Heart failure (HF) is the terminal stage of most cardiovascular diseases with high morbidity and mortality [
Tingli Dazao Xiefei decoction (TDXD) originated from Zhang Zhongjing’s “Synopsis of Golden Chamber” and has been widely used in clinical practice for more than 1000 years. TDXD, composed of Tinglizi (Semen Lepidii) and Dazao (Jujube), is a classic prescription for the treatment of HF with a definite curative effect [
Network pharmacology integrates systems biology and computer technology to construct the multidimensional “components-targets-pathways” network to reveal the pharmacological effects of TCM and the molecular mechanism of disease treatment, which is the research hotspot of TCM [
TDXD dry powder was provided by the Chinese Pharmacy of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. Captopril tablets (25 mg/tablet) were purchased from Beijing Jingfeng Pharmaceutical Group Co., Ltd. (Beijing, China). Hematoxylin-eosin dye (G1003), Masson dye (G1006), and TUNEL kit (G1501) were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). BNP ELISA kit (BPE30445), IL-6 ELISA kit (BPE30646), IL-1
The active components of Tinglizi and Dazao were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP,
GeneCards (
The overlapping targets of TDXD and HF were acquired by the Venny 2.1 online tool (
The overlapping targets of TDXD and HF were inputted into the STRING 11.0 database (
The Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the overlapping targets was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID v6.8,
AutoDock 4.2.6 and PyMOL software were used to dock the core targets with the main active ingredients of TDXD [
All animal experiment protocols involved in this study were approved by the Animal Protection Committee of Shanghai University of Traditional Chinese Medicine with the approval number “PZSHUTCM210903018” and the experiments were performed in compliance with the guide for “Animal Research: Reporting of In Vivo Experiments (ARRIVE).” Forty SPF Wistar male rats (200 ± 10 g) were purchased from Beijing Weitong Lihua Co., Ltd. (Beijing, China) and raised in the Experimental Animal Center of Shanghai University of TCM. After 7 days of acclimation to the laboratory, 40 rats were randomized into four groups (
Administration dosages of TDXD (0.675 g/kg·d) and captopril (4.5 mg/kg·d) in rats were determined according to the equivalent patient dose [
After the last administration, the rats were fasted for 12 hours and anesthetized with isoflurane inhalation [
The heart tissues of rats in each group were fixed in 4% paraformaldehyde for more than 24 hours, then dehydrated, and embedded in paraffin. Next, 5
First, the slices were dewaxed with water, and antigenic repair was performed with protease K. After room temperature equilibrium, TUNEL color reaction solution was added and incubated for 2 hours at 37°C. Then, DAPI was added to the slices and incubated for 10 min. Finally, the slices were sealed with an antifluorescence quenching agent and observed under a fluorescence microscope. ImageJ software was used to count the number of TUNEL-positive cardiomyocyte nuclei. TUNEL-positive (%) = apoptotic nuclei number/total nuclei number × 100% [
After the abdominal aorta blood was collected, it was left standing at room temperature for 2 h and centrifuged at 3000 rpm/min at 4°C for 15 min, and the supernatant was taken. The serum levels of BNP, LDH, CK-MB, IL-6, IL-1
The myocardial tissue in the marginal zone of left ventricular infarction was homogenized, and the total protein concentration was determined by the BCA protein detection kit. The equivalent protein samples were separated by 10%–12% SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane, followed by western blotting as previously described [
SPSS 23.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis, and data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Bonferroni or Dunnett’s T3 post hoc test was used for multiple comparisons, and value of
With OB ≥30% and DL ≥0.18 as screening conditions, 39 active ingredients of TDXD were screened, including 12 compounds from Tinglizi and 29 compounds from Dazao. Quercetin and beta-sitosterol were the common active ingredients (Supplementary Materials, Table
Gene targets of HF were obtained through GeneCards, taking the top 500 targets with the highest correlation according to the “Relevance score,” and then merging them with the results retrieved from TTD, OMIM, and DrugBank. After deleting duplicates values, 960 HF-related targets were identified. The Venn diagram was used to take the intersection of drug targets and disease targets, and 66 overlapped targets were screened (Figure
Venn diagram of TDXD targets and HF targets.
The 66 overlapping targets correspond to 23 active ingredients. In Cytoscape 3.7.2, the overlapping targets and corresponding active ingredients were introduced to construct the “active compounds-disease targets” network (Figure
Network of active compounds-disease targets. Green regular triangle nodes represent the Tinglizi and Dazao. Purple round nodes represent the active components of TDXD. Red inverted triangle node represents HF. Yellow rhombic nodes represent the gene targets.
The 66 overlapping targets were uploaded to STRING to obtain the interaction diagram, after which Cytoscape 3.7.2 was used to visualize and construct the PPI network (Figure
The PPI network of overlapping targets. Circular nodes represent the gene targets; the darker the node color and the larger the graph, the greater the degree value. Black lines represent the interaction between nodes, and the thicker the line, the closer the connection.
GO function analysis included three parts: biological process (BP), cell component (CC), and molecular function (MF). There were 294 BP entries, including aging, positive regulation of nitric oxide biosynthetic process, response to hypoxia, response to the drug, and so on; 30 CC entries, including extracellular space, extracellular region, plasma membrane, lysosome, and others; and 47 MF entries, including enzyme binding, protein binding, cytokine activity, and protein homodimerization activity. According to the sequencing of
Enrichment analysis of TDXD against HF: (a) GO analysis of overlapping targets. The figure lists the top 10 GO pathways in each category. (b) Top 20 pathways of KEGG enrichment analysis. Bubble size represents the number of genes in this pathway, and the color reflects the −log10 (
Based on the results of the literature search and KEGG pathway enrichment, there were 8 pathways related to HF in the first 20 pathways (Supplementary Materials, Table
The top 5 core targets (IL-6, VEGFA, TNF, AKT1, and MAPK1) in the PPI network were docked with the main active components (quercetin, kaempferol, beta-carotene, isorhamnetin, and beta-sitosterol) of TDXD in treating HF and the positive drug (captopril) for HF. Because beta-carotene has no free hydrogen bond, it cannot stably bind to the target through hydrogen bond, so it is eliminated. It is generally believed that when the binding energy of ligand and receptor is <−4 kcal/mol, there is potential binding activity between them; binding energy <−5 kcal/mol suggests a significant binding ability between them [
The binding energy of molecular docking (kcal/mol).
Quercetin | Kaempferol | Isorhamnetin | Beta-sitosterol | Captopril | |
---|---|---|---|---|---|
IL-6 (1iL6) | −6.09 | −6.17 | −6.36 | −8.72 | −6.64 |
VEGFA (4KZN) | −5.34 | −5.61 | −5.86 | −5.91 | −4.96 |
TNF (6OP0) | −6.95 | −8.15 | −7.54 | −8.36 | −5.38 |
AKT1 (1UNP) | −6.27 | −6.46 | −6.49 | −7.18 | −6.33 |
MAPK1 (4FV8) | −6.68 | −6.07 | −6.68 | −7.04 | −6.74 |
Molecular docking structure diagram. (a) IL-6-quercetin, (b) IL-6-kaempferol, (c) IL-6-isorhamnetin, (d) IL-6-beta-sitosterol, (e) VEGFA-quercetin, (f) VEGFA-kaempferol, (g) VEGFA-isorhamnetin, (h) VEGFA-beta-sitosterol, (i) TNF-quercetin, (j) TNF-kaempferol, (k) TNF-isorhamnetin, (l) TNF-beta-sitosterol, (m) AKT1-quercetin, (n) AKT1-kaempferol, (o) AKT1-isorhamnetin, (p) AKT1-beta-sitosterol, (q) MAPK1-quercetin, (r) MAPK1-kaempferol, (s) MAPK1-isorhamnetin, and (t) MAPK1-beta-sitosterol.
As shown in Figures
TDXD improved cardiac function in rats with HF. (a) Representative echocardiography images in each group, (b) EF%, (c) FS%, (d) LVIDd, and (e) LVIDs,
Under normal circumstances, BNP, LDH, and CK-MB are located in the cytoplasm of cardiomyocytes. The increase of their levels in serum is usually considered as the diagnostic markers of HF and is positively correlated with the severity of HF [
Ventricular remodeling caused by myocardial fibrosis is the main pathological mechanism of HF [
TDXD reduced myocardial fibrosis in rats with HF. (a) Representative images of H&E staining in different groups,
Cardiomyocyte apoptosis is one of the important pathological mechanisms of myocardial injury, which is closely related to the severity of HF, inflammation, and myocardial fibrosis [
TDXD inhibited apoptosis and reduced the level of inflammatory cytokines. (a) Representative images of TUNEL staining in different groups, scale bar: 20
Moderate regulation of the inflammatory response after MI is very important for the prognosis of HF. IL-6, IL-1
On the basis of network pharmacology analysis and literature research, in order to explore the underlying mechanism of TDXD in HF, PI3K/Akt, and MAPK signaling pathways were investigated. The PI3K/Akt signaling pathway can affect cardiac function through various mechanisms such as regulation of cardiomyocyte apoptosis, energy metabolism, oxidative stress, inflammatory response, and autophagy [
TDXD inhibited the activation of PI3K/AKT and MAPK signaling pathways. (a–d) Representative western blotting pictures and quantitative analysis of PI3K, p-Akt/AKT, and p-MAPK/MAPK,
HF has always been a hot and difficult problem in the field of cardiovascular research because of its high morbidity and mortality [
Network pharmacology can reveal the material basis of TCM’s ability to treat diseases and provide a reference for follow-up research. The results showed that quercetin, kaempferol, beta-carotene, isorhamnetin, and beta-sitosterol were the most important compounds. Studies have found that quercetin can downregulate the TLR4/NF-
HF is associated with both local and systemic activation of inflammatory signaling cascades [
PI3K/Akt is a classical signal transduction pathway, which can directly regulate the expression of Bcl-2/Bax and plays an important role in regulating cardiomyocyte survival, myocardial remodeling, and inflammation [
In summary, this study clarified the therapeutic effect and underlying mechanism of TDXD against HF through network pharmacology and in vivo experiments. It verified that TDXD inhibits the phosphorylation of PI3K/Akt and MAPK signaling pathways, thereby alleviating myocardial inflammation, fibrosis, apoptosis, and improving heart function in rats with HF. The results of this study provide a theoretical basis for further research and clinical application of TDXD.
The data used to support the findings of this study are available from the corresponding author upon request.
Dan-dan Zhao and Xiao-qing Zhang are the co-first authors.
The authors declare that there are no conflicts of interest regarding the publication of this study.
Dan-dan Zhao and Xiao-qing Zhang designed and wrote the manuscript. Tao Yang and Qian Liu analyzed the data. Zhen-zhen Lan and Xiao-li Yang made all tables and pictures. Hua Zhou and Hui-yan Qu reviewed the manuscript. Hua Zhou and Hui-yan Q are the corresponding authors.
This work was supported by the National Natural Science Foundation of China (nos. 81673753 and 81973656) and the Science and Technology Support Project of Shanghai Science and Technology Commission (no. 18401932800).
Table S1: basic information on active ingredients in TDXD. Table S2: 221 gene targets of TDXD. Table S3: topological parameter of top 10 compounds in active compounds-disease targets network. Table S4: topological parameter of top 10 targets in PPI network. Table S5: parameter information of 8 pathways related to HF in the top 20 KEGG pathways.