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Table of Contents
Abstract
Drug Design, Development And Therapy Dovepress
Introduction
Materials
Results
Discussion
Limitation
QSHT.
Ethics Statement
Funding
Disclosure
Abstract
1The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2Department of Cardiology, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 3State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4Guangzhou Key Laboratory of Chirality Research on Active Components of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 5Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 6State Key Laboratory of Traditional Chinese Medicine Syndrome, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 7Chinese Medicine Guangdong Laboratory, Guangdong Hengqin, People’s Republic of China
O R I G I N A L R E S E A R C H Integrating Network Pharmacology and Experimental Validation to Investigate the Mechanism of Qushi Huatan Decoction Against Coronary Heart Disease Chunxia Yin1,*, Taohua Lan2,3,*, Yunshan Wu4,5, Jing Cai1, Haoxiang Li1, Xiaolan Kuang1, Lin Jiao1, Xiaomin Ou1, Hua Yang2, Bo Liu 3–5, Weihui Lu 2,6,7 1The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2Department of Cardiology, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 3State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 4Guangzhou Key Laboratory of Chirality Research on Active Components of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, People’s Republic of China; 5Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 6State Key Laboratory of Traditional Chinese Medicine Syndrome, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 7Chinese Medicine Guangdong Laboratory, Guangdong Hengqin, People’s Republic of China *These authors contributed equally to this work Correspondence: Weihui Lu, Bo Liu, The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Email weihui.lu@gzucm.edu.cn; doctliu@gzucm.edu.cn Purpose: This study was designed to evaluate the effect and mechanism of the Qushi Huatan (QSHT) decoction against coronary heart disease (CHD) through network pharmacology and experimental verification. Methods: In the present study, the active ingredients of the QSHT decoction were identified by ultra performance liquid chromatography/ tandem mass spectrometry (UPLC/MS), then the potential ingredients and coronary heart disease targets were predicted using the SwissTarget Prediction database and the database of Genecards and OMIM database, respectively. A herb-compound-target network was constructed using Cytoscape. GO and KEGG enrichment analysis were performed using the ClusterProfiler data package of R software. Molecular docking was used to predict the core targets of QSHT against CHD. In addition, we used a myocardial infarction (MI) and high-fat diet ApoE−/− mice model to investigate the cardioprotective effects of QSHT. Western blotting and immunochemistry were used to verify the core targets and the signaling pathway. Results: A total of 68 active ingredients were found in the QSHT decoction. Network pharmacology indicated 28 targets and 147 signal pathways, including AKT1, HIF-1α, GSK-3β, TLR4 and NF-κB, those key targets were also verified by molecular docking. The results of GO and KEGG enrichment analysis showed that the targets of QSHT against CHD were largely associated with inflammatory and oxidative stress, and AKT/HIF-1α and TLR4/NF-κB pathways might be key functional pathways. In vivo, QSHT significantly improved cardiac function and attenuated fibrosis and inflammation. Furthermore, QSHT could significantly inhibit the expression of HIF-1α, TLR4, phosphorylation of AKT1, GSK-3β and NF-κB after MI in ApoE−/− mice. Conclusion: Based on network pharmacology, molecular docking and experimental verification, this study demonstrated that QSHT could improve cardiac function and attenuate cardiac fibrosis by regulating TLR4/NF-κB and AKT/HIF-1α signaling pathway in postMI and high-fat diet ApoE−/− mice. Keywords: network pharmacology, molecular docking, Qushi Huatan decoction, coronary heart disease Drug Design, Development and Therapy 2024:18 4033–4049 4033 © 2024 Yin et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
Drug Design, Development and Therapy Dovepress
open access to scientific and medical research Open Access Full Text Article Received: 5 April 2024 Accepted: 17 August 2024 Published: 11 September 2024 D ru g D es ig n, D ev el op m en t a nd T he ra py d ow nl oa de d fr om h ttp s: //w w w .d ov ep re ss .c om / F or p er so na l u se o nl y.
Introduction
Coronary heart disease (CHD) is characterized by an inadequate supply of oxygen-rich blood to the myocardium due to arterial plaques that narrow the coronary arteries, with manifested episodic or persistent angina.1 Epidemiological research showed that CHD is one of the leading causes of death worldwide, and its incidence population tends to be younger, which threatens human life and health.2 In recent decades, the effects of western medicines have encountered a bottleneck, clinical practice has found that the effect of simple western medicine is not ideal, and long-term medication also increases the risk of adverse drug reactions and drug resistance.3,4 Therefore, it is necessary to seek alternative medicines to treat CHD. Traditional Chinese medicine (TCM), as a type of alternative drug, has shown the merits of safety and effectiveness. TCM has been shown to have a significant clinical effect in the treatment of CHD.5 On the basis of the theory of TCM, CHD belongs to the category of “chest obstruction or heart pain”. The pathogenesis of CHD is closely related to phlegmdampness, which blocks meridians and resulting in heart pain.6 Therefore, clinical medication should focus on removing phlegm-dampness and relieving pain. Qushi Huatan (QSHT) decoction was developed on the basis of Pingwei San by Keji Chen, who is the famous TCM expert and academician of Chinese Academy of Sciences. In composition, the QSHT decoction consists of six Chinese herbal medicines, including three herbs from Pingwei San (rhizoma atractylodis, Magnolia officinalis and dried orange peel), and the other three herbs are Rhizoma Curcumae, Acorus gramineus and Rhizoma Pinellinae Praeparata. More recently, QSHT decoction was applied in clinical to treat CHD and is effective. However, the active compounds, potential targets, and signaling pathways involved in anti-CHD have not been elucidated. With the development of bioinformatics and system biology, network pharmacology provides us with useful tools to screen potential active ingredients and elucidate therapeutic mechanisms by constructing disease-gene-target-drug networks.7,8 In recent years, the network pharmacology-based method has been used to manifest the “multicompound, multi-target” characteristics of TCM. However, there exist limitations in the traditional Chinese medicinerelated database, including incompleteness of data, lacking detailed descriptions about the compounds, genes, and diseases.9 The comprehensive and complete information of ingredients of TCM is critical for accurate assessments of network pharmacology, therefore, we achieve active ingredients of QSHT decoction by ultra performance liquid chromatography/mass spectrum (UPLC/MS) analysis but not database. In the current study, a comprehensive method is developed that combines UPLC/MS analysis with network pharmacology and experimental validation. The flow chart is illustrated in Figure 1. Briefly, (1) UPLC/MS technology was used to determine the main active components of QSHT decoction; (2) Bioinformatic analysis is used to establish the “herbs-active components-targets and pathways” network for screening potential major targets of QSHT decoction in the treatment of CHD; (3) The therapeutic effects of QSHT decoction and some key targets were experimentally validated in post-MI and high-fat diet ApoE−/− mice.
Materials
Chemicals and Atractylodin (Lot. 5098), Magnolol (Lot. 5672), Honokiol (Lot. 9355), Hesperidin (Lot. 7340), Nobiletin (Lot. 8005), Curdione (Lot. 9697), Curcumol (Lot. 3834), Germacrone (Lot. 7447), α-asarone (Lot. 8009), β-Asarone (Lot. 4391), Ammonium Glycyrrhizinate (Lot. 7435) were purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China). Methanol and acetonitrile (HPLC grade) were purchased from Merck Co., Ltd. The water was prepared using the RODI-200B1 water purification system (RSJ Scientific Instruments Co., ltd, Xiamen, China). Magnolia officinalis (Lot. 220201) and Rhizoma Curcumae (Lot. 220301) were purchased from yulin materia medica Chinese herbal medicine Co., Ltd. Dried orange peel (Lot. 220401681) and rhizoma pinelliae (Lot. 22010404) were purchased from Kangmei Pharmaceutical Co., LTD, rhizoma atractylodis (Lot. 2112003) were purchased from Lingnan Chinese medicine yinpian co., LTD, Acorus gramineus (Lot. C12201094) were purchased from China Pharmaceutical Group co., LTD. https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4034 Powered by TCPDF (www.tcpdf.org) Drug Preparation A dose of QSHT consists of rhizoma atractylodis (20 g), Magnolia officinalis (20 g), dried orange peel (10 g), Rhizoma Curcumae (15 g), Acorus gramineus (15 g) and Rhizoma Pinelliae Praeparata (15 g). Eight doses of QSHT were immersed with 8-fold water for 30 min, then decocted twice of 45 min for each time. All QSHT decoctions were combined and condensed to approximately 160 mL using a rotary evaporator. A part of the decoction was freeze-dried to obtain powder for UPLC/MS analysis. The powder was dissolved with 50% (v/v) aqueous methanol. Identification of QSHT Ingredients by UPLC–HRMS Analysis Thermo Fisher Q Exactive Orbitrap LC–MS/MS equipped with electrospray ionization (ESI, Thermo Fisher Scientific, Waltham, MA, USA) in positive and negative ion modes, which was controlled by Thermo Xcalibur 3.0.63 (Thermo Fisher Scientific). Drug Design, Development and Therapy 2024:18 https://doi.org/10.2147/DDDT.S463054 DovePress 4035 Powered by TCPDF (www.tcpdf.org) An ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) was used to separate the sample with the temperature set at 40 °C. Mobile phase A was H2O with 0.1% formic acid, and mobile phase B was 100% acetonitrile with a flow rate of 0.3 mL/min. The gradient was set as follows: 0–1 min (5% B); 1–20 min (5% B-15% B); 20–50 min (15–75% B); 50–60 min (75–95% B), 60–70 min (95% B), 70–75 min (95–5% B), The injection volume was established as 2 μL. Full MS and data-dependent tandem mass spectrometry 2 (ddMS2) in both positive and negative ion mode were used to acquire full scan MS and MS data from the samples. The scan mass range 100 to 1500 Da was selected. High resolution full-scan MS and MS2 data were collected at resolving power of 70,000 and 17,500, respectively. The HCD collision energy was set to 10, 20, 40%. The capillary temperature was set at 300 °C, and the spray voltage was 3.0 kV for negative ion mode and 3.4 kV for positive ion mode. The flow rates of the sheath gas (N2) and the auxiliary gas (N2) were 50 and 10 arb, respectively. The data obtained were analyzed by Thermo Xcalibur 4.0.27 Qual Browser (Thermo Fisher Scientific). After collecting MS and MS/MS data from the QSHT test solution, structural characterization and confirmation of QSHT constituents were performed using Compound Discoverer 3.1 software (Thermo Fisher Scientific). The main parameters of this software were set as follows: alignment retention times, maximum shift 2 min; mass tolerance, 5 ppm; detect compound, mass tolerance 5 ppm; intensity tolerance, 30%; minimal peak intensity, 500,000; data sources, mzCloud, mzVault, MassList, and ChemSpider Search; and S/N threshold, 3, after which compounds searching and matching were conducted. Then the software search report was combined with the fragmentation rule of reference MS as well as the corresponding characteristic fragment ion information. Collection of Drug Targets and CHD Targets After achieving the active ingredients of QSHT by UPLC/MS analysis, the sdf structures of the ingredients were downloaded from Pubchem (http://pubchem.ncbi.nlm.nih.gov), then the sdf formats of the active ingredients were uploaded into the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to predict the targets. The Genecards database (https://www.genecards.org/) and OMIM (https://omim.org) were searched with the keyword of “coronary heart disease” to obtain disease targets. Construction of the PPI Network with Active Ingredients-Target Active ingredient and disease targets were imported to the Venny 2.1.0 mapping platform (http://bioinfogp.cnb.csic.es/ tools/venny/) to filter shared targets. The PPI, protein-protein interaction, of every target was generated through the STRING database (https://string-db.org/). The active ingredients target network was constructed with Cytoscape software (v3.8.2) and the CytoNCA (v2.1.6) plugin was used to filter the core targets. GO and KEGG Enrichment Analyses To achieve the potential mechanism of QSHT in the treatment of CHD. GO and KEGG enrichment analysis was performed using the ClusterProfiler data package of R software. P < 0.05 indicated that the enrichment was statistically significant. Pathways with low P-values were selected for experimental verification. Molecular Docking The protein structures and 3D structures of the QSHT ingredients were retrieved from the RCSB Protein Data Bank and Pubchem, respectively. Molecular Docking calculations were performed on the Yinfo Cloud Platform (https://cloud. yinfotek.com). The Dock6.9 program was used to perform flexible docking and the grid scoring evaluated affinities. Animal Experiments Male ApoE−/− mice (6–8 weeks of age) and male wild-type C57BL/6 (C57) mice were obtained from the Guangdong Yaokang Biotechnology Company. All animal experiments were carried out in accordance with the Experimental Animal Use Ethics Committee of the Guangdong Hospital of Traditional Chinese Medicine (Approval No. 2022125). All animal experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4036 Powered by TCPDF (www.tcpdf.org) published by the Ministry of Science and Technology of China. ApoE−/− mice were fed a high-fat diet for 2 months, and wild-type C57 mice were only fed a chow diet. All ApoE−/− mice were performed by ligating the left coronary artery anterior descending branch (LAD). In briefly, male ApoE−/− mice were anesthetized and received small animal ventilator (Rovent Jr, Kent Scientific, America) at a respiratory rate of 39 mL/min. After the pericardium was separated, the heart was exposed and the left branch of the coronary artery was ligated with a 8–0 nylon silk suture below the level of the left auricle. The sham group C57 mice (Sham, n =10) underwent the same procedure without ligation of the left coronary artery. Then, ApoE−/− mice were randomly divided into three groups: the MI group, the MI+QSHT -L group (low dose) and MI+QSHT -H group (high dose). ApoE−/− mice in the MI + L group and the MI + H group were administered the equivalent clinical dose in humans and 2 times the clinical dose of QSHT for 4 weeks. Those in the sham group and the MI group were treated with the same dose of water for 4 weeks. Echocardiogram Serial echocardiography was performed using a high-resolution echocardiographic system (Vevo 2100; VisualSonics Inc, Canada). All mice were lightly anesthetized with isoflurane. Transthoracic echocardiography was performed with a 18-38 MHz phased array probe. The ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic dimension (LVED), and left ventricular end-systolic volume (LVEV) were measured and calculated from the M-mode recordings. The M-mode recordings were performed at the level of the papillary muscles. HE and Masson Staining Staining with hematoxylin-eosin (HE) and masson’s trichrome (Masson) was performed to evaluate the morphological changes of the ventricles. After deparaffinization of the sections, all procedures were performed with the HE and Masson kit. The stained sections were then observed under a microscope (BX53+DP72, Olympus, Japan) and quantified with ImageJ software. Immunohistochemistry Staining After the sections were deparaffinized, sodium citrate buffer high-pressure boiling method was used for antigen retrieval. After washing with PBS 3 times, the sections were blocked with bovine serum albumin for 45 min. The sections were then incubated with primary antibody against TLR4 or HIF-1α overnight at 4 °C followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody. After washing with PBS 3 times, the cell nucleus was incubated with hematoxylin at room temperature for 3 min. After staining, the sections were observed under a microscope (IX51, Olympus Corporation, Tokyo, Japan) and positive areas were quantified with ImageJ software. Western Blotting The procedures for protein preparation and Western blot were performed as described in our previous studies.10 In briefly, the total protein of the infarct border zone was extracted by RIPA lysis buffer with a protease and phosphatase inhibitor cocktail. Protein concentrations were determined by the instruction of the BCA assay kit. Protein samples were separated by 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with skim milk for 1 h, PVDF membranes were labeled with primary antibodies against AKT (CST,1:1000), p-AKT (CST,1:1000), GSK-3β (CST,1:1000), p-GSK-3β (CST,1:1000), NF-κB (CST,1:1000), p- NF-κB (Santa Cruz, 1:100), and GAPDH (CST,1:8000) followed by horseradish peroxidase (HRP) conjugated secondary antibody (CST, 1:2000). The signals of the Western blot bands were detected by Enhanced Chemiluminescence Plus. Bands quantification was carried out by Image J (NIH, USA). The protein was normalized with GAPDH. Statistical Analysis Data were expressed as mean ± standard deviation of mean and all experiments in this study were replicated independently at least three times. SPSS Statistics 26 was used to analyze all data. The analysis of variance (ANOVA) test was performed to determine statistical significance for multiple comparisons with LSD (equal variance) or Dunnett (unequal variance). A P value <0.05 was considered statistically significant. Drug Design, Development and Therapy 2024:18 https://doi.org/10.2147/DDDT.S463054 DovePress 4037 Powered by TCPDF (www.tcpdf.org)
Results
UPLC/MS Analysis of the Chemical Constituents of QSHT To identify the components of QSHT decoction, the test solution was analyzed by UPLC/MS. As shown in Figure 2 and Supplementary Table S1, a total of 68 significant constituents were found in the QSHT test solution. Targets of QSHT in Treatment of Coronary Heart Disease As a result, a total of 1079 drug targets were obtained from the SwissTargetPrediction database, and a total of 10120 target for coronary heart disease were obtained after the duplicate targets were excluded. Finally, 696 common genes were identified by Venn analysis (Figure 3A). Network Construction Based on a string database, the PPI network was constructed using cytoscape software, which consisted of 731 nodes. Core targets were selected through a filter. The CytoNCA (v2.1.6) plug-in and 28 nodes were obtained (Figure 3B). The top 28 core targets based on the degree are listed in Table 1. NF-κB, TLR4, AKT1, TNF, IL-6, CASP3, GSK-3β and HIF1α were closely related to oxidative stress and inflammation. To clarify the relationship among the ingredients of QSHT and core targets, an ingredient - targets network were constructed by Cytoscape, which comprised 96 nodes (28 core targets and 68 active ingredients) and 257 edges (Figure 3C). Go Enrichment and KEGG Pathway Analysis The GO enrichment and KEGG pathway analysis were employed by the R software Cluster Profiler package. As a result, 2122 BP entries, 56 CC entries, and 136 MF entries were selected. In the BP category, the core targets are mostly enriched in cellular response to chemical stress, response to oxidative stress, and neuron death (Figure 4A). A total of 147 pathways were significantly enriched (p<0.05). The top 20 pathways are shown in Figure 4B. Lipid and atherosclerosis, the IL-17 signaling pathway, and the HIF-1 signaling pathway were the major pathways. Molecular Docking Analysis To evaluate the affinities between the QSHT ingredients and the core targets, AKT1, HIF-1α, GSK-3β, TLR4 and NF-κB was used to perform molecular docking. The results are shown in Table 2 and Figure 5. Generally speaking, the grid score lower than −70 kcal/mol indicated that the component could strongly bind to the protein, the grid score is between −40 kcal/mol and −70 kcal/mol indicated that the binding force is moderate. According to the results, AKT1, HIF-1α, GSK-3β and NF-κB had a lower grid score (lower than −60 kcal/mol), among them, AKT1 and NF-κB had the strongest https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4038 Powered by TCPDF (www.tcpdf.org) binding with the active ingredients of QSHT (lower than −70 kcal/mol), suggesting that they could be crucial targets for the treatment of CHD by QSHT. Besides, we re-docked validation with co-crystallized ligands to confirm the protein selection and molecular docking method are corrected, the results are shown in Table S2 and Figure S1, all of grid scores are lower −57 lower kcal/mol.
4 TP53 0.628227194 303 16,980.22267
7 ALB 0.615514334 278 17,798.98569
Powered by TCPDF (www.tcpdf.org) QSHT Improves Cardiac Function We explored the effect of QSHT on coronary heart disease in Apoe−/− mice (Figure 6A). All mice were conducted echocardiogram tests at the end of 12 weeks. Compared to the Sham group, EF and FS decreased remarkably in the model group after 12 weeks, meanwhile, LVEDS and LVEVS increased significantly. These indicate heart dysfunction after MI in high-fat diet fed ApoE−/− mice. On the contrary, the low and high dose of QSHT significantly improved these heart dysfunctions and dramatically improved EF and FS, and significantly decreased LVDS and LVEVS (Figure 6B–F). QSHT Attenuated Myocardial Inflammation and Fibrosis Normally, cardiac dysfunction is related to cardiac hypertrophy. Compared to the Sham group, the model group had an increased ratio of heart weight to body weight, which was substantially attenuated by the low and high dose of the QSHT group (Figure 7B). The protection of QSHT was further confirmed by HE staining and Masson staining of the heart section. The results of HE staining indicate severe inflammatory cell infiltration and myocardial structural disorder in the model group, while low and high doses of QSHT could reduce the infiltration of inflammatory cells, indicating that QSHT could protect the heart by anti-heart inflammation (Figure 7A). The results of masson trichrome staining showed significant collagen deposition in the model group. While the low and high doses of QSHT markedly
15 GAPDH 0.650623886 340 28,920.38635
17 IL6 0.637554585 318 17,790.31023
18 TNF 0.638670166 321 19,769.03815
19 ESR1 0.590614887 231 10,077.31781
21 AKT1 0.651204282 342 24,922.59837
22 CTNNB1 0.598360656 250 11,071.53655
23 PPARG 0.583067093 217 9781.200496
24 CXCR4 0.549285177 156 3620.508434
25 IL2 0.545590433 141 3373.502238
27 TLR4 0.557251908 173 3201.724218
https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4040 Powered by TCPDF (www.tcpdf.org) reduced myocardial interstitial fibrosis (Figure 7A and C). Those data demonstrated that QSHT substantially protects ApoE−/− mice against high-fat diet and MI-induced myocardial injury. QSHT Regulated the TLR4/NF-κB and AKT/HIF-1α Singnaling Pathway in ApoE−/− Mice Based on network pharmacology and molecular docking, TLR4/NF-κB and AKT/HIF-1α singnaling pathway might be the major pathways. To verify the mechanisms of QSHT reduced heart injury, we performed Western Blot and immunohistochemistry to detect protein expression of key targets of TLR4/NF-κB and AKT/HIF-1α pathway. Powered by TCPDF (www.tcpdf.org) Compared to the sham group, the expression of TLR4, p-NF-κB, p-AKT, HIF-1α and p-GSK-3β was increased in the model group, while the tendency could be reversed by a low and high dose of QSHT (Figures 8 and 9).
Discussion
As the top of traditional Chinese cultural treasures, TCM has particular advantages in the treatment of CHD. In recent years, great progress has been made in the treatment of CHD, which can effectively improve the cardiac function of patients and enhance the quality of life of patients.11 The QSHT decoction was developed by the famous TCM expert of Keji Chen based on Pingwei San. A recent study showed that Pingwei San improved chronic colitis in mice by suppressed NF-κB pathway and NLRP3 inflammasome activation.12 However, the effect and mechanism of QSHT or Pingwei San on CHD remain unknown. In the present study, we performed network pharmacology analysis and related experimental verification to study the effect and mechanism of QSHT on CHD. The major results are as follows: (1) Active ingredients of QSHT decoction were screened through UPLC/MS, which target a total of 1079 genes. Cross-contrasting the disease targets and the ingredients targets resulted in 696 common genes. (2) Go and KEGG enrichment analysis revealed that the anti-inflammatory and anti-oxidation of QSHT might be the main mechanisms in CHD. Among them, TLR4/NF-κB and AKT/HIF-1α signaling pathways might be the key pathways for the anti-inflammatory and anti-oxidation action of QSHT. (3) The key genes of the network include NF-κB, TLR4, AKT1, GSK-3β and HIF-1α. Furthermore, molecular docking verified the affinities between the ingredients of QSHT and those core targets. (4) Animal experimental results demonstrated that QSHT improved cardiac function, attenuated myocardial inflammation, and cardiac fibrosis after MI in ApoE−/− mice. (5) QSHT improved cardiac function might be through regulating TLR4/NFκB and AKT/HIF-1α signaling pathways. In the drug-ingredient-target network, Atractylenolide II, honokiol, magnolol, 3-Methoxynobiletin, naringenin, p-coumaric acid and baicalein have more therapeutic targets for CHD than other ingredients, therefore, those https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4042 Powered by TCPDF (www.tcpdf.org) ingredients might be the core effector components of QSHT in treatment of CHD. Atractylenolide II is the main ingredient in rhizoma atractylodis, researches have shown that atractylenolide II has remarkable anti-cancer, antifibrosis and anti-oxidation activities.13–15 It showed a significant antitumor effect through elevated antiinflammatory activity by inhibiting the NF-κB pathways and modulating AKT/ERK signaling pathways.16,17 Furthermore, Atractylenolide II could improve cardiac function in spontaneous hypertensive rats by attenuating cardiac fibrosis and oxidative stress.14 Honokiol and magnolol are compounds of magnolia officinalis. Recently, research has shown that honokiol and magnolol could suppress tumor, alleviate oxidative stress, fibrosis, and inflammation as well as protect nerve.18–22 Samant et al showed that honokiol blocks cardiac hypertrophic and cardiac fibrosis via activating sirt3.19 Magnolol mediated biological functions implicated many signaling pathways, such as the NF- κB/MAPK, Nrf2/HO-1, and PI3K/AKT pathways.23 3-Methoxynobiletin could alleviate obesity induced by a high-fat diet, lower blood triglyceride, total cholesterol and low-density lipoprotein.24 Naringenin could prevent nonalcoholic fatty liver disease via down-regulating NLRP3/NF-κB pathway, then reducing inflammation in mice.25 Naringenin could also have significant therapeutic potential in cardiovascular disease through anti-inflammatory, anti-oxidative and anti-apoptotic activity.26 P-coumaric acid is the main ingredient of dried orange peel and Rhizoma Pinellinae Praeparata, it has many pharmacological activities, including antioxidant, inhibiting lipid peroxidation, immune regulation and anti-inflammatory action.27–29 P-coumaric acid could also protect cardiomyocytes by regulating macrophage polarization and inhibiting inflammation in myocardial ischemia/reperfusion mice.30 Baicalein could attenuate isoproterenol-induced cardiac hypertrophy by clearing reactive oxygen species and promoting autophagy by binding to the transcription factor FOXO3a.31 Furthermore, recent researches have discovered several pharmacological functions of baicalein, such Drug Design, Development and Therapy 2024:18 https://doi.org/10.2147/DDDT.S463054 DovePress 4043 Powered by TCPDF (www.tcpdf.org) as antioxidant, anti-cancer and anti-inflammatory effects.32,33 Recently, baicalein has been shown to protect the heart and attenuate cardiac hypertrophy involved several signaling pathways, such as AKT/mTOR, NF-κB and calcineurin signaling pathway.34 All in all, those active ingredients have potent anti-inflammatory and antioxidant activity, therefore, they might be the key components of QSHT in the treatment of CHD. Furthermore, Atractylenolide II, honokiol and magnolol,35 Naringenin36 and baicalein31 could improve cardiac function in different pathological model, they may become novel therapeutics with a better effect than QSHT, we will take further experiments to prove their effect on CHD in future. Go enrichment analysis showed that QSHT treats CHD by regulating biological responses such as oxidative stress, chemical stress, and neuron death. KEGG enrichment analysis results showed that lipid and atherosclerosis, the IL-17 signaling pathway and the HIF-1 signaling pathway may be the potential key pathway of QSHT in the treatment of coronary heart disease. Previous studies have shown that disturbed lipid metabolism, increased oxidative stress, and inflammation are associated with the occurrence and development of CHD.37,38 TLR4 plays a key role in facilitating the inflammatory response during heart disease. The expression of TLR4 and NF-κB was increased in the heart of MI mice.39 Treatment with TLR4 inhibitor significantly reduced the size of the infarct area and improved cardiac https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4044 Powered by TCPDF (www.tcpdf.org) function in MI mice.40 Furthermore, inhibition of TLR4/NF-κB pathway could regulate lipid peroxidation and inflammasome activation in high-fat diet-fed golden hamsters.41 HIF-1α has been shown to play an important protective role in the pathophysiological progress of ischemic heart disease.42 HIF-1α deficiency may result in congenital heart defects in human.43 The PI3K/AKT pathway is important for regulating HIF-1α protein levels to hypoxia, Phosphorylated AKT could regulate HIF-1α activity.44 In the present study, we demonstrated that QSHT could improve cardiac function in CHD mice. To explore the mechanism of QSHT in the treatment of CHD, we compared the difference in protein expression of key targets in mouse hearts by Western blot and immunohistochemistry. The experimental results showed that compared to the sham group, the expression of TLR4, NF-κB, p-AKT and HIF-1α was significantly increased in the model group, while the expression of those proteins was significantly reduced in the QSHT group. Additionally, molecular docking analysis also demonstrated that AKT1, HIF-1α, GSK-3β, TLR4 and NF-κB are might be crucial targets for QSHT treating CHD. These results are in agreement with the predicted results of network pharmacology. To our knowledge, this research is the first to study the efficacy and mechanism of QSHT in the treatment of CHD. By integrating network pharmacology and experimental verification, we demonstrated that QSHT could improve cardiac function and attenuate cardiac fibrosis by regulating TLR4/NF-κB and AKT/HIF-1α signaling Drug Design, Development and Therapy 2024:18 https://doi.org/10.2147/DDDT.S463054 DovePress 4045 Powered by TCPDF (www.tcpdf.org) pathway after MI in ApoE−/− mice, suggesting its utility in the treatment of CHD. However, the key active ingredients and more detailed mechanism of QSHT in the treatment of CHD still need to be studied in further detail in future research.
Limitation
In this study, we only demonstrated that the effect and mechanism of QSHT decoction in the treatment of CHD, but not the metabolite isolation from the QSHT decoction. In future, we need to verify the effect of metabolites by vitro experiments, and compare whether those metabolites can become novel therapeutics with a better effect than
QSHT.
Ethics Statement
This study is involving human data from public database OMIM and Genecards. Due to OMIM and Genecards databases are publicly accessible and users can freely download relevant data for research and publish related articles, the Ethics committee of Guangdong Hospital of Traditional Chinese Medicine confirms that the need for ethics approval for the aspect of this study involving public database has been waived. https://doi.org/10.2147/DDDT.S463054 DovePress Drug Design, Development and Therapy 2024:18 4046 Powered by TCPDF (www.tcpdf.org)
Funding
The present work was supported by Guangzhou Basic and Applied Basic Research Foundation (Grant Nos. 202201020348, 202201020488, and 2023A03J0742), National Natural Science Foundation of China (Grant No. 82074369), the Specific Fund of State Key Laboratory of Dampness Syndrome of Chinese Medicine (Grant Nos. SZ2021ZZ46, SZ2021ZZ12, SZ2021ZZ26, SZ2021ZZ33, SZ2023ZZ13 and SZ2022KF23), and the Traditional Chinese Medicine Bureau of Guangdong Province (Grant No. 20225019), Natural Science Foundation of Guangdong Province (Nos. 2022A1515010103, and 2023B1212060063).
Disclosure
The authors report no conflicts of interest in this work.
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