The atherosclerosis-ameliorating effects and molecular mechanisms of BuYangHuanWu decoction

Biomedicine & Pharmacotherapy 123 (2020) 109664

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The atherosclerosis-ameliorating effects and molecular mechanisms of BuYangHuanWu decoction

T

Bo Liua,b,c,d,1, Zhenyan Songa,*,1, Jingping Yua, Ping Lia, Yuan Tangd, Jinwen Gea,* a

Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases, College of Integrated Traditional Chinese and Western Medicine, Hunan University of Chinese Medicine, China b Research Institute of Zhong Nan Grain and Oil Foods, China c Hunan Grain Group Co., Ltd, China d College of Pharmacy, Hunan University of Chinese Medicine, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Atherosclerosis BuYangHuanWu decoction Network pharmacology NF-κB Inflammation

Atherosclerosis (AS) is one of the leading causes of cardiovascular disease and has a high rate of morbidity and mortality. Traditional Chinese Medicine (TCM) supplied many therapies for AS treatment for centuries. Among these treatments, BuYangHuanWu decoction (BYHWD) is a classic prescription. In this study, we analyzed the mechanisms of BYHWD in the treatment of AS by using a network pharmacology method. Our results revealed the mechanisms of BYHWD in treating AS, which is highly related to inflammation and apoptosis pathways, moreover, the genes including IL1β, TGFB1, TNF, IL6, NFκB1 are proved to be the key pharmacological targets for the treatment of AS. Furthermore, an AS rat model was established and the rats in the treatment group received different amounts of BYHWD. Serum lipid levels (TC/TG/HDL-C/LDL-C) and tissue oxidative stress levels (SOD, GSH-Px, CAT and MDA) were ameliorated in a dose-dependent manner. The morphology of the aortic intima in the BYHWD-treated groups was improved. Real-time PCR and Western blot analysis results indicated that inflammatory cytokines were suppressed and that the NF-κB signaling pathway was blocked by BYHWD. All of this evidence suggested that BYHWD is an ideal prescription for treating AS.

1. Introduction Cardiovascular disease (CVD) is a universal problem threatening human health. Atherosclerosis (AS) is one of the leading causes of CVD and results in a high rate of morbidity and mortality. It has been recognized that dyslipidemia participates in the initiation of atherosclerosis development [1]. First, lipoproteins accumulate in dysfunctional endothelial cell, which causes the initial lipid deposition and inflammatory response in the intima [2,3]. Second, additional lipids are deposited in the intima, which attracts apoptotic or necrotic cell debris, and all of these deposits are within the vessel wall [4,5]. Dyslipidemia promotes oxidative stress and the inflammatory response in plaques, such as the secretion of inflammatory cytokines and chemokines [6]. Statins have become a ubiquitous part of the clinical treatment for atherosclerotic disease. However, many patients experience the side effects of statins; furthermore, statins should be used for long time and withdrawal from these drugs may lead to increased cardiovascular or neurovascular events [7]. Consequently, there is a great clinical need

for therapies that can reduce or even reverse the progression of atherosclerotic vascular disease. Traditional Chinese medicine (TCM) supplied many therapies to treat atherosclerosis for centuries. Among these therapies, a classical therapy called BuYangHuanWu decoction (BYHWD) is widely used. BYHWD was first recorded in an ancient Chinese medical book YiLinGaiCuo (Correction on Errors in Medical Classics) and is a classic traditional Chinese herbal prescription that has been commonly used for the treatment of cardiovascular disease and ischemic stroke for more than two centuries in China. The decoction is composed of Radix Astragali Membranacei (RAM), Radix Paeoniae Rubra (RPR), RhizomaChuanxiong (RC), Radix Angelicae Sinensis (RAS), Lumbricus (L), Semen Pruni Persicae (SPP), and Flos Carthami Tinctorii (FCT). Studies have found that BYHWD can slow heart rate, reduce blood pressure [8], expand coronary arteries, ameliorate ischemic stroke [9], reduce myocardial oxygen consumption [10], improve cerebral ischemia/reperfusion injury [11], and ameliorate atherosclerosis [12]. As a newly emerging field of pharmacology, network pharmacology

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Corresponding authors. E-mail addresses: [email protected] (Z. Song), [email protected] (J. Ge). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109664 Received 27 August 2019; Received in revised form 1 November 2019; Accepted 7 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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targets, a GO online analysis tool called DAVID (https://david.ncifcrf. gov/) was performed, and the GO enrichment information was analyzed with P-value < 0.5 and FDR < 0.5 [17]. Based on pathway categorization information from the public database KEGG (https://www. genome.jp/kegg/), the online pathway analysis tool Omicshare (http:// www.omicshare.com) was run to enrich information with P-value＜0.5 and FDR＜0.05 as a parameter by pathway [18]. Finally, the targetspathway (T-P) network diagram was set up by Cytoscape-v3.6.1.

emphasizes the concept of a "multicomponent, multitarget therapeutic network" and highlights the overall thoughts of TCM [13]. Network pharmacology provides a new idea for studying the multitargeted mechanism of the treatment of complex diseases, including atherosclerosis, with TCM [14]. In this study, we analyzed the mechanism of BYHWD in the treatment of atherosclerosis by using a network pharmacology method. First, the effective components of BYHWD, the traditional Chinese medicine prescription, were identified from the TCMSP database. Second, network pharmacology was used to analyze the interactions in the compounds-targets-diseases network; bioinformatic analysis was used to elucidate the multi-target multi-pathway mechanism of BYHWD in the prevention and treatment of atherosclerosis. Finally, molecular pharmacology was performed to verify the signaling pathways elucidated by network pharmacology.

2.6. TNF signaling pathway

2. Methods

To further elucidate the mechanism underlying the treatment of atherosclerosis with BYHWD, the selected target genes were added to the TNF signaling pathway (KEGG number: map04668) to identify genes associated with the pathway. Finally, a diagram was drawn by the KEGG parser, a plug-in for Cytoscape.

2.1. Establishment of a drug database

2.7. BYHWD preparation

All of the information regarding the compounds in BYHWD were obtained from the online public database Traditional Chinese Medicine Systems Pharmacology (TCMSP) (http://lsp.nwu.edu.cn/tcmsp.php) and TCM Database@Taiwan (http://tcm.cmu.edu.tw/). According to the relevant parameters of the pharmacokinetic properties of the compound (AMED), the active compounds of BYHWD were screened based on OB ≥ 30 % and DL ≥ 0.18 [15], and then the active compounds omitted from the supplementary part were excavated by reading the literature. All of the compound information was standardized according to "Canonical SMILES", which is based on the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).

The decoction formula was prepared according to Dou B [19] and Zheng XW [20]; BYHWD is composed of RAM, RPR, RC, RAS, L,SPP, and FCT at a ratio of 120:6:4.5:3:3:3:3 (dry weight). All herbs were purchased from the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine (Changsha, China). The mixture was boiled in distilled water at 100 °C for 1 h twice. The combined filtrate was concentrated to 2 g/ml (equivalent to the dry weight of the raw materials). 2.8. BYHWD quality control 2.8.1. Chromatographic and mass spectrometry conditions The analysis were performed on an Agilent 1260 Infinity HPLC coupled to an Agilent 6460 Tripe-Quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface (Agilent Technologies, USA). Chromatographic separations were performed on an Agilent Poroshell 120 EC-C18 (100 × 2.1 mm, 2.7 μm particles) (Agilent Technologies, USA). The column temperature was maintained at 35℃. The mobile phase was 0.1 % ammonium acetate solution (solvent A) and acetonitrile (solvent B) with a run time of 30 min in negative mode. The gradient program for negative mode was as follows: 0−15 min, 20–90 % B; 15−24 min, remain 90 % B, 24.01−30 min, returned to 20 % B for reequilibration. The mobile phase was delivered at a flow rate of 0.4 ml/min. The injection volumes for both samples and standard solutions were 5 μl. The mass spectrometer was operated in negative ESI mode. The drying gas temperature was 350 ℃ and the flow rate was 12 L/min. The nebulizer pressure was 35 psi (0.35 MPa), the capillary voltage was 4000 V for negative mode. The mass spectrometer was operated in a multiple reaction monitoring (MRM) mode. The scan durations were set according to the retention time of each analyte so as to shorten the cycle time and increase sensitivity. Instrument control, data acquisition and quantification were performed by MassHunter Workstation software B. 04. 00 (Agilent Technologies, USA).

2.2. Target prediction of active compounds Active compound prediction was mainly performed through database screening, searching the literature and target prediction based on ligand structure characteristics. First, we obtained the target information of active compounds from TCMSP. Second, through the website SEA (http://sea.bkslab.org) and the Binding Database (http://www. bindingdb.org) and based on similarity predictions of chemical structures, we predicted the targets of the active compounds. Finally, UniProt (http://www.UniProt.org/) was used to standardize all the target information. 2.3. Screening of genes related to atherosclerosis Disease-related gene screening was performed using the following free public databases: TTD (https://db.idrblab.org/ttd/), Drugbank (https://www.drugbank.ca/), and DisGeNET (http://www.disgenet. org/web/DisGeNET/menu/home). We screened disease-related genes with the keyword "atherosclerosis". The target sites for atherosclerosis were mapped to the related target networks predicted by BYHWD, and the overlapping targets in the two networks were considered to be the atherosclerosis treatment targets for the Chinese herbal components of BYHWD.

2.8.2. Preparation of calibration standard and BYHWD test solution 1 mg reference standards (Ferulic Acid, Amygdalin, Paeoniflorin and Astragaloside IV) were dissolved in 1 ml methanol to prepare individual stock solutions. These stock solutions were mixed and then serially diluted to obtain calibration standard stock solutions. 0.5 g samples (accurate to 0.0001 g) were accurately weighed and dissolved in 5.0 ml 50 % methanol, and then centrifuged at 10,000 r/min for 5 min, the supernatant was absorbed to prepare the BYHWD test stock solution. The BYHWD test stock solution was diluted 10 times and 100 times, respectively, and then the diluents were filtered by a 0.25um filter membrane to be the test solutions. All solutions were sealed and stored at -20℃ until use.

2.4. Visualization network construction The relationships between the active compounds of BYHWD and the predicted targets, as well as the relationships among the active compounds of BYHWD were visualized by the compound-target-disease network(C-T-D) built by the software Cytoscape-v3.6.1 [16]. 2.5. Gene ontology analysis and pathway analysis To understand the biological functions and signaling pathways of the TCM ingredients in BYHWD in the treatment of atherosclerotic 2

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from the aortic tissue with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd., cat. P0028, Shanghai, China), and total protein extraction was performed according to the instructions of a RIPA kit (Beyotime Biotechnology Co., Ltd., cat. P0013B, Shanghai, China). The protein concentration was measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific Technology (China) Co., Ltd., cat. 23227, Shanghai, China). The proteins were denatured by boiling with SDS loading buffer, separated by SDS-PAGE and transferred to 0.45 μm PVDF membranes (Millipore Sigma Inc., IPVH00010, Billerica,USA). The membranes were then blocked with 5 % bovine serum albumin (BSA) (Sigma-Aldrich, Inc., cat. V900933, Billerica, USA) at room temperature for 1 h and incubated with primary antibody (1:1000) overnight at 4 °C. The membranes were incubated with secondary antibody (1:10,000) at room temperature for 2 h. Then, the membranes were incubated with ECL Plus™ Western Blotting Substrate (Thermo Scientific Technology (China) Co.Ltd., cat. 32132, Shanghai, China), and the signals were assessed by an imaging system (ChemiDoc™ XRS+, Bio-Rad, California, USA). The primary antibodies against NF-κB p65(cat. bs-0465R) and Histone H1t (cat. bs-1413R) and β-actin (cat. bs-0061R) were purchased from Bioss BiotechnologyCo., Ltd. (Beijing, China). The goat anti-rabbit IgG antibody (cat. AP132 P) was purchased from Sigma-Aldrich, Inc. (Billerica, USA).

2.9. Animal treatment The Animal Experimental Ethics Committee of Hunan University of Chinese Medicine approved all experimental procedures, which meet the standards and guidelines set forth in the Guidelines for Animal Experiments of the Chinese Medical Ethics Committee. Fifty adult male Sprague-Dawley (SD) rats (220 ± 30 g) were purchased from the Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). The experimental animal production license was No. SCXK (Xiang) 20160002. The animals were housed in a specific pathogen-free (SPF) animal room on a 12 h circadian cycle. The animals were reared at 45–55% relative humidity and 22 ± 2 °C with ample food and water. The AS rat model was established by a combination of a high-fat diet (typical diet supplemented with 40 % saturated fatty oil and 5 % cholesterol, Hunan SJA Laboratory Animal Co., Ltd, Changsha, China) for 6 weeks and a single intraperitoneal injection of vitamin D3 (600,000 IU/kg). After 7 days of adaptation, the rats were divided into the following 5 groups (n = 10 each): control group (Con); atherosclerotic (model) group (AS); high dose BYHWD treatment group (H-BYHWD, BYHWD: 16 g/kg.bw); middle dose BYHWD treatment group (M-BYHWD, BYHWD: 8 g/ kg.bw); and low dose BYHWD treatment group (L-BYHWD, BYHWD: 4 g/kg.bw). The rats in the control group were fed a sustaining fodder, while those in the model group were fed a high-fat diet. Eight weeks later, the level of blood lipids in the serum and the pathological changes in the aorta were detected to determine whether the model was successful. All treatments were delivered by oral gavage. The dose of BYHWD used in this study was based on previous reports of its effectiveness in animals with ischemia/reperfusion (I/R) injury [20]. At the end of the experimental period, the rats were fasted overnight, anesthetized with pentobarbital sodium, and subsequently sacrificed by cervical dislocation. The serum obtained after centrifugation (1500×g for 10 min at 4 °C) was used to estimate various serum biochemical assays. The aortic tissues were excised from the rats, immediately frozen in liquid nitrogen, and stored at −80 °C for further histopathological and Western blot analyses.

2.13. Real-time PCR Total RNA from tissue and cells was isolated using Invitrogen™ TRIzol reagent (cat. 15596-026, Thermo Scientific Technology (China) Co.Ltd., China). Reverse transcription was performed using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser (cat. RR047A, Takara, Japan). SYBR Premix Ex Taq II (cat. RR820 L, Takara, Japan) and CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, California, USA) were used for Real-time PCR according to manufacturer's instructions. Real-time PCR primers were designed by Primer Primer5 software. Relative expression was calculated using the 2−ΔΔCT method [21] and normalized to the expression of β-actin. The primer sequences were as follows: β-actin forward, 5′-TCAGCAAGCAGGAGTACGATG-3′; β-actin reverse, 5′-GTGTAAAACGCAGCTCAGTAACA-3′; TNF-α forward, 5′-CCCCAAAGGGATGAGAAGTT-3′; TNF-α reverse, 5′-CACTTGGTGGTTTGCTACGA-3′; IL-1β forward, 5′-GGATGAGGACATGAGCACCT-3′; IL-1β reverse, 5′-AGCTCATATGGGTCCGACAG-3′; IL-6 forward, 5′-CCGGAGAGGAGACTTCACAG-3′; IL-6 reverse, 5′-CAGAATTGCCATTGCACAAC-3′; iNOS forward, 5′-CAGCTGGGCTGTACAAACCTT-3′; and iNOS reverse, 5′-CATTGGAAGTGAAGCGTTTCG-3′.

2.10. Biomarker measurements The serum was separated from whole blood according to the instructions in the commercial detection kit (Wuhan Cusabio Biological Technology Co. Ltd, Wuhan, China). A colorimetric analysis method was used to analyze the amount of total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) in the serum. Well-ground 10 % homogenate of aortic tissue was centrifuged at 3000 r/min for 15 min at 4 °C to prepare a supernatant for further determination of biochemical indicators. Super oxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) level were all determined by a commercial detection kit (Wuhan Cusabio Biological Technology Co. Ltd, Wuhan, China).

2.14. Statistical analysis All results in this study were displayed as means ± standard deviation (S.D.) from at least three independent repeats of the experiment, were analyzed using Prism Graphpad 6.0 software (GraphPad Software, San Diego, CA, USA). Comparisons of two or more data sets were analyzed using Student’s t test or one-way analysis of variance (ANOVA) with post hoc Tukey's tests. Significance between groups was considered as *P < 0.05 and **P < 0.01.

2.11. Histopathological morphology of rat aorta The aortas were fixed in 4 % formaldehyde for 24 h, embedded in paraffin, and sectioned at 4 μm on a microtome (Leica RM2016, Wetzlar, Germany), and the sections were stained with hematoxylin and eosin (H&E, Cat. No. BA4025, Baso Diagnostics Inc., Guangdong, China), according to the manufacturer’s protocol. Pictures were taken with a Matic biological microscope (M150, Motic Software Engineering Co., Ltd., Xiamen, China) and Motic Image 2000 (Motic Software Engineering Co., Ltd., version 1.3, Xiamen, China).

3. Results 3.1. Active compound screening Using the established filter conditions, OB ≥ 30 % and DL ≥ 0.18, 91 active compounds were identified in seven traditional Chinese medicines in BYHWD from the TCMSP and TCM Database@Taiwan databases (six compounds were discarded because the target information was not available in the databases). Additionally, nine active

2.12. Western blotting One hundred milligrams of aortic tissue was fragmented using liquid nitrogen, and the nuclear and cytoplasmic proteins were separated 3

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Fig. 1. Visualization network construction for the treatment of atherosclerosis with BYHWD was established by network pharmacology. a: Herb-Compound (H-C) network: The 95 active compounds in BYHWD were derived from 7 herbs, and the purple nodes represent herbs, the pink nodes represent compounds. b: The targets related to atherosclerosis in the targets predicted by BYHWD were selected, and Red represents incipient, Green represents moderate. c: Compound-Target-Disease (C-T-D) network: A compound-target-atherosclerosis network and the purple nodes represent targets, the yellow nodes represent targets, and the lines between the nodes represent the interactions between the compound and the target.

4

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biological processes among the potential targets of BYHWD, showing that the mechanism of atherosclerosis treatment by BYHWD may be closely related to the inflammation and apoptosis pathways and that the above genes may be important targets.

medical compounds were identified from articles [22–27]. Finally, 95 active compounds in BYHWD were identified and used for follow-up analysis (as shown in Fig. 1a and Table S1). 3.2. Predicted targets of the active compounds

3.5. KEGG pathway enrichment analysis As described in the Materials and Methods, section 2.2, a total of 879 predicted targets (as shown in Table S2) for the 95 active compounds were mapped to 1268 gene networks related to atherosclerosis in three databases (TTD, Drugbank and DisGeNET). Additionally, 265 potential targets were identified from 879 targets (as shown in Fig. 1b and Table S3) as the targets of the Chinese herbal ingredients in BYHWD for treating atherosclerosis.

A total of 265 BYHWD predicted targets associated with atherosclerosis imported into the Omicshare online analysis software, and the enrichment statistics of the target genes relative to background genes were obtained (as shown in Table S5). Additionally, we selected the top 20 most enriched entries to make bubble charts (as shown in Fig. 2c). The results showed that many target points were enriched in the PI3KAkt signaling pathway, MAPK signaling pathway, and TNF signaling pathway. Most of the enriched signaling pathways were associated with inflammation and apoptosis, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, TNF signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, etc., indicating that BYHWD may mainly treat atherosclerosis by regulating inflammatory and apoptosis signaling pathways.

3.3. Constructing networks The C-T-D network diagram was constructed based on the interactions among the 7 herbs, 95 active compounds and 265 targets associated with BYHWD (as shown in Fig. 1c). The network consists of 350 nodes (representing active compounds and targets) and 2,080 edges (representing the interaction between the active compound and the target); each node is associated with 11.86 adjacent nodes on average. The degree in the network refers to the number of strips of edges associated with the node, and the higher the degree is, the more targets there are [28]. Of the 95 active compounds, 31 of them have more than 20 targets; for example, quercetagetin (M88, degree = 114), 2, 3-didehydro GA77(M9, degree = 90), GA122-isolactone (M50, degree = 90), (1S,2S,4R)-trans-2-hydroxy-1,8-cineole-B- D-glucopyranoside (M2, degree = 89), and GA121-isolactone (M49, degree = 87) had a high degree values and were located at central positions in the network. Thus, these components could be the main effective substances in BYHWD. Additionally, 213 of the 265 potential targets were connected to at least 2 compounds, such as PTGS2 (degree = 44), ESR1 (degree = 35), PTGS1 (degree = 28), IL2 (degree = 26), ESR2 (degree = 25), DPP4 (degree = 24), NR1H4 (degree = 22), PPARG (degree = 22), SHBG (degree = 22), and NOS2 (degree = 21). These targets had a high degree in the network; moreover, the higher the degree of the target is, the more biological functions in the body it participates in and the more biological importance it has. Thus, these targets could be the main target genes on which BYHWD acts in the treatment of atherosclerosis.

3.6. TNF signaling pathway From the results of the network pharmacology analysis, we found that the inflammatory signaling pathway may be an important signaling pathway for the treatment of atherosclerosis by BYHWD, and the TNF signaling pathway was the most relevance pathway (minimum p-value number, Fig. 2c).We mapped 265 potential targets to this pathway in the KEGG database (KEGG number：map04668), revealing a total of 27 protein targets in the TNF signaling pathway network (as shown in Fig. 3). 3.7. Multi-components determination of BYHWD by LC–MS/MS A liquid chromatography- tandem mass spectrometry (LC–MS/MS) method was established to determine the content of 4 quality standards chemicals in BYHWD water extract. The content of all four chemicals were detected in BYHWD (as shown in Fig. 4a). Chromatograms and mass spectrograms of the four standards in BYHWD were uploaded to the supplementary data (Supplementary S6). BYHWD sample chromatogram results indicated that the retention time of Ferulic acid, amygdalin, paeonflorin and astragaloside IV were separately 0.889 min, 2.494 min, 5.562 min and 10.263 min. Quantitative results of 4 standards in BYHWD were obtained by mass spectrometry (Fig. 4b), which is separately 1.292 mg/g, s 4.724 mg/g, 12.440 mg/g and 0.154 mg/g.

3.4. Gene ontology analysis GO is a bioinformatics analysis tool that defines the input genes by describing the function of the gene and the relationship between these concepts. It includes the following three parts: cellular component (CC), molecular function (MF), and biological process (BP). Among them, BP can best reflect changes in biological function within the body [29].The GO enrichment analysis of 265 potential targets related to atherosclerosis (as shown in Fig. 2a) showed that the main biological processes enriched in the GO second class were immune system process, metabolic process, signaling and biological regulation. To further understand the changes in these biological functions, we selected the top 20 biological processes in the third GO category to make bubble charts (as shown in Fig. 2b). The results showed that the effect of BYHWD on atherosclerosis was mainly related to inflammatory response, hypoxia response, positive regulation of nitric oxide biosynthetic process, apoptotic process, positive regulation of NF-κB transcription factor activity, positive regulation of MAP kinase activity, glucose and lipid metabolic processes and so on. Among these processes, 47 genes were enriched in the biological process of "inflammatory response", 33 genes were enriched in the biological process "hypoxia response", and 31 genes were enriched in "apoptotic process"(as shown in Table S4). PPARD, SOD2, PTGS2, VEGFA, TLR2, IL1β, TGFB1, TNF, IL6, NFκB1, and CCL2 were the most associated genes involved in these

3.8. Effect of BYHWD on serum lipids Compared to the blood lipid profile of the control group, the AS model group had significantly increased serum levels of TC, TG, HDL-C and LDL-C (as shown in Fig. 4a-d). The TC content of the model group was 7.52 ± 0.15, which was significantly higher than that of the control group at 1.76 ± 0.11 (P < 0.01). Different concentrations of BYHWD differentially decreased TC levels, namely, 6.34 ± 0.11, 5.70 ± 0.27, and 2.90 ± 0.32 in the low, middle, and high dose groups, respectively. The TG content in the control group was 1.20 ± 0.07, while the TG content in the model group was 1.84 ± 0.11, a much higher level than in the control group. Compared with the model group, the L-BYHWD group had a slightly decreased TG level, but the difference was not significant (P＞0.05); M-BYHWD and H-BYHWD were 1.38 ± 0.08 (P < 0.05) and 1.18 ± 0.08 (P < 0.01), respectively, and were both significantly lower than the model group. The LDL-C content in the control group was 0.44 ± 0.05, while that in the model group was 1.78 ± 0.08. Compared with the model group, the L-BYHWD group had decreased LDL-C content (1.26 ± 0.09, P < 0.05). Moreover, the LDL-C content of the M5

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Fig. 2. GO enrichment analysis and KEGG pathway enrichment analysis. a: GO second class enrichment analysis of 265 potential targets related to atherosclerosis, xaxis represent the GO terms: Red represents the Biological Process (BP) terms, Green represents the Cellular Component (CC) terms, and Blue represents the Molecular Function (MF) terms. y-axis shows the gene number enriched in the GO terms. b: Top 20 biological processes (BP) of gene ontology (GO) terms sorted by Pvalue < 0.01. Counts of genes and P-value related to each BP terms are shown. The y-axis represents BP terms, and the x-axis shows counts of genes were annotated to the BP terms. c: Top 20 of pathway enrichment. x-axis shows the gene number in the given gene set that were annotated to the certain pathways, y-axis represents B level classification of pathways.

BYHWD and H-BYHWD groups were 1.06 ± 0.11 and 0.78 ± 0.08, respectively, both values significantly lower than the LDL-C content of the model group (P < 0.01). BYHWD treatment sharply decreased the HDL-C level, to only 0.28 ± 0.08, while the control group was 1.82 ± 0.08. These values were reversed by BYHWD; the high, medium, and low dose groups were 0.54 ± 0.05, 1.16 ± 0.11, 1.26 ± 0.09, respectively (P < 0.01).

SOD, GSH-Px, and CAT and the concentration of MDA. HFD significantly reduced the antioxidant capacity of the rats, as evidenced by a sharp increase in MDA levels and a decrease in SOD, CAT and GSHP × . BYHWD was determined to decrease oxidative stress in a dosedependent manner (as shown in Fig. 4e-h).

3.9. Effects of BYHWD on SOD, GSH-Px, CAT, and MDA

As shown in Fig. 5a-c, the aorta intima of the control group was intact with typical, healthy morphology, and the endothelial cells were arranged in an orderly manner. In contrast, in the model group, the

3.10. Effects of BYHWD on histopathology

The oxidative stress level of tissues is indicated by the activity of 6

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Fig. 3. TNF signaling pathway enrichment analysis. 265 target genes were mapped into the TNF pathway (map04668) from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to confirm the possible effect pathway of BYHWD on atherosclerosis.

the regulatory mechanism of complex networks, which appropriately represents the overall diagnosis and treatment concepts of TCM [31]. This study demonstrated that BYHWD, a well-known traditional Chinese herbal prescription, possessed the ability to prevent AS. In recent years, some studies on the treatment of AS with Chinese herbs and prescriptions have been reported using network pharmacological methods. Li et al. found that flavonoids and saponins were the main active compounds of tongsaimai tablets in the treatment of AS and predicted that there were 22 targets of these compounds, which were closely related to inflammation regulation, plaque stabilization, vascular endothelial cell protection, and blood lipid regulation, by using molecular docking and molecular target protein network analysis methods [32]. Lee et al. identified 21 compounds and 57 predicted targets in yijin-tang by network pharmacological analysis, revealing that the main pathways were related to hyperlipidemia and atherosclerosis [15]. Moreover, research on the therapeutic effect of BYHWD on cerebral infarction using network pharmacology has been reported [33]. In this study, we explored the relationship between the active compounds of BYHWD and atherosclerotic therapeutic targets from the perspectives of pharmacokinetics, gene ontology, network analysis and pathway enrichment analysis based on network pharmacology. We identified 95 active compounds from seven traditional Chinese medicines in BYHWD. A total of 879 predicted targets of the 95 active compounds were mapped to 1268 gene networks related to AS in three databases (Fig. 7). In addition, a series of important signaling pathways were identified, including all inflammatory-associated pathways, such as the PI3KAkt signaling pathway, MAPK signaling pathway and NF-κB signaling pathway. Finally, we conducted animal experiments to verify the antiinflammatory effect of BYHWD based on the predicted results of network pharmacology. The main effective components of BYHWD have been reported to exert anti-inflammatory and anti-atherogenic effects. Astragaloside IV is the main active component of Radix Astragali Membranacei (RAM), Astragaloside IV was determined as an anti-atherosclerosis chemical

aortic endothelial cells proliferated and the foam cells accumulated, and the aortic intimal media was significantly thicker in the model group than in the control group. The aortic intima morphology of the LBYHWD group was improved, but this group still exhibited proliferated endothelial cells. In addition, the morphology of M-BYHWD and HBYHWD were significantly improved (as shown in Fig. 5d-e). The morphology of the M-BYHWD and H-BYHWD groups was normal, and the endothelial cells were regular. 3.11. Effects of BYHWD on inflammatory cytokines and signaling pathways To explore the anti-inflammatory mechanism of BYHWD, the mRNA expression levels of cytokines were analyzed by using real-time PCR. Compared to the control group, the AS model group had dramatically increased mRNA expressions of inflammatory cytokines such as TNF-α, IL-1β, IL-6 and iNOS. The aberrant mRNA expression of the cytokines was inhibited by BYHWD (as shown in Fig. 6a-d). Furthermore, Western blot analysis revealed that NF-κB P65 was highly expressed in the nucleus but was expressed at low levels in the cytoplasm of the model group. BYHWD significantly increased the cytoplasmic content and reduced the nuclear content. These results demonstrated that BYHWD could reduce the inflammation induced by HFD by blocking the NF-κB signaling pathway (as shown in Fig. 6e-f). 4. Discussion As a complex system with multiple components and multiple targets, traditional Chinese medicine and the mechanism of multicomponent therapeutics are difficult to study from the perspective of modern medicine. In recent years, network pharmacology has become a new pharmacological field that integrates pharmacology, biochemistry, genomics and bioinformatics [30]. Network pharmacology provides a new powerful tool for visualizing and analyzing complex interactive data of herbs, compounds, targets and diseases based on computer modeling analysis and target prediction. It comprehensively analyzes 7

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Fig. 4. LC–MS/MS chromatogram and mass spectrometry of four candidate ingredients. a: HPLC chromatogram of four candidate ingredients. (1) Ferulic acid, (2) Amygdalin, (3) Paeoniflorin, (4) Astragaloside IV. b: Retention time, chromatogram and mass spectrogram of Ferulic acid, Amygdalin, Paeoniflorin and Astragaloside IV.

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Fig. 5. BYHWD decreased the serum lipid content and tissue oxidantive stress. The TC, TG and LDL-C in model group were much higher than the control group, while the HDL-C was lower than the control group. The tissue oxidantive stress was evaluated by SOD, GSH-Px, CAT and MDA, the SOD, GSH-Px and CAT were obviously suppresed in modle group while the MDA was significantly higher than the control group. BYHWD could reverse these trends in a dose dependent manner. * represent p < 0.5 and ** represent p < 0.1.

inhibiting the TLR4 / MyD88 / NF-κB signal pathways [35]. Amygdalin is the key medical component of another BYHWD ingredient called Semen Pruni Persicae. Amygdalin ameliorated atherosclerosis by the regulation of regulatory T cells (Tregs), which was proved to be pivotal in the regulation of T cell‑mediated immune responses in atherosclerosis in apolipoprotein E-deficient (ApoE/‑) mice. The results also indicated that amygdalin regulated the formation of atherosclerosis and

which suppressed the expression of PPAR-γ and inflammation-associated cytokines including TNF-α, IL-18, IL-6, ICAM-1 and VCAM-1, Furthermore, the inflammtory associated signal pathways NF-κB/PPARγand p38 MAPK were also blocked by Astragaloside IV [34]. Paeonflorin is the main active component and also the only quality evaluation standard (Chinese pharmacopoeia) of Radix Paeoniae Rubra (RPR). Paeonflorin was determined as an effective drug in treating AS through 9

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Fig. 6. The treating effects of BYHWD on blood vessel. The model group aortic endothelial cells proliferated, foam cells accumulated and the aortic intimal media was significantly thicker than the aortic intima in control group. Different content of BYHWD decreased the foam cells accumulation and proliferation of aortic endothelial cells. High BYHWD group could significantly reverse the high fat diet induced vascular disorders.

amygdalin, paeoniflorin and ferulic acid were separately 1.095 mg/g, 2.10 mg/g and 0.091 mg/g [39]. Which are far more lower than our results. NF-κB activation is essential for the regulation of key cytokines involved in the inflammatory processes that play critical roles in atherogenesis [40] and macrophages [41]. Release of proinflammatory cytokines, including TNF-ɑ, IL-1β, IL-6, and mediated cytoplasmic NF-κB activation and shift to the nucleus. Activation of the NF-κB signaling pathway further stimulates the transcription and expression of pro-inflammatory cytokines and other markers, thereby exacerbating the inflammatory response [42,43]. One of the highlights of this study is that we confirmed that BYHWD inhibited the nuclear translocation of NFκB. Western blot analysis indicated the downregulation of NF-κB p65 in

stabilized the plaque by suppressing inflammatory cytokines including IL-1β, IL-6 and TNF-α [36,37]. Ferulic acid is the quality maker of RhizomaChuanxiong and Radix Angelicae Sinensis. It is determined to exhibit anti-oxidant and anti-inflammatory preperties, besides, it was proved to decrease the level of oxidized low density lipoprotein (oxLDL), which is a hallmark of early atherosclerosis and results in foam cell and plaque formation in the arterial wall [38]. In China Pharmacopoeia (2015) (Ch. P. (2015)), Ferulic Acid, Amygdalin, Paeoniflorin and Astragaloside IV were chosen as quantitative markers for the quality control of BYHWD. In this study, the water extract of BYHWD was quality controlled by LC–MS/MS, and four main effective components were detected. Before our research, Wang et al. detected three of the standards by HPLC, they reported that the content of 10

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Fig. 7. BYHWD decreased the mRNA expression of inflammatory cytokines and the protein expression of the NF-κB signal pathway. a-d: Compared with control group, the mRNA of TNF-α, IL-1β, IL-6 and iNOS in model group increased dramatically. BYHWD inhibit this trend in a dose-dependent manner. e-f: The nucleus NFκB p65 was dramatically increased; besides, the cytoplasm p65 was significantly drecresed. BYHWD dose-dependently reversed this trend. * represent p < 0.5 and ** represent p < 0.1.

Furthermore, GSH-Px and CAT, were also commonly used as biomarkers reflecting the production of free radicals and can prevent oxidative damage cooperatively at different sites during ROS metabolic pathways [46]. In the current study, serum GSH-Px, SOD and CAT activities decreased significantly after the HFD, and this phenomenon was reversed by BYHWD. The results were in accordance with those reported in previous articles [47]. In summary, this study not only comprehensively analyzed relevant BYHWD compounds and found potential targets by using systematic pharmacological methods but also explained the mechanism of atherosclerosis treatment by BYHWD from the perspective of anti-inflammatory effects with animal experiments. These results clearly elucidate the effectiveness and the mechanisms of BYHWD. More importantly, these results provide an example for future studies of the treatment of complex diseases with TCM.

the cytoplasm and its upregulation in the nucleus in the AS group; however, BYHWD administration reversed this effect. The above effects clarified that BYHWD significantly suppresses NF-κB transcription and reduce the expression of pro-inflammatory cytokines, the principle event that contributes to the inflammatory reaction. This phenomenon was observed when BYHWD was used to treat other diseases; for example, BYHWD inhibited nuclear factor-κB (NF-κB) activation in the ischemic brain [19]. Beside the pro-inflammatory signaling pathways and expression of cytokine/chemokine, the pathogenesis of atherosclerosis involves activation of increased oxidative stress. Oxidative stress is the imbalance in favor of increased generation of reactive oxygen species (ROS) and/or reduced body’s innate anti-oxidant defense systems. The major antioxidant systems in the vascular wall include SOD, GSH-Px, CAT, and MDA. SOD converts superoxide to hydrogen peroxide which is further degraded by glutathione peroxidases, catalases, and thioredoxins [44]. Excess MDA can oxygenate and modify LDL-C to form MDA-LDL-C, which can cause the degeneration and necrosis of endothelial cells, inflammatory reactions and disordered antioxidant systems [44,45].

Author contributions Yuan Tang, Ping Li and Jingping Yu performed the major 11

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experiments, Zhenyan Song analyzed the data and contributed to the manuscript. Bo Liu and Jinwen Ge supervised the project and the writing of the manuscript. All authors have seen the manuscript and approved to submit to this journal.

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Data availability The data used to support the findings of this study are available from the corresponding author upon request. Declaration of Competing Interest The authors declare no competing interests. Acknowledgments The research is supported by the National Natural Science Foundation of China (Grant No. 81774174); the Postdoctoral Research Foundation of China (Grant No. 2019M652784); the Natural Science Foundation of Hunan Province (Grant No. 2019JJ50441); the Scientific Research Foundation of Hunan Provincial Education Department (Grant No. 18B246; No. 18C0400) and Hunan Administration of Traditional Chinese Medicine Science Foundation (Grant No. 201825). Hunan province traditional Chinese medicine decoction piece standardization and function engineering technology research center open foundation (Grant No, 201806). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109664. References [1] D. Zysset, B. Weber, S. Rihs, J. Brasseit, S. Freigang, C. Riether, Y. Banz, A. Cerwenka, C. Simillion, P. Marques-Vidal, A.F. Ochsenbein, L. Saurer, C. Mueller, TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis, Nat. Commun. 7 (2016) 13151. [2] R. Ross, L. Harker, Hyperlipidemia and atherosclerosis, Science 193 (4258) (1976) 1094–1100. [3] S. Xu, P.J. Little, T. Lan, Y. Huang, K. Le, X. Wu, X. Shen, H. Huang, Y. Cai, F. Tang, H. Wang, P. Liu, Tanshinone II-A attenuates and stabilizes atherosclerotic plaques in apolipoprotein-E knockout mice fed a high cholesterol diet, Arch. Biochem. Biophys. 515 (1–2) (2011) 72–79. [4] D.A. Chistiakov, Y.V. Bobryshev, A.N. Orekhov, Macrophage-mediated cholesterol handling in atherosclerosis, J. Cell. Mol. Med. 20 (1) (2016) 17–28. [5] L. Zheng, T. Wu, C. Zeng, X. Li, X. Li, D. Wen, T. Ji, T. Lan, L. Xing, J. Li, X. He, L. Wang, SAP deficiency mitigated atherosclerotic lesions in ApoE (-/-) mice, Atherosclerosis 244 (2016) 179–187. [6] N. Pothineni, S.K. Karathanasis, Z. Ding, A. Arulandu, K.I. Varughese, J.L. Mehta, LOX-1 in atherosclerosis and myocardial ischemia: biology, genetics, and modulation, J. Am. Coll. Cardiol. 69 (22) (2017) 2759–2768. [7] M. Blanco, F. Nombela, M. Castellanos, M. Rodriguez-Yáñez, M. García-Gil, R. Leira, I. Lizasoain, J. Serena, J. Vivancos, M.A. Moro, A. Dávalos, J. Castillo, Statin treatment withdrawal in ischemic stroke: a controlled randomized study, Neurology 69 (9) (2007) 904–910. [8] H. Chen, H. Song, X. Liu, J. Tian, W. Tang, T. Cao, P. Zhao, C. Zhang, W. Guo, M. Xu, R. Lu, Buyanghuanwu Decoction alleviated pressure overload induced cardiac remodeling by suppressing Tgf-β/Smads and MAPKs signaling activated fibrosis, Biomed. Pharmacother. 95 (2017) 461–468. [9] W.W. Zhang, F. Xu, D. Wang, J. Ye, S.Q. Cai, Buyang Huanwu Decoction ameliorates ischemic stroke by modulating multiple targets with multiple components: in vitro evidences, Chin. J. Nat. Med. 16 (3) (2018) 194–202. [10] J. Shen, Y. Zhu, K. Huang, H. Jiang, C. Shi, X. Xiong, R. Zhan, J. Pan, Buyang Huanwu Decoction attenuates H2O2-induced apoptosis by inhibiting reactive oxygen species-mediated mitochondrial dysfunction pathway in human umbilical vein endothelial cells, BMC Complement. Altern. Med. 16 (2016) 154. [11] Z.Q. Zhang, J.Y. Song, Y.Q. Jia, Y.K. Zhang, Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury: mechanisms of brain tissue repair, Neural Regen. Res. 11 (3) (2016) 435–440. [12] H.Z. Zhang, L. Li, R. Jiao, Y. Zhang, Y. Qian, Effect of Buyang Huanwu Decoction on mRNA Expressions of Aorta Rho Kinase and NF-κB p65 in Atherosclerosis Model Rats, Zhongguo Zhong Xi Yi Jie He Za Zhi 35 (12) (2015) 1495–1500. [13] C. Huang, C. Zheng, Y. Li, Y. Wang, A. Lu, L. Yang, Systems pharmacology in drug discovery and therapeutic insight for herbal medicines, Brief. Bioinformatics 15 (5)

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