The binding affinity of a drug with the target protein is a critical parameter for evaluating its impact on the disease mechanism [ 19 ]. This study selected naringenin as the molecular docking object, representing the most significant node in the CRP-AS-potential target gene network. We hypothesized that naringenin could potentially prevent the onset and progression of AS by interacting with MMP9 and MAPK3. Molecular docking tests indicated that two of the eight potential targets had the strongest binding affinity with naringenin. Matrix Metalloproteinases (MMPs) are a family of zinc-dependent proteases that can degrade protein substrates. They have the ability to modify chemokines and cytokines, participate in regulating cell proliferation, adhesion, and migration, and play a key role in vascular wall remodeling [ 20 ]. Previous research has demonstrated that MMP9, when highly expressed and active in VSMCs, can promote phenotypic transformation, migration, vascular calcification, and matrix remodeling, thereby aggravating the progression of AS [ 21 ]. CRP has been found to inhibit the expression of MMP9 to prevent the occurrence and development of AS [ 22 ]. However, the role of CRP in regulating AS development through MAPK3 remains unknown, hence our focus on verifying the involvement of MAPK3. MAPK3, a serine/threonine kinase, is a crucial signaling molecule in the ERK/MAPK pathway and is involved in regulating apoptosis, cell proliferation, and migration [ 23 ]. Previous studies have demonstrated the involvement of MAPK and AKT in lipid metabolism and controlling the liver secretion of VLDL, LDL-C, and TG levels [ 24 ]. Our experiment further confirmed that CRP could inhibit the expression of MAPK3 and lipid accumulation induced by Ox-LDL treatment. In conclusion, naringenin, the primary active ingredient in CRP, has the potential to decrease MAPK3 expression in VSMCs. This effect could slow the onset and progression of AS by weakening the MAPK cascade reaction, downregulating downstream signaling pathways, and inhibiting lipid formation. These findings provide a solid scientific basis for further research on the impact of naringenin and citrus peel on AS. However, our study has several limitations. Due to variations in data storage across databases, continuous data updates, and the complexity of drug-target protein interactions, we were only able able to validate a fraction of the naringenin anti-AS mechanism. In the future, more comprehensive research will be conducted. 5. Conclusion Our study aimed to explore CRP's potential target and mechanism of action in treating AS using network pharmacological prediction and molecular docking. The in vitro experiment results supported the inhibitory effect of naringenin on MAPK3 expression. These findings offer insights and a scientific foundation for further investigation into the mechanism of action of traditional Chinese medicine in treating AS. Abbreviations AS: Atherosclerosis TCM: Traditional Chinese Medicine CRP: Citri Reticulatae Pericarpium VSMCs: Vascular smooth muscle cells OB: Oral bioavailability DL: Drug-likeness PPI: Protein-protein interaction GO: Gene Ontology KEGG: Kyoto Encyclopedia of Genes and Genomes BP: Biological process CC: Cellular component MF: Molecular function TCMSP: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform MMPs: Matrix Metalloproteinases MOVAS: Mouse aortic vascular smooth muscle cells Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Chongqing key project of technological innovation and application development (cstc2020jscx-msxmX0070) and Chongqing talent plan (CQYC201903166). Authors' contributions PYM and WP conceptualized and designed the study. WYL and YLM contributed to the experiments and data analysis. LYY and LCJ helped with the data collection, methodology, and software. YC contributed to the design of the work, funding acquisition, and revision. All authors have read and agreed to the published version of the manuscript. Acknowledgements We would like to thank the support of Chongqing Key Laboratory for Pharmaceutical Metabolism Research on this project. References Herrington W, Lacey B, Sherliker P, Armitage J, Lewington S. Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease. Circ Res. 2016;118:535-46. Corrigendum to: European Society of Cardiology: Cardiovascular Disease Statistics 2019. Eur Heart J. 2020;41:4507. 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Supplementary Files A2label.tif AKT1label.png AKT11.png AKT12.png AKT13.png AKT1.pse AKT11unq2naringenin.pdbqt CASP32dko2naringenin.pdbqt HSP90AA15j802naringenin.pdbqt MA2label.png MA2label.tif MAPK3MOCK2.tif MAPK3MOCK2.png MAPK3Naringenin2.tif MAPK3Naringenin2.png MAPK82xrw2naringenin.pdbqt Moleculardockingresults.xls PPARG6ms72naringenin.pdbqt TP533d062naringenin.pdbqt casp31.png casp32.png casp33.png casp3label.png casp3lable.png casp3.pse hsp901.png hsp902.png hsp903.png hsp90lable.png hsp90.pse mapk81.png mapk82.png mapk83.png mapk8.png mapk8.pse pparg1.png pparg2.png pparg3.png pparglabel.png pparg.pse tp531.png tp532.png tp533.png tp53.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4241694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316760981,"identity":"e7da2135-e475-461d-bda8-5d52e2c9293e","order_by":0,"name":"Yumeng Pan","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yumeng","middleName":"","lastName":"Pan","suffix":""},{"id":316760982,"identity":"197b8601-6aa5-4df1-a755-14a8b8a58792","order_by":1,"name":"Ping Weng","email":"","orcid":"","institution":"Chongqing Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Yueyue","middleName":"","lastName":"Li","suffix":""},{"id":316760986,"identity":"728f062e-5121-4db8-85f0-2aa8d5a18991","order_by":5,"name":"Chengju Li","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chengju","middleName":"","lastName":"Li","suffix":""},{"id":316760987,"identity":"e8c47879-bb5a-4c5e-97dc-2994202e5cec","order_by":6,"name":"Chao Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHACNiC2QWITqSWNdC2HSdBicCP52YMfFefl+a6dMWD4UHaYgX92AyEtaeaGPWduG868nWPAOOPcYQaJOwcIaclhk+Btu51gANTCzNt2mMFAIoGwFsm/becgWv4Sq0Wat+0ARAsjMVokzzwzk5Y5kwz0S1rBwZ5z6TwSNwho4Tue/EzyTYWdPN/t5I0PfpRZy/HPIKBF4QCMdQCMGHjwqwcC+QYkLaNgFIyCUTAKsAIAj8pEaiznyvoAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-04-09 12:01:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4241694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4241694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59441178,"identity":"d8a48066-bcbd-4b23-8ea8-10d7e31f1f2f","added_by":"auto","created_at":"2024-07-01 21:13:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1501185,"visible":true,"origin":"","legend":"\u003cp\u003eThe workflow to study the molecular mechanism of Chenpi in AS.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/e123eec074fdfa95dea53788.png"},{"id":59441353,"identity":"c3dbc8b0-118a-448b-af00-6c0bc2cb3b7b","added_by":"auto","created_at":"2024-07-01 21:21:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":349066,"visible":true,"origin":"","legend":"\u003cp\u003eThe Venn diagram shows the targets of the CRP and AS, with 54 overlapping genetic symbols between disease and drug.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/e31adc241618a72f4a5747d1.png"},{"id":59440776,"identity":"7afc15dc-8a6b-43ce-8743-3b2988dd063d","added_by":"auto","created_at":"2024-07-01 21:05:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6455294,"visible":true,"origin":"","legend":"\u003cp\u003ePPI map of 54 overlapping genes associated with CPR and AS.The number of nodes:54.The number of edges:421.(The edges represent protein-protein associations, and the node size is proportional to the degree of interaction.)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/c6dd767dbca0ebfbe04be21d.png"},{"id":59440734,"identity":"4ee1dba1-2ba0-42e5-b5df-177028655f73","added_by":"auto","created_at":"2024-07-01 21:05:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163586,"visible":true,"origin":"","legend":"\u003cp\u003eThe core targets of PPI.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/ab4847653f26f1354fe2fb6e.png"},{"id":59440774,"identity":"f8318abb-a379-479d-8609-674f7712a238","added_by":"auto","created_at":"2024-07-01 21:05:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2020217,"visible":true,"origin":"","legend":"\u003cp\u003eThe potential target gene network of CRP-AS.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/0e14eed091089fc44c1d91c3.png"},{"id":59441160,"identity":"a179855b-f173-4f0d-b65e-c6bca1e29497","added_by":"auto","created_at":"2024-07-01 21:13:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3114494,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of the GO enrichment analysis are presented as follows:\u003c/p\u003e\n\u003cp\u003eA. A bubble diagram illustrating the molecular functions (MF) enrichment analysis.\u003c/p\u003e\n\u003cp\u003eB. A bubble diagram illustrating the biological processes (BP) enrichment analysis.\u003c/p\u003e\n\u003cp\u003eC. A bubble diagram illustrating the cellular components (CC) enrichment analysis.\u003c/p\u003e\n\u003cp\u003eD. The GO enrichment analysis consists of the first ten analyses, with green, orange, and purple representing the enrichment analysis of BF, CC, and MF, respectively.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/cbd177f17109af28d167e211.png"},{"id":59441164,"identity":"7c2c5738-f4e5-4a2b-a045-359eb7714aab","added_by":"auto","created_at":"2024-07-01 21:13:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":477528,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of KEGG enrichment analysis. The top 20 pathways were shown.\u003c/p\u003e\n\u003cp\u003eA. Histogram of the top 20 pathways based on KEGG enrichment analysis.\u003c/p\u003e\n\u003cp\u003eB. Bubble diagram of the top 20 pathways based on KEGG enrichment analysis. The top 20 pathways are arranged by the enrichment ratio from high to low.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/e630001b1322033fefa8cd2f.png"},{"id":59440742,"identity":"3d89b525-574f-4f6e-95b8-1059d0b32400","added_by":"auto","created_at":"2024-07-01 21:05:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4399431,"visible":true,"origin":"","legend":"\u003cp\u003ePresents the results of molecular docking analysis on the interaction between naringenin and the target protein.\u003c/p\u003e\n\u003cp\u003eA. Among the tested interactions, the absolute binding energy between naringenin and MMP9 exhibited the highest value, indicating a strong affinity with a score of -10.1 kcal/mol.\u003c/p\u003e\n\u003cp\u003eB. Following MMP9, the binding energy between naringenin and MAPK3 showed the second highest absolute value, indicating a substantial affinity with a score of -9.3 kcal/mol\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/068f2197172138bcc52239dc.png"},{"id":59440751,"identity":"27b97bad-3a61-4b0a-af65-2750bf129424","added_by":"auto","created_at":"2024-07-01 21:05:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3003259,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Result\u003c/p\u003e\n\u003cp\u003eA. The safety of MOVAS cells treated with different concentrations (0μM, 10μM, 25μM, 50μM, and 100μM) of naringenin was determined using the CCK8 assay. The optical density (OD) value was measured at 450nm,n=5.\u003c/p\u003e\n\u003cp\u003eB. The thermal stability test was conducted to evaluate the expression of MAPK3 in the MOCK group and the group treated with 10μM naringenin. The test was carried out at temperatures ranging from 37 to 77℃, with 5℃ intervals.\u003c/p\u003e\n\u003cp\u003eC. The expression of MAPK3 in the groups treated with Ox-LDL (100μg/ml) with and without naringenin (10μM) was detected using Western blotting (WB).\u003c/p\u003e\n\u003cp\u003eD. The lipid accumulation in the control group treated with DMSO (CTL), Ox-LDL group (100μg/ml), and Ox-LDL group (100μg/ml) + naringenin group (10μM) was assessed by immunofluorescence assay. The scale used for imaging was 100μm.\u003c/p\u003e\n\u003cp\u003eE. The fluorescence intensity quantization mapping of panel D was performed to quantify the fluorescence intensity,n=5.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/99dbde25346a4e58c0bbb6fb.png"},{"id":64215755,"identity":"6d7488c2-9a16-43d2-a7d0-8408ae324554","added_by":"auto","created_at":"2024-09-10 09:25:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21889455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/fbd358ea-53e1-4f40-bfba-f5544fcbd4d1.pdf"},{"id":59440738,"identity":"40bcc921-545c-42c2-86a9-9531a2fb753f","added_by":"auto","created_at":"2024-07-01 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21:13:17","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"supplement","size":8227083,"visible":true,"origin":"","legend":"","description":"","filename":"mapk81.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/3d78dbd768b4964e04296f67.png"},{"id":59441355,"identity":"8b5836ca-ea57-4004-9011-41726d241004","added_by":"auto","created_at":"2024-07-01 21:21:16","extension":"png","order_by":44,"title":"","display":"","copyAsset":false,"role":"supplement","size":8460522,"visible":true,"origin":"","legend":"","description":"","filename":"mapk82.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/b5a5cb92c4f7e71c213b5bfc.png"},{"id":59440758,"identity":"99f78fb8-f76e-4928-ad2a-d57b9bc937a9","added_by":"auto","created_at":"2024-07-01 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21:05:17","extension":"png","order_by":53,"title":"","display":"","copyAsset":false,"role":"supplement","size":6247788,"visible":true,"origin":"","legend":"","description":"","filename":"tp531.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/741cf778af1a9c4718b3ee7c.png"},{"id":59440754,"identity":"c0c79b00-0b72-4750-8507-b5c1e4073b54","added_by":"auto","created_at":"2024-07-01 21:05:16","extension":"png","order_by":54,"title":"","display":"","copyAsset":false,"role":"supplement","size":6294706,"visible":true,"origin":"","legend":"","description":"","filename":"tp532.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/c0c4c45a62c492529d60fb48.png"},{"id":59441173,"identity":"2e81bd65-76e1-4af0-8300-0b2a7b212ed0","added_by":"auto","created_at":"2024-07-01 21:13:17","extension":"png","order_by":55,"title":"","display":"","copyAsset":false,"role":"supplement","size":6048030,"visible":true,"origin":"","legend":"","description":"","filename":"tp533.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/c3be5ec56073e6e16222f28d.png"},{"id":59440752,"identity":"d7499dbc-b1db-4a61-b56b-c1a3d8e40cb0","added_by":"auto","created_at":"2024-07-01 21:05:16","extension":"png","order_by":56,"title":"","display":"","copyAsset":false,"role":"supplement","size":379888,"visible":true,"origin":"","legend":"","description":"","filename":"tp53.png","url":"https://assets-eu.researchsquare.com/files/rs-4241694/v1/a9f8e66b3b5716125e52cf07.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Explore the mechanism of Citri Reticulatae Pericarpium (Chenpi) in atherosclerosis Based on Network Pharmacology, Molecular Docking and Experimental Evidence","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAtherosclerosis (AS) is a chronic progressive disease characterized by the deposition of lipids and infiltration of inflammatory cells. It presents significant clinical manifestations, including ischemic heart disease, ischemic stroke, and peripheral artery disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. AS has emerged as the primary cause of cardiovascular illnesses and is increasingly prevalent among younger individuals, now serving as the leading cause of global mortality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, effective control of AS occurrence and development is paramount in reducing cardiovascular disease incidents. Dyslipidemia, particularly elevated levels of plasma LDL-cholesterol, marks the beginning of AS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Lipid-lowering drugs, such as statins, are widely used in the clinical treatment of AS for primary and secondary prevention of atherosclerotic cardiovascular diseases. They aim to improve vascular risk factors and exert anti-inflammatory effects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the clinical utilization of statins is limited due to adverse effects, including Statin-associated muscle symptoms (SAMS) and liver damage, and may even lead to increased cardiovascular adverse outcomes [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Hence, it is imperative to conduct further research to identify more effective and safe lipid-regulating drugs.\u003c/p\u003e \u003cp\u003eThe theory of ancient Traditional Chinese Medicine (TCM) suggests that the dysfunction of body organs leads to the stagnation of blood, which in turn results in the formation of AS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. TCM has a long history of application due to its positive therapeutic effects, including minimal side effects and superior curative outcomes for AS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne particular component of TCM, CRP, commonly known as Chenpi in Chinese, belongs to the citrus genus and Rutaceae family. It is a dried and ripe orange peel [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CRP consists of various active components that possess multiple functions, such as reducing blood lipids, anti-inflammatory properties, anti-thrombotic effects, and inhibition of oxidative stress. As a result, CRP has been widely used as a health food and dietary supplement for the prevention and treatment of cardiovascular diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the target and specific pharmacological mechanism of CRP in preventing and treating AS have not been fully established, considering the complex formation process of AS, the intricate composition of traditional Chinese medicine components, and the ambiguity surrounding the target of action [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNetwork pharmacology is a powerful technology used to investigate the practical components and mechanisms of traditional Chinese medicine in treating various diseases. This approach involves extensive database mining and analyzes the intricate relationship between drug components, action targets, action pathways, and diseases. By constructing a visual network, it systematically and comprehensively reveals the action mechanism of multi-component drug plants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, the network pharmacology approach, combined with molecular docking, was adopted to create a network of \"drug composition - target of action - pathway of action.\" In vitro experiments were then conducted to confirm and predict the probable target of CRP for AS and its mode of action. These findings serve as the foundation for further basic research and offer a more comprehensive outlook on the clinical application of CRP. The workflow to study the molecular mechanism of Chenpi in AS was exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Data Preparation\u003c/h2\u003e \u003cp\u003eThe components of CRP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database (TCMSP, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tcmspw.com/index.php\u003c/span\u003e\u003cspan address=\"https://tcmspw.com/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This retrieval and collection process aimed to acquire the oral bioavailability (OB) and drug-likeness (DL) values of the compounds. Screening conditions were set based on the ADME principle, with OB\u0026thinsp;\u0026ge;\u0026thinsp;30% and DL\u0026thinsp;\u0026ge;\u0026thinsp;0.18 serving as criteria for identifying active ingredients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The target proteins were named using the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Online Mendelian Inheritance in Man (OMIM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omim.org/\u003c/span\u003e\u003cspan address=\"https://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were utilized to search for and collect information related to the keyword \"atherosclerosis.\" The obtained data were then summarized and collated to intersect with the targets of CRP components. The VennDiagram package of R 4.2.1 software was employed to process the data and generate a Venn diagram, which allowed for the identification of common disease targets and potential drugs. These findings suggest that CRP has the potential to be a treatment for atherosclerosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Network Construction\u003c/h2\u003e \u003cp\u003eThe STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was chosen as a resource to obtain protein interactions. The process involved introducing the disease-drug interaction protein, selecting \"Homo sapiens\" as the organism, setting the minimum required interaction score to be greater than 0.4, and hiding disconnected nodes in the network. This resulted in the generation of a protein-protein interaction (PPI) network. The protein interaction network file was then imported into Cytoscape 3.8.0 software for visual analysis, allowing for the construction of a network diagram representing the \"drug component-target-pathway\" interactions and identifying potential core targets. Additionally, the connection results were enriched using R 4.2.1 software, which facilitated the creation of visual bar plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Enrichment Analysis\u003c/h2\u003e \u003cp\u003eThe R 4.2.1 software was utilized to convert the gene symbols of CRP and AS into Entrez ID. Subsequently, various R packages were employed to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, resulting in the generation of bar and bubble charts. The Perl computer program language was used to screen the GO enrichment results and extract the functions of the corresponding genes. For further analysis, only the GO enrichment results with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant and retained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Molecular Docking\u003c/h2\u003e \u003cp\u003eProteins were obtained from the RCSB database, and protein receptor pretreatment was carried out using AutoDockTools 1.5.6. The pretreatment involved dehydrating, hydrogenating, removing the primary ligand, and setting the proteins as receptors. Small molecules were acquired from the PubChem database. A slight energy minimization was performed on the small molecules using OpenBabel 2.4.1 with the MMFF94 force field. The small molecule ligands were then processed using AutoDockTools 1.5.6, hydrogenated, and the number of rotatable bonds was set. These prepared ligands were used in molecular docking conducted with the software Autodock Vina 1.1.2. The docking process involved generating ten docking conformations, and the conformation with the most favorable absolute value of binding energy was selected from the docking results. Finally, the selected conformation was analyzed and mapped using Discovery Studio 2019 Client.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experimental verification\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Cytotoxic assay\u003c/h2\u003e \u003cp\u003eMouse aortic vascular smooth muscle cells (MOVAS) were seeded in 96-well plates one day prior to the experiment. After a 12-hour serum starvation treatment, the control group was supplemented with DMEM/12F medium containing 10% FBS, while the treatment group received naringenin at concentrations of 10 \u0026micro;M, 25 \u0026micro;M, 50 \u0026micro;M, and 100 \u0026micro;M, respectively. The cells were then incubated in a CO2 incubator at 37\u0026deg;C for 24 hours. Following incubation, the medium was removed, and the cells were washed twice with PBS to remove any residual substances. Next, a mixture of CCK-8 medium was added at a 10% concentration, and the cells were further incubated for 1 hour in the incubator. The optical density (OD) at 450 nm was measured using an enzymometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Nile Red staining\u003c/h2\u003e \u003cp\u003eThe cells were seeded in 96-well plates one day prior to the experiment. After a 12-hour serum starvation treatment, the control group was supplemented with DMEM/12F medium containing 1% FBS. Meanwhile, the treatment group received 100 \u0026micro;g/ml Ox-LDL\u0026thinsp;+\u0026thinsp;DMSO and 100 \u0026micro;g/ml Ox-LDL\u0026thinsp;+\u0026thinsp;10 \u0026micro;M naringenin, respectively. The cells were then incubated for an additional 24 hours. Following incubation, the medium was discarded, and the cells were washed twice with PBS. They were then fixed with 4% paraformaldehyde for 15 minutes, followed by three washes with PBS. Subsequently, the cells were stained with 1 \u0026micro;M Nile Red/PBS for 30 minutes, followed by three more washes with PBS. Additionally, the cells were stained with DAPI for 10 minutes, followed by three more washes with PBS. Finally, fluorescence quenching was performed using an anti-fluorescence quencher under a Leica inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Western Blot Analysis\u003c/h2\u003e \u003cp\u003eFor cell culture and grouping, please refer to step 2.5.2. Following removing the medium, the cells were rinsed twice with PBS and then treated with RIPA lysate containing 1% PMSF for cell lysis. This lysate was collected into a 1.5ml EP tube and heated in a metal bath at 100℃ for 5 minutes. The subsequent Western blotting procedure was conducted: 30 \u0026micro;g of total protein lysate was loaded and separated by 10% SDS\u0026ndash;polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in TBST for 2 hours at room temperature, the membranes were incubated overnight at 4℃ with various primary antibodies, including anti-MAPK3. Secondary antibodies were added to the membrane at room temperature for 2 hours. β-actin was used as both the loading control and for normalization purposes. The expression of the MAPK3 antibody (Proteintech, 11257-1-AP) was finally detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Thermal Stability Test\u003c/h2\u003e \u003cp\u003eFor details, see [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1.Data Preparation\u003c/h2\u003e \u003cp\u003eThe components of CRP were obtained by retrieving and collecting data from TCMSP database. Active components and their corresponding targets were screened, and the names and serial numbers of the identified drug targets were recorded. A total of 60 drug targets were identified. Using OB\u0026thinsp;\u0026ge;\u0026thinsp;30% and DL\u0026thinsp;\u0026ge;\u0026thinsp;0.18 as screening criteria, five active ingredients were obtained: sitosterol, naringenin, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one, Citromitin, and nobiletin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genes obtained from the GeneCards and OMIM databases were excluded, resulting in a final list of 6015 AS-related genes. The R 4.2.1 software was employed to create a Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), revealing 54 common targets shared by both the disease and the drug. These targets represent potential candidates for CRP treatment of AS, serving as a basis for further research.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe ID, OB, and DL of five active compounds in CRP.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecule ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecule name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecule weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOB(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMOL000359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esitosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e414.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMOL004328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enaringenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e272.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMOL005100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e302.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMOL005815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCitromitin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e404.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e86.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMOL005828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enobiletin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e402.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Network Construction\u003c/h2\u003e \u003cp\u003eThe protein-protein interaction (PPI) networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were constructed by importing 54 target genes into the STRING database.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PPI map of the 54 overlapping genes associated with CRP and AS consists of 54 nodes and 421 edges. Each node represents a gene, and the size of the node is proportional to the degree of interaction of that gene with other genes in the network.\u003c/p\u003e \u003cp\u003eThe connectivity results were enriched using R 4.2.1 software, and a bar chart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was generated to display the top twenty proteins based on their degrees. The bar chart reveals that the targets with higher degree values include AKT1, TP53, CASP3, PPARG, PPARA, PTGS2, ESR1, MAPK3, MMP9, HSP90AA1, CAT, CREB1, MAPK8, MAPK1, and ADIPOQ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, this data was imported into Cytoscape 3.8.0 software for visual analysis and the construction of the CRP-AS-potential target gene network (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). 113 connections were identified, enabling the examination of the target genes and essential chemicals. It is worth noting that a node's core density increases with its size. The image demonstrates the significance of naringenin, the primary constituent of CRP (MOL004328), in combating illnesses and influencing its targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the network, the size of each node represents its degree value, indicating the number of connections it has with other nodes. The gray connecting lines indicate the interconnectedness between the nodes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Enrichment Analysis\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. GO enrichment analysis\u003c/h2\u003e \u003cp\u003eThe data of disease drug targets was transformed using the R software package BiocManager to display their gene IDs, facilitating later data enrichment analysis. The GO enrichment analysis encompassed three aspects: biological process (BP), cellular component (CC), and molecular function (MF).\u003c/p\u003e \u003cp\u003eThe function of the GO gene was enriched using R 4.2.1 software, resulting in the identification of 1685 significant GO items (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To determine the roles of the corresponding genes, the enrichment results were screened using the Perl computer programming language. The gene GO ontology entries were sorted based on their p-values, and the top 10 scatterplots and histograms for BP, CC, and MF were selected for plotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe top twenty GO entries for BP, CC, and MF were displayed in the plot. Regarding BP, the entries were primarily enriched in response to oxidative stress and nutrient levels. For CC, the entries were primarily enriched in the endoplasmic reticulum lumen. The MF entries specifically included DNA-binding transcription factor binding. These results highlight the crucial role of CRP targets in treating AS across BP, CC, and MF domains.\u003c/p\u003e \u003cp\u003eThe plot's x-axis represents the gene ratio, and the y-axis displays enriched GO entries. The size of each dot corresponds to the number of genes associated with that entry. The dot color indicates the degree of enrichment, ranging from red to green, corresponding to decreasing Q values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. KEGG pathway analysis\u003c/h2\u003e \u003cp\u003eThe potential target of CRP in treating AS was analyzed through KEGG pathway analysis using R software, which enabled the creation of a signal pathway map for a deeper understanding of CRP's mechanism of action. The analysis identified and screened 152 significant signal pathways (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To provide a visual representation, a chart (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) was generated to display the top 20 crucial signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy examining the path annotation in the KEGG database, it was found that the lipid and atherosclerosis pathway is the most well-known pathway, involving 17 genes. Among the 15 targets with the most significant potential effects of CRP (as suggested in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), eight targets, namely AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8, are part of this central pathway. These findings suggest that these targets may play a key role in the therapeutic effects of CRP.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Molecular docking\u003c/h2\u003e \u003cp\u003eThe molecular docking technique was employed to assess the strength of naringenin's binding ability to the eight targets mentioned earlier: AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8. The results obtained from this analysis helped identify the most likely targets for naringenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, supplementary materials).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTypically, a higher absolute value of binding energy signifies a stronger binding affinity. A binding energy of -4.6 kcal/moL suggests primary binding, while values below \u0026minus;\u0026thinsp;5 kcal/moL indicate excellent binding, and values below \u0026minus;\u0026thinsp;7 kcal/moL indicate a good binding effect.\u003c/p\u003e \u003cp\u003eIn the visualization provided (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e of the supplementary materials), small molecular ligands are represented by green structures, and binding bonds are represented by dotted lines. The alpha-number type notes represent residue names for proteins, while pure number notes represent bond lengths.\u003c/p\u003e \u003cp\u003eUpon observing the figure, it becomes evident that naringenin forms multiple binding bonds with the two target proteins, MMP9 and MAPK3, which exhibit the best binding affinity. This suggests that naringenin and these two target proteins are closely bound to each other.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Experimental verification\u003c/h2\u003e \u003cp\u003eNext, we conducted experimental verification using MOVAS. Initially, we used CCK-8 to determine the safe concentration range of naringenin (Figure. 9A). Based on these results, we selected a concentration of 10 \u0026micro;M for further treatment. Subsequently, thermal stability experiments demonstrated that naringenin could affect the stability of MAPK3 (Figure. 9B), suggesting a direct interaction between the two. Furthermore, naringenin exhibited a significant reduction in the expression of MAPK3 induced by Ox-LDL (Figure. 9C).\u003c/p\u003e \u003cp\u003eWe then investigated the lipid-lowering effect of naringenin, which was assessed using Nile Red staining. Our findings revealed a notable reduction in lipid accumulation in the naringenin group compared to the Ox-LDL group (Figure. 9D-E). Additionally, KEGG analysis predicted the lipid-lowering effect of naringenin, suggesting that it may inhibit lipid accumulation in smooth muscle cells and impede the occurrence of atherosclerosis by suppressing the expression of MAPK3 during smooth muscle cell foam formation.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAtherosclerosis is a complex disease characterized by lipid aggregation, vascular endothelial cell damage, and the activation of inflammatory factors. Within the pathophysiology of AS, the accumulation of lipids in the arterial wall, particularly in foam cells, is a hallmark lesion.\u003c/p\u003e \u003cp\u003eUsing TCMSP database, we found 5 active ingredients of CRP. By intersecting these compounds with AS-related targets, we identified 54 potential action targets. Subsequently, a protein-protein interaction (PPI) analysis was performed, revealing naringenin as the primary core component and 15 core targets (AKT1, TP53, CASP3, PPARG, PPARA, PTGS2, ESR1, MAPK3, MMP9, HSP90AA1, CAT, CREB1, MAPK8, MAPK1, and ADIPOQ).\u003c/p\u003e \u003cp\u003eFurther examination of KEGG data indicated that CRP influences the progression of AS through various pathways. Notably, the \"Lipid and atherosclerosis (hsa05417) pathway\" emerged as a significant pathway, suggesting that CRP likely plays a crucial role in AS by affecting lipid metabolism. The \"Lipid and atherosclerosis (hsa05417) pathway\" overlapped with eight of the top 15 PPI targets, validating the accuracy of the network pharmacological analysis. Consequently, eight fundamental regulatory targets\u0026mdash;AKT1, TP53, CASP3, PPARG, MAPK3, MMP9, HSP90AA1, and MAPK8\u0026mdash;were identified.\u003c/p\u003e \u003cp\u003eThe binding affinity of a drug with the target protein is a critical parameter for evaluating its impact on the disease mechanism [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This study selected naringenin as the molecular docking object, representing the most significant node in the CRP-AS-potential target gene network. We hypothesized that naringenin could potentially prevent the onset and progression of AS by interacting with MMP9 and MAPK3. Molecular docking tests indicated that two of the eight potential targets had the strongest binding affinity with naringenin.\u003c/p\u003e \u003cp\u003eMatrix Metalloproteinases (MMPs) are a family of zinc-dependent proteases that can degrade protein substrates. They have the ability to modify chemokines and cytokines, participate in regulating cell proliferation, adhesion, and migration, and play a key role in vascular wall remodeling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous research has demonstrated that MMP9, when highly expressed and active in VSMCs, can promote phenotypic transformation, migration, vascular calcification, and matrix remodeling, thereby aggravating the progression of AS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. CRP has been found to inhibit the expression of MMP9 to prevent the occurrence and development of AS [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the role of CRP in regulating AS development through MAPK3 remains unknown, hence our focus on verifying the involvement of MAPK3.\u003c/p\u003e \u003cp\u003eMAPK3, a serine/threonine kinase, is a crucial signaling molecule in the ERK/MAPK pathway and is involved in regulating apoptosis, cell proliferation, and migration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Previous studies have demonstrated the involvement of MAPK and AKT in lipid metabolism and controlling the liver secretion of VLDL, LDL-C, and TG levels [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our experiment further confirmed that CRP could inhibit the expression of MAPK3 and lipid accumulation induced by Ox-LDL treatment.\u003c/p\u003e \u003cp\u003eIn conclusion, naringenin, the primary active ingredient in CRP, has the potential to decrease MAPK3 expression in VSMCs. This effect could slow the onset and progression of AS by weakening the MAPK cascade reaction, downregulating downstream signaling pathways, and inhibiting lipid formation. These findings provide a solid scientific basis for further research on the impact of naringenin and citrus peel on AS.\u003c/p\u003e \u003cp\u003eHowever, our study has several limitations. Due to variations in data storage across databases, continuous data updates, and the complexity of drug-target protein interactions, we were only able able to validate a fraction of the naringenin anti-AS mechanism. In the future, more comprehensive research will be conducted.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study aimed to explore CRP's potential target and mechanism of action in treating AS using network pharmacological prediction and molecular docking. The in vitro experiment results supported the inhibitory effect of naringenin on MAPK3 expression. These findings offer insights and a scientific foundation for further investigation into the mechanism of action of traditional Chinese medicine in treating AS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAS: Atherosclerosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTCM: Traditional Chinese Medicine\u003c/p\u003e\n\u003cp\u003eCRP: Citri Reticulatae Pericarpium\u003c/p\u003e\n\u003cp\u003eVSMCs: Vascular smooth muscle cells\u003c/p\u003e\n\u003cp\u003eOB: Oral bioavailability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDL: Drug-likeness\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePPI: Protein-protein interaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGO: Gene Ontology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBP: Biological process\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCC: Cellular component\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMF: Molecular function\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTCMSP:\u0026nbsp;Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform\u003c/p\u003e\n\u003cp\u003eMMPs:\u0026nbsp;Matrix Metalloproteinases\u003c/p\u003e\n\u003cp\u003eMOVAS: Mouse aortic vascular smooth muscle cells\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Chongqing key project of technological innovation and application development (cstc2020jscx-msxmX0070) and Chongqing talent plan (CQYC201903166).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePYM and WP conceptualized and designed the study. WYL and YLM contributed to the experiments and data analysis. LYY and LCJ helped with the data collection, methodology, and software. YC contributed to the design of the work, funding acquisition, and revision.\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the support of Chongqing Key Laboratory for Pharmaceutical Metabolism Research on this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHerrington W, Lacey B, Sherliker P, Armitage J, Lewington S. Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease. Circ Res. 2016;118:535-46.\u003c/li\u003e\n\u003cli\u003eCorrigendum to: European Society of Cardiology: Cardiovascular Disease Statistics 2019. 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Cell. 2019;179:1276-88.e14.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atherosclerosis, Citri Reticulatae Pericarpium, Network pharmacology, Chinese traditional medicine, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-4241694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4241694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCitri Reticulatae Pericarpium (CRP), a traditional Chinese medicine, is extensively used to prevent and treat cardiovascular diseases. However, the exact target and pharmacological mechanism of CRP remain unclear. This study aims to investigate the potential mechanism of CRP in treating atherosclerosis (AS) using network pharmacology, molecular docking, and experimental verification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe chemical constituents and targets of CRP were retrieved, collected, and screened in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database. Potential AS targets were obtained from GeneCards and OMIM databases. Subsequently, the STRING database was used to establish a protein-protein interaction network, and Cytoscape was employed to construct the CRP-AS-potential target gene network to identify core targets. After GO and KEGG enrichment analysis, naringenin and core targets were selected for molecular docking simulation. Finally, the anti-AS mechanism of naringenin was validated through cell experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Five potential active components of CRP were identified, and 54 common targets of the disease and drugs, including 15 core targets (such as MAPK3 and MMP9), were obtained. Lipid and atherosclerosis were found to be the most prominent pathways of action. Molecular docking demonstrated the strong binding of naringenin with MMP9 and MAPK3. In vitro experiments, it was revealed that naringenin might inhibit lipid accumulation in smooth muscle cells and slow down the occurrence of atherosclerosis by decreasing the expression of MAPK3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThrough network pharmacological analysis, molecular docking, and experimental verification, this study found that naringenin, the core active ingredient of CRP, may inhibit the occurrence of smooth muscle cell foam by reducing the expression of MAKP3 in vascular smooth muscle cells (VSMCs)and play an anti-AS role, providing a new idea for further research on CRP and naringenin in the prevention and treatment of AS.\u003c/p\u003e","manuscriptTitle":"Explore the mechanism of Citri Reticulatae Pericarpium (Chenpi) in atherosclerosis Based on Network Pharmacology, Molecular Docking and Experimental Evidence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-01 21:05:08","doi":"10.21203/rs.3.rs-4241694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2a4ecce-07f0-49c9-9d14-79f1a66524f7","owner":[],"postedDate":"July 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-10T09:17:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-01 21:05:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4241694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4241694","identity":"rs-4241694","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}