Abstract

Background

   Leonurus japonicus Houtt. (LJH) has multiple pharmacological effects.

Objective

   To investigate the potential mechanism of LJH in the treatment of
   myocardial ischemia-reperfusion injury (MIRI) using network
   pharmacology, molecular docking technology, and in vitro experiments.

Methods

   Herbs for ischemic heart disease were identified with the help of
   herb-disease databases. The TCMSP database was used to find the
   potential targets of LJH. Disease targets of MIRI were identified with
   the help of Disgenet, Genecard, Alliance of Genome Resources databases.
   The common targets were obtained with the help of VENN diagram, and the
   common targets were analyzed by GO function and KEEG pathway enrichment
   to predict the potential mechanism of action of LJH in treating MIRI.
   With the help of STRING database and Cytoscape software, we constructed
   a visual protein-protein interaction (PPI) network model to screen the
   core targets and then docked the core targets with the corresponding
   ligand molecules. AC16 cells were used to simulate MIRI by
   glucose-oxygen deprivation, and apoptosis was detected by Annexin
   V-FITC/PI double staining; protein expression was detected by Western
   blot.

Results

   LJH was one of the herbal remedies for the treatment of ischemic heart
   disease. LJH had 247 potential targets of action and 26 targets in
   common with MIRI. These 26 targets were enriched in the TNF signaling
   pathway and NF-kappa B signaling pathway, and the core targets screened
   by the PPI results included TNF, VCAM1, and MMP9. Molecular docking
   results showed that the compounds in LJH docked well with the core
   target proteins. In vitro experiments showed that LJH could inhibit the
   elevation of TNF, VCAM1, and MMP9 after MIRI, reduce apoptosis, and
   inhibit inflammation.

Conclusion

   The mechanism of LJH in the treatment of MIRI was mainly related to the
   activation of TNF signaling pathway and NF-kappa B signaling pathway,
   and the regulation of TNF, VCAM1, and MMP9 protein expression.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13019-025-03425-7.

   Keywords: Leonurus japonicus Houtt., Myocardial ischemia-reperfusion
   injury, TNF-α, NF-κB

Introduction

   Acute myocardial infarction (AMI) is a serious clinical emergency
   [[32]1]. AMI is a myocardial necrosis caused by acute and persistent
   ischemia and hypoxia of the coronary artery, characterized by acute
   onset, multiple complications, and a high mortality rate [[33]2]. With
   the development of China’s economy and changes in people’s lifestyles,
   the mortality rate of AMI has shown an upward trend in recent years
   [[34]3]. For patients with myocardial infarction, the preferred
   treatment method to reduce injury is thrombolytic therapy or one-stage
   percutaneous coronary intervention to achieve timely and effective
   myocardial reperfusion [[35]4]. However, when the myocardium undergoes
   ischemia and re-establishes blood supply, local inflammation and
   increased production of reactive oxygen species can lead to secondary
   damage [[36]5]. In many clinical practices, it has been found that AMI
   patients who recover blood flow reperfusion through percutaneous
   coronary intervention do not completely reverse the pathological
   process of AMI. Instead, further exacerbate myocardial injury and
   myocardial cell death usually results in enlarged myocardial infarction
   area, arrhythmia, and other symptoms, which is clinically defined as
   myocardial ischemia-reperfusion injury (MIRI) [[37]6]. The recovery of
   blood flow and oxygen transport further activates the inflammatory
   signaling pathway usually induces inflammation during myocardial
   ischemia [[38]5]. Currently, an increasing number of studies have found
   that interleukin (IL) and inflammasomes are key inflammatory mediators
   and processes leading to abnormal damage [[39]7]. The pathogenesis of
   MIRI is complex. Effective prevention and treatment for MIRI are
   limited, so it is necessary to develop new treatment methods.

   Traditional Chinese medicine has been used in China for thousands of
   years and has accumulated a long experience in treating cardiovascular
   diseases. At present, the databases on the ingredients, targets, and
   disease relationships of Chinese herbal medicine are relatively
   complete [[40]8]. In recent years, traditional Chinese medicine has
   played an important role as an alternative therapy in the clinical
   treatment of MIRI. Herbs have become a research hotspot due to their
   high efficacy and few side effects. Herbs can participate in different
   signaling pathways and exert pharmacological effects through these
   different pathways, which is in line with the complex pathological
   mechanisms of MIRI [[41]9]. The multi-target characteristics of Chinese
   herbal medicine stem from its complex chemical composition. Modern
   research suggests that the possible cardioprotective mechanisms of
   herbs may be multifactorial, including clearing inflammation,
   inhibiting cell apoptosis, and regulating vascular remodeling [[42]10].

   In current research, we explored frequently-used herbs in ischemic
   heart disease based on herb-disease databases. Then, using network
   pharmacology methods, Leonurus japonicus Houtt. (LJH) was used as the
   research object for in-depth target and pathway prediction. On the
   basis of PPI, we screened the hub gene and its corresponding components
   for molecular docking. Cell experiments validated the findings from
   network pharmacology.

Materials and methods

Searching for herb to be studied

   Herbs related to ischemic heart disease were identified by Traditional
   Chinese Medicine Systems Pharmacology Database and Analysis Platform
   (TCMSP) (using ‘ischemic heart disease’ as a key word), HERB (using
   ‘Acute ischemic heart disease’ as keyword, P-value < 0.05), and
   TCM-Suite (using ‘Ischemic Heart Disease’ as keyword) databases. The
   obtained herbs from these three databases were subjected to VENN
   diagram for common herbs.

Searching for possible pharmacological components and potential targets for
LJH

   The TCMSP database was used to obtain the chemical compositions of LJH
   using LJH as the search term. For the chemical constituents of LJH,
   oral bioavailability (OB) ≥ 20% and drug-like properties (DL) ≥ 0.1
   were set as the screening conditions to obtain the potential
   pharmacologically active components [[43]11]. The corresponding target
   proteins were queried according to the active ingredient ID through
   TCMSP database. The collected target proteins were imported into
   UniProt database for name standardization, to obtain the target
   proteins corresponding to LJH.

   MIRI-related genes were searched using the human disease-gene
   correlation databases, including DisGeNET, GeneCards, and Alliance of
   Genome Resources databases. The search term used for the Disgenet
   database was ‘Myocardial Ischemia’, the search term for GeneCards
   database was ‘myocardial ischemia reperfusion’ (Relevance score > 1),
   and the Alliance of Genome Resources database used the search term
   ‘heart disease’. The obtained MIRI-related genes were imported into the
   UniProt database for name normalization and then intersected with
   LJH-targeting proteins. In this way, the LJH-targeting proteins against
   MIRI were obtained.

Construction of compound-disease target network and target annotation

   LJH active compounds and the genes of target proteins targeted by these
   compounds were imported into Cytoscape software and visualized to
   construct a compound-MIRI target network. In the network diagram,
   compounds and targets were represented by nodes, and the interactions
   between nodes were represented by edges. Proteins were imported into
   the STRING database to analyze protein-protein interaction
   relationships at a high confidence of 0.700. Cytoscape software was
   used to screen the top three proteins using Closeness, Degree, MCC, and
   MNC methods. The target proteins were subjected to Gene Ontology (GO)
   functional enrichment analysis and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis by clusterProfiler and
   pathview R packages, with P < 0.05 as the screening condition. The
   proteins involved in TNF signaling pathway and NF-kappa B signaling
   pathway were traced for the compound source.

Molecular docking validation

   The obtained hub proteins were used as receptors and their
   corresponding compounds as ligands for molecular docking analysis.
   Firstly, the 3D structure of compounds was obtained through PubChem
   database. The crystal structures of the proteins were obtained in the
   RCSB Protein Data Bank (PDB) database, with the H[2]O and small
   molecule ligands removed using PyMOLWin.exe software. The processed
   receptor and ligand files were subjected to protein-ligand blind
   docking at the detected candidate pockets with AutoDock Vina.
   Protein-compound pairs with excellent Vina scores were validated for
   cellular experiments.

Preparation of lyophilizate from standard LJH decoction

   LJH slices (200 g) were boiled twice with water. For the first boil, 12
   times the amount of water was added and soaked for 30 min. After
   boiling on high heat, a slight boil was maintained for 30 min. The
   decoction liquid was filtered through a 350-mesh sieve. Then, 10 times
   the amount of water was added for the second boiling. The two filtrates
   were combined and concentrated under reduced pressure (temperature:
   65℃, vacuum: -0.10 MPa) to 150 mL of liquid extract, which was divided
   into vials. After dispensing, the vials were transferred to a vacuum
   freeze-dryer for freeze-drying.

Cell culture

   AC16 (ATCC, USA) were differentiated into mature cardiomyocytes using
   Dulbecco’s Modified Eagle Medium (DMEM)/F-12 medium, with the addition
   of 1× Insulin, Transferrin, Selenium Solution (Gibco, USA) and 2% Horse
   Serum (Gibco, USA) to make the medium complete. Hypoxia-glucose
   deprivation was performed on AC16 cells for simulating ischemia; then,
   oxygen-glucose supply was restored to AC16 cells for simulating
   reperfusion. Briefly, AC16 cells were incubated for 1 h using a
   pre-saturated ischemia-simulated solution of 95% N[2] + 5% CO[2]
   instead of medium. The simulated ischemia solution was then discarded
   and AC16 cells were incubated for 3 h using DMEM/F12 to simulate
   reperfusion solution. All groups except the control group received the
   ischemia-reperfusion procedure described above.

   To investigate the effect of LJH on MIRI through TNF signaling pathway
   regulation, TNF-α was added to the culture medium to activate the TNF
   signaling pathway. LJH lyophilizate was used at concentrations of 20
   (low), 50 (medium), and 100 (high) ng/mL. For pretreatment, the drug
   was used 24 h before MIRI modeling.

Western blot analysis

   Protein expression in AC16 cells was determined by western blotting.
   After the cells were digested and lysed, the total protein
   concentration was determined using the Bradford protein assay (BioRad,
   USA). Aliquots of protein samples were loaded on 10–15% SDS
   polyacrylamide gel electrophoresis for separation and then transferred
   to polyvinylidene difluoride (PVDF) membranes. PVDF was blocked and
   incubated with primary antibodies overnight. The primary antibodies
   included anti-TNF-α (1:1000, Abcam), anti-MMP-9 (1:1000, Abcam),
   anti-VCAM1 (1:1000, Abcam), phospho-IKKα/β at its autophosphorylation
   site (Ser176/180) (Cell Signaling Technology), NF-κB p65 (1:1000, Cell
   Signaling Technology), anti-collagen type I (COL1) (1:1000,
   ThermoFisher Scientific), anti-αSMA (1:1000, Cell Signaling), and
   anti-osteopontin (OPN) (1:1000, Merck Darmstadt, Germany). After
   washing, the membrane was incubated with the second antibody conjugated
   with horseradish peroxidase. Imprint was incubated in Immunobilion ^®
   Forte Western blotting of HRP substrates (Millipore, USA) to form
   luminescent bands for detection. The intensity of the bands was
   analyzed using ImageJ software and standardized to β-actin.

Enzyme linked immunosorbent assay (ELISA)

   After treatment, the supernatant of cell culture was collected and
   centrifuged at 1000 g (3750 rpm) for 20 min at 4℃. The cell supernatant
   reacted and was tested using human TNF-α, IL-1β, and IL-6 ELISA kits
   according to each supplier’s instruction.

Statistical analysis

   All statistical analyses were conducted using GraphPad software.
   One-way Analysis of Variance with post-hoc Bonferroni test.

Results

Collection of active ingredients in LJH and construction of ingredient-target
network

   Based on TCMSP, HERB, and TCM-Suite database queries, LJH was one of
   the main herbs used to treat ischemic heart disease (Supplementary
   Fig. [44]1). After querying the TCMSP database, LJH contained 51
   compounds. Among them, there were 18 compounds that complied with
   OB ≥ 20% and DL ≥ 0.1. Target prediction was performed on these active
   ingredients, and 231 target proteins were obtained after name
   normalization through UniProt. The network composed of these components
   and target proteins included 247 nodes and 445 edges (Fig. [45]1), with
   an average number of neighbors of 3.6. Therefore, LJH had the advantage
   of being multi-component and multi-target in the treatment of disease.

Fig. 1.

   [46]Fig. 1
   [47]Open in a new tab

   The “compound-target” network of components and targets in LJH. Among
   them, the green square represents compounds; The yellow circle
   represents the target of action. After analysis, the network presents
   shapes of different sizes according to the degree values

Annotation for targets of LJH

   After searching through DisGeNET, GeneCards, and Alliance of Genome
   Resources, we collected MIRI-related genes. Then, LJH can target 26
   MIRI-related genes (Fig. [48]2A, Supplementary Fig. [49]2). These
   targets were enriched in TNF signaling pathway, NF-kappa B signaling
   pathway, etc. (Fig. [50]2B). The GO terms enriched by these 26 targets
   were a response to lipopolysaccharide, regulation of cell-cell
   adhesion, regulation of mononuclear cell proliferation, cytokine
   activity, receptor ligand activity, growth factor activity, etc.
   (Fig. [51]2C).

Fig. 2.

   [52]Fig. 2
   [53]Open in a new tab

   Annotation of common targets between Leonurus japonicus Houtt. and
   MIRI. (A) A PPI network for common targets of Leonurus japonicus Houtt.
   and MIRI. (B) KEEG pathway enrichment analysis of common targets
   between Leonurus japonicus Houtt. and MIRI. (C) GO enrichment analysis
   of common targets between Leonurus japonicus Houtt. and MIRI

Key pathways of LJH

   The enriched TNF signaling pathway and NF-kappa B signaling pathway
   aroused our interest. We backtracked the involved targets and compounds
   in these two pathways. TNF, VCAM1, IL6, MMP9, ICAM1, CXCL2, TNFRSF1B,
   and SELE were enriched in TNF signaling pathway, while TNF, BCL2,
   PRKCB, VCAM1, CD40LG, ICAM1, and CXCL2 were enriched in NF-kappa B
   signaling pathway (Fig. [54]3A). The protein-protein interactions among
   these eleven targets were shown in Fig. [55]3B. Using the hub gene
   screening methods, TNF, VCAM1, and MMP9 were identified as hub genes.
   After molecular docking, MMP9-apigenin pair showed the best Vina score
   (-9.7 kcal/mol) (Table [56]1).

Fig. 3.

   [57]Fig. 3
   [58]Open in a new tab

   TNF/NF-kappa B signaling pathway. (A) Sankey plots for the targets and
   compounds involved in TNF/NF-kappa B signaling pathway. (B) A PPI
   network for targets involved in TNF/NF-kappa B signaling pathway

Table 1.

   Molecular Docking results
   Protein Ligand CurPocket ID Vina score (kcal/mol) Cavity volume (Å3)
   Center (x, y, z)
   MMP9 apigenin C1 -9.7 705 39, 22, 141
   C2 -9.2 460 63, 31, 114
   MMP9 quercetin C1 -9.6 705 39, 22, 141
   C2 -9.4 460 63, 31, 114
   TNF apigenin C1 -8.3 1593 -5, 84, 27
   C4 -7.6 243 -17, 72, 36
   C2 -7.1 337 8, 67, 12
   TNF quercetin C1 -8.8 1593 -5, 84, 27
   C2 -7.7 337 8, 67, 12
   C4 -7.3 243 -17, 72, 36
   TNF kaempferol C1 -7.9 1593 -5, 84, 27
   C4 -7.3 243 -17, 72, 36
   C2 -7.1 337 8, 67, 12
   VCAM1 quercetin C1 -8.4 1365 37, 15, 8
   C3 -7.8 1096 22, 8, 16
   C4 -7.7 691 15, 11, 3
   C5 -7.7 336 21, 18, 17
   C2 -7.5 1112 6, 15, 25
   VCAM1 kaempferol C3 -8.2 1096 22, 8, 16
   C1 -8.1 1365 37, 15, 8
   C5 -7.9 336 21, 18, 17
   C2 -7.5 1112 6, 15, 25
   C4 -7.4 691 15, 11, 3
   [59]Open in a new tab

LJH targeted to TNF, VCAM1, and MMP9

   We conducted in vitro experiments using a human myocardial cell line
   (AC16) to further validate the network pharmacology prediction results
   of LJH in MIRI. Firstly, we stimulated AC16 cells with different
   concentrations of LJH and measured the expression TNF levels in cells
   and cell culture. The results showed that LJH reduced the expression
   levels of TNF-α in the AC16 I/R model (Fig. [60]4A) and its release in
   the cell culture medium (Fig. [61]4B). LJH reduced the expression level
   of MMP9 protein in the AC16 I/R model, but the addition of TNF-α
   re-increased this level (Fig. [62]4C). LJH also reduced the expression
   level of VCAM1 protein in the AC16 I/R model, but the addition of TNF-α
   re-increased this level (Fig. [63]4D). Moreover, LJH regulated the
   protein levels in the NF kappa B signaling pathway, including IKKα/β
   (Figs. [64]4E) and NF-κB protein levels (Figs. [65]4F).

Fig. 4.

   [66]Fig. 4
   [67]Open in a new tab

   Effects of Leonurus japonicus Houtt. on TNF-α, VCAM1, and MMP9
   expression levels in AC16 cells. (A) TNF-α protein levels in AC16 cells
   were evaluated by western blot analysis with β-actin as a loading
   control. (B) TNF-α release in AC16 cell culture were evaluated by
   ELISA. (C) MMP9, (D) VCAM1, (E) p-IKKα/β, and (F) NF-κB protein
   expression in AC16 cells were evaluated by western blot analysis.
   (n = 5; ***P < 0.001, one-way Analysis of Variance)

Effects of LJH on inflammatory response and vascular remodeling during MIRI

   Our results indicate that compared to the control group, the apoptosis
   rate in the MIRI AC16 cell model was significantly increased; LJH can
   reduce the apoptosis rate of AC16 cells under MIRI state, but the
   addition of TNF-α can induce cell apoptosis again (Fig. [68]5A). Next,
   we investigated the levels of IL-1β in cell culture medium and the
   expression level of NLRP3 protein in cells using ELISA and Western
   blotting. Compared with the control group, LJH reduced the levels of
   IL-1β and NLRP3 protein expression induced by MIRI, while TNF-α could
   increase the levels of these factors (Fig. [69]5B and C). Moreover, LJH
   significantly inhibited the expression of vascular fibrosis marker COL1
   in MIRI-induced AC16 cells (Fig. [70]5D). Furthermore, LJH
   significantly inhibited MIRI-induced OPN protein expression
   (Fig. [71]5E), but did not affect αSMA protein level (Fig. [72]5F),
   indicating that LJH inhibited phenotypic switching of AC16 cells under
   MIRI.

Fig. 5.

   [73]Fig. 5
   [74]Open in a new tab

   Effects of Leonurus japonicus Houtt. on AC16 cell function. (A) AC16
   cell apoptosis was determined using Flow cytometry. (B) IL-1β release
   in AC16 cell culture were evaluated by ELISA. (C) NLRP3, (D) COL1, (E)
   αSMA, and (F) OPN protein expression in AC16 cells were evaluated by
   western blot analysis. (n = 5; ***P < 0.001, one-way Analysis of
   Variance)

Discussion

   In China, the incidence of AMI is gradually showing a younger trend. At
   present, the lack of effective treatment methods and drugs for MIRI
   after clinical treatment is an urgent problem that needs to be solved
   in clinical practice. Traditional Chinese medicine has certain
   advantages in preventing and treating MIRI. After database screening,
   this study found that LJH is one of the commonly used traditional
   Chinese medicines for the treatment of ischemic heart disease. LJH is a
   traditional Chinese medicine with a long history of development and
   application in China. Especially, recent studies have shown that LJH
   has various effects. The oxidative stress effects of LJH have been
   demonstrated in ischemic hearts [[75]12]. LJH contains various active
   ingredients, including alkaloids, flavonoids, diterpenoids, and fatty
   acids. Recent studies have shown that these molecules play beneficial
   roles in coronary artery disease and cerebral ischemia [[76]13,
   [77]14]. Therefore, these molecules may become new candidates for drug
   discovery and development. Based on database analysis, we found that
   LJH contained multiple pharmacologically active ingredients, including
   apigenin, leojaponin, melatonin, LJH, kaempferol, etc. These compounds
   targeted various targets and pathways, including TNF, VCAM1, MMP9, TNF
   signaling pathway, and NF-kappa B signaling pathway. We validated the
   regulatory effects of LJH on TNF, VCAM1, MMP9, and its rescue effects
   on MIRI cell damage through cell experiments.

   Previous studies have found that an increase in TNF-α in the infarcted
   area after chronic myocardial infarction. In TNF-α-knockout mice, it
   has been observed that survival rate increased after chronic myocardial
   infarction, along with decreased heart rupture, inflammatory cell
   infiltration and cytokine expression [[78]15]. Moreover, a decrease in
   cytokine expression is also associated with an increase in matrix
   metalloproteinase-9 (MMP-9) in the infarcted myocardium [[79]16]. TNF-α
   inhibition may be an effective strategy to reduce MIRI. It is known
   that pharmacological inhibition of NF-κB or TNF-α can significantly
   improve the formation of myocardial infarction [[80]17]. VCAM1, TNF-α,
   and MMP-9 have been reported to be involved in inflammation and
   vascular remodeling [[81]18]. Here, we found that LJH can inhibit TNF-α
   expression and release in AC16 under MIRI. During MIRI, excessive ROS
   leads to high permeability by disrupting the stability of the
   endothelial barrier. At the same time, adhesion molecules such as VCAM1
   are upregulated, which leads to neutrophils blocking capillaries and
   infiltrating infarcted tissue [[82]19]. We discovered that LJH can
   suppress the expression of VCAM1 in AC16 cells under MIRI. The
   phenotype transition of myocardial cells is considered a key mechanism
   of myocardial remodeling. The increased protein expression of the
   contraction marker αSMA and the synthetic marker OPN confirms that MIRI
   can induce phenotype transition in AC16 cells. LJH can significantly
   inhibit the expression of OPN protein and inhibit MIRI-induced
   phenotype transition.

   Activation of inflammasomes is associated with myocardial I/R injury.
   The inhibition of NLRP3 by small interfering RNA can prevent
   inflammasome activation and cardiac cell death, thereby improving
   myocardial remodeling in MI mouse models [[83]20]. Previous research
   has shown that NLRP3 is mainly localized in fibroblasts of the left
   ventricle and mediates its secretion of IL-1β for lipopolysaccharide,
   indicating that NLRP3 may be the initial receptor involved in
   inflammasome activation [[84]19]. Here, LJH was found to inhibit NLRP3
   in AC16 cells under MIRI, suggesting that LJH may be a promising
   therapeutic approach for preventing or reducing myocardial I/R injury.
   However, our study was limited to the in vitro experiments. In vivo
   validation is needed in future studies.

   In summary, the results of this study indicate that LJH, as a commonly
   used herb for the treatment of ischemic heart disease, can exert
   pharmacological effects through multi-target and multi-pathway modes.
   LJH can reduce inflammation in MIRI state, which may be mediated by
   targeting TNF-α, VCAM1, and MMP9 to participate in the TNF/NF-κB
   pathway. Our study provides evidence for LJH as an effective
   therapeutic drug for MIRI.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [85]13019_2025_3425_MOESM1_ESM.tif^ (11.9MB, tif)

   Supplementary Material 1: Supplementary Fig. 1. Common herbs of TCMSP,
   HERB, and TCM-Suite for treating Ischemic heart disease
   [86]13019_2025_3425_MOESM2_ESM.tif^ (10.4MB, tif)

   Supplementary Material 2: Supplementary Fig. 2. Common targets of
   Leonurus japonicus Houtt. and MIRI

Acknowledgements