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. 2025 Jul 21;73(31):19373–19385. doi: 10.1021/acs.jafc.4c10675

Theogallin Protects Myocardial Ischemia-Reperfusion Injury by Inhibiting the Interleukin-17 Signaling Pathway

Feng Zhu †, Huijun Cheng †,‡, Zixian Luo †, Anqi Cao †, Tianxing Yu †, Huifang Ge †, Shanshan Hu †, Zhongwen Xie, Daxiang Li †,*
PMCID: PMC12333330  PMID: 40690952

Abstract

Tea polyphenols have been shown to prevent cardiovascular disease through antioxidant and anti-inflammatory mechanisms. Theogallin, a phenolic acid derived from gallic and quinic acids, is a unique polyphenolic compound found in teas. Its preventive effect on myocardial ischemia-reperfusion (I/R) injury remains unclear. This study utilized an ex vivo model of I/R injury in isolated rat hearts through Langendorff perfusion and an in vivo myocardial injury model through left anterior descending coronary artery (LAD) ligation. In isolated hearts, theogallin pretreatment at both 100 and 200 ng/mL significantly improved hemodynamic parameters following I/R injury. Additionally, in vivo, pretreatment with 20 mg/kg theogallin in LAD ligation rats for 7 days significantly enhanced myocardial contractile function, reduced the release of myocardial enzymes, and decreased infarct size and fibrosis in LAD ligation rats. These protective effects may be attributed to theogallin’s ability to inhibit the expression of interleukin-17 (IL-17) receptor A, transcription factor Jun-B (JUNB), Fos-related antigen 1 (FRA1), and matrix metalloproteinase 9 (MMP9) in the IL-17 signaling pathway. These findings suggest that theogallin exerts cardioprotective effects by downregulating the IL-17 signaling pathway in rats with myocardial I/R injury.

Keywords: theogallin, ischemia-reperfusion injury, fibrosis, IL-17RA, MMP9

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1. Introduction

Cardiovascular diseases are a significant threat to global health. A recent study found that ischemic heart disease has the highest global age-standardized disability-adjusted life years among all diseases worldwide. The most effective treatments for restoring blood flow to the heart, such as percutaneous coronary intervention and coronary artery bypass grafting, work by reopening blocked coronary arteries. , However, these interventions are not always successful. This is largely due to myocardial ischemia-reperfusion (I/R) injury, a condition in which restoring blood flow leads to further damage in the heart tissue, affecting both myocardial cells and the surrounding vascular network. , Despite decades of intensive research, many potential innovative therapies have failed to effectively reduce reperfusion injury.

Myocardial I/R injury is a complex and multifaceted process, with inflammation playing a central role. Over time, excessive deposition of extracellular matrix (ECM) proteins and collagen in the myocardium leads to myocardial fibrosis, exacerbating cardiac damage and potentially causing heart failure. Recent studies have highlighted the importance of the interleukin-17 (IL-17) signaling pathway, which drives inflammation and contributes to the development of I/R injury. IL-17 promotes inflammation, leukocyte recruitment, cell proliferation, and collagen production. , The IL-17 receptor (IL-17R) family consists of five members, including IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE. When IL-17A, IL-17F, or their combination (IL-17A + F) binds to these receptors, they trigger the activation of nuclear factor-κB (NF-κB) or activator protein 1 (AP-1). This activation leads to increased expression of matrix metalloproteinases (MMPs) in cardiac fibroblasts, promoting fibroblast migration and contributing to myocardial remodeling. Among the MMPs, MMP9 plays a key role in breaking down ECM components such as collagen, elastin, and fibronectin. Under normal conditions, MMP9 activity is tightly controlled to maintain the structural integrity and function of the heart. However, excessive activation of MMP9 can disrupt myocardial tissue structure and lead to an overproduction of collagen fibers, contributing to the progression of myocardial fibrosis. Given the limitations of current therapeutic approaches and the variability in patient response, the need for novel preventive strategies is critical.

Green tea is well-known for its many health benefits, primarily due to its high levels of natural antioxidants and anti-inflammatory compounds, including polyphenols. One of its unique polyphenols is theogallin, which constitutes approximately 1–2% of the dry weight of green tea. Theogallin is formed through the esterification of gallic acid with the (5R)-hydroxy group of (−)-quinic acid. Currently, research on theogallin’s health effects is limited. One study has shown that, when combined with theanine, it can improve depression and cognitive function. To date, no studies have explored the effects of theogallin on cardiac I/R injury. However, previous research has shown that its precursor, gallic acid, can prevent myocardial I/R injury. − The protective mechanism of gallic acid involves reducing inflammation and oxidative stress and inhibiting the opening of the mitochondrial permeability transition pore. Therefore, this study aims to investigate whether theogallin can reduce myocardial I/R injury through the IL-17 signaling pathway and potentially alleviate myocardial fibrosis.

2. Materials and Methods

2.1. Materials

Theogallin was synthesized in the laboratory with a purity of 95%, and its structure was further confirmed by proton nuclear magnetic resonance (1H NMR and 13C NMR). Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), sodium bicarbonate (NaHCO3), potassium dihydrogen phosphate (KH2PO4), magnesium sulfate heptahydrate (MgSO4·7H2O), glucose, and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (Darmstadt, Germany). Creatine kinase (CK) kit (cat. no. R006), creatine kinase isoenzyme-MB (CK-MB) kit (cat. no. R007), lactate dehydrogenase (LDH) kit (cat. no. R009), and α-hydroxybutyrate dehydrogenase (HBDH) kit (cat. no. R010) were purchased from Meikang Company (Ningbo, China). RIPA lysis buffer and bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime (Shanghai, China). The antibodies used were anti-IL-17RA (cat. no. DF3602), antitranscription factor Jun-B (JUNB) (cat. no. AF7742), and anti-Fos-related antigen 1 (FRA1) (cat. no. DF3096) from the Affinity Company (Melbourne, Australia). Anti-MMP9 (cat. no. 380831) was purchased from the Zen-Bioscience Company (Chengdu, China). Anti-p-drosophila mothers against decapentaplegic (SMAD)­2 (cat. no. 4511), anti-SMAD2 (D13E1) (cat. no. 8690), anti-p-SMAD3 (cat. no. 9520), anti-SMAD3 (cat. no. 9523), and anti-β-actin (13E5) (cat. no. 4970) were procured from the CST Company (Danvers, MA, USA).

2.2. Animals

Male Sprague–Dawley (SD) rats (age, 7 weeks; weight: 200–250 g) were purchased from SPF (Suzhou) Biotechnology Co., Ltd. (Suzhou, China). The animals (4 per cage) were acclimated for 1 week before the experiment and were allowed to freely consume standard rat chow and water in a temperature-controlled room with a 12- h light-dark cycle. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University (Ethical approval code: AHAU2022011).

2.3. I/R Injury in Langendorff Isolated Heart

Before surgery, rats were injected with sodium heparin and were anesthetized using sodium pentobarbital (100 mg/kg). Once anesthesia was confirmed, the heart was excised through a sternotomy and immediately immersed in a precooled Krebs–Henseleit (K–H) solution. The composition of the K–H solution was as follows: NaCl, 118.3 mM; KCl, 4.7 mM; CaCl2, 2.0 mM; NaHCO3, 25.0 mM; KH2PO4, 1.2 mM; MgSO4·7H2O, 1.2 mM; and glucose, 11.1 mM. The buffer was saturated with 95% O2 and 5% CO2 and maintained at pH 7.4 and 37 °C. The heart was then retrogradely mounted on a Langendorff perfusion apparatus (ADInstruments, NSW, Australia) via the aorta. A latex balloon, connected to a pressure transducer, was inserted into the left ventricle through a mitral valve. The balloon was filled with saline to maintain a left ventricular end-diastolic pressure (LVEDP) of 5–10 mmHg, and its volume was maintained constant throughout the experiment. After a stabilization period of 18 min, the hearts were subjected to the experimental protocols outlined in Figure A. Data were recorded using the LabChart system and included the heart rate (HR), LVEDP, left ventricular developed pressure (LVDP), the maximal uprising velocity of left ventricular pressure (+dp/dtmax), the maximal decreasing velocity of left ventricular pressure (−dp/dtmax), coronary flow (CF), and rate pressure product (RPP = HR × LVDP).

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Theogallin restored hemodynamics after myocardial I/R injury in isolated rat hearts. (A) Schematic of I/R injury in the Langendorff isolated heart model, (B) heart rate, (C) LVDP, (D) +dP/dtmax, (E) −dP/dtmin, (F) coronary flow, (G) RPP, and (H) RPP recovery. All data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 vs I/R group. LVDP, left ventricular developed pressure; + dP/dtmax, maximal rate of left ventricular pressure rise; −dP/dtmin, minimal rate of left ventricular pressure fall; RPP, rate pressure product (LVDP × heart rate); I/R, ischemia/reperfusion; TOG, theogallin.

After an initial 15-min stabilization period, hearts were excluded from the study if they met any of the following criteria: LVEDP higher than 15 mmHg, LVDP less than 70 mmHg, intrinsic heart rate less than 250 beats per minute or irregular, or evidence of aortic regurgitation. Following this screening, the remaining hearts were randomly assigned to one of three experimental groups: [1] I/R group, [2] I/R pretreated with 100 ng/mL theogallin group, or [3] I/R pretreated with 200 ng/mL theogallin group. Each group included three hearts (n = 3).

2.4. Rat Myocardial I/R Injury

The rats were evenly divided into four groups: [1] Sham, [2] I/R, [3] theogallin intervention group at a dose of 5 mg/kg per day (mg/kg/day), and [4] theogallin intervention group at a dose of 20 mg/kg per day (mg/kg/day), with ten rats in each group (n = 10). Theogallin, at either 5 mg/kg/day or 20 mg/kg/day , dissolved in saline, was intraperitoneally injected into SD male rats daily for 7 days. The sham group and I/R group received equal volumes of saline by intraperitoneal injection. After finishing the treatment, the rats were anesthetized using inhaled isoflurane (1.5% v/v), intubated, and ventilated with a small animal respirator (Matrx, Ohio, USA). A left thoracotomy was then performed to expose the heart, and the left anterior descending coronary artery (LAD) was ligated with a 6.0 surgical silk suture for 30 min to induce ischemia, followed by 24 h of reperfusion. The sham-operated rats underwent the same procedure, except the suture was placed under the LAD without ligation. After 24 h of reperfusion, transthoracic echocardiography was performed on the rats to evaluate their cardiac function, followed by euthanasia. The hearts of half of the rats were collected for TTC staining, while the remaining rats were used for serum collection and histological analysis.

2.5. Echocardiographic and Hemodynamic Analysis of LAD Ligation Rats

Transthoracic echocardiography was conducted following a previously published protocol. Briefly, the rats were anesthetized with isoflurane (1.5%, v/v) and mechanically ventilated. Cardiac function was assessed after 24 h of reperfusion using an echocardiography system (VINNO 6VET, VINNO, Suzhou, China). M-mode images were obtained from the long axis of the left ventricle at the level of the papillary muscles, allowing for the measurement of the left ventricular ejection fraction (EF) and left ventricular shortening (FS).

2.6. Determination of Myocardial Injury Indexes in the Serum of LAD Ligation Rats

Blood was collected from the left carotid artery, stored in tubes, and immediately centrifuged at 3500 rpm for 10 min at 4 °C. The resulting serum was stored at −80 °C for later analysis. Serum concentrations of CK,CK-MB, LDH, and HBDH were measured using an automatic biochemical analyzer (Automatic Analyzer 3000, HITACHI, Japan), following the manufacturer’s instructions.

2.7. Determination of Heart Infarct Size in LAD Ligation Rats

TTC staining was performed to evaluate the infarct size, as previously described. The heart was removed, sliced, and incubated with 1% TTC for 15 min at 37 °C. The myocardial infarct size (INF) for each heart slice was analyzed by using Image-Pro Plus software.

2.8. Heart Histopathological Analysis in LAD Ligation Rats

The infarcted heart tissue was fixed with a 4% paraformaldehyde solution. The slices were subsequently embedded in paraffin and then cut into 5 μm thick sections (HistoCore BIOCUT, Hesse State, Germany). The myocardium sections were taken for hematoxylin and eosin (H&E) and Masson staining. Stained sections were observed and photographed with a light microscope (Pannoramic SCAN, 3DHISTECH CaseViewer, Hungary). Using Masson staining, muscle fibers were stained red, while collagen fibers were stained blue. The collagen volume fraction was quantified from the Masson-stained images and expressed as a percentage of the total left ventricular myocardial volume, serving as an indicator of myocardial fibrosis.

2.9. Microarray Data Processing and DEG Identification

The microarray gene expression data sets related to myocardial I/R injury and matched controls were obtained from the Gene Expression Omnibus (GEO) Data Sets (https://www.ncbi.nlm.nih.gov/geo/). “Myocardial ischemia-reperfusion injury” was used as a keyword for retrieval in the GEO data set. “” was used as a filter condition for “Organism.” The selection criteria for microarray data sets of myocardial I/R injury were as follows: [1] tissue samples of the left ventricle from mice in the I/R group and sham group, [2] gene expression profiling of mRNA, and [3] a sample count of at least three for each group. According to these screening criteria, three myocardial I/R injury microarray gene expression data sets were included in this study: GSE58486 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE58486), GSE193997 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE193997), and GSE214122 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE214122).

The raw data in CEL format and the probe annotation files were downloaded from GEO. R packages “affy” and “affyPLM,” provided by the Bioconductor project, were used to assess chip quality. The raw data were preprocessed via background correction, quantile normalization, and expression calculation using the robust multiarray average algorithm. Gene expression fold changes (FC) were computed by subtracting the mean of normalized log2-based expression levels of the respective control groups from each subject’s normalized log2-based expression levels. The R package “limma” was utilized to identify differentially expressed genes (DEGs) between myocardial I/R injury and matched controls in each data set. The Benjamini–Hochberg method was used to adjust original p values, and adjusted p < 0.05 was used to filter DEGs. Subsequently, the Robust Rank Aggregation method was applied to rank the DEGs of these three data sets to find robust DEGs based on the criteria of Padj < 0.05 and gene expression values of | log2 FC | > 1.

2.10. Functional Enrichment Analysis of DEGs

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to predict the potential functions of robust DEGs using the R package “Clusterprofiler.” GO terms and KEGG pathways with Padj < 0.05 were considered statistically significant.

2.11. Protein–Protein Interaction (PPI) Network of DEGs and Hub Genes Identification

The PPI network of robust DEGs was constructed by the Search Tool for the Retrieval of Interacting Genes (STRING database, v11.0; http://string-db.org/) to predict protein functional associations. Subsequently, the network was visualized using Cytoscape software (V3.8.0; http://cytoscape.org/). The Cytoscape plugin CytoHubba was then employed to evaluate the “degree” of each node in the interactive network. In this network, a node represents a protein (gene), and lines represent interactions between the proteins. The “degree” of each node is equal to the number of nodes interacting with that node. The higher the degree, the closer the connections are to other nodes, indicating a higher importance of the node within the network. The top ten genes, as ranked by degree, were considered the hub genes of myocardial I/R injury, which would be further researched in LAD ligation rats.

2.12. Molecular Docking

AutoDock Vina 1.1.2 was utilized to analyze the interaction between the theogallin and the core targets. To further verify the above results of the bioinformatics analysis, the core targets of the IL-17 signaling pathway, IL-17RA (UniProt ID: Q60943), JUNB (UniProt ID: P09450), FRA1 (UniProt ID: P48755), and MMP9 (UniProt ID: P41245), based on the KEGG analysis, were chosen as the receptor proteins, and their crystal structures were further downloaded from the Unified Protein Database (https://www.uniprot.org/).

Pymol 2.3.0 was used to remove protein crystal water and original ligands and to import the protein structure into AutoDockTools 1.5.6 for hydrogenation, charge calculation, charge allocation, specifying atomic types, and saving in “PDBQT” format. The 3D structure of theogallin was downloaded from the PubChem database in SDF format. It was imported into ChemBio3D Ultra 14.0 for energy minimization. The minimum RMS gradient was set to 0.001, and the small molecule was saved in mol2 format. The optimized small molecule was imported into AutoDockTools 1.5.6 for hydrogenation, charge calculation, charge assignment, and setting rotatable keys, and then saved in “PDBQT” format.

POCASA 1.1 (https://g6altair.sci.hokudai.ac.jp/g6/service/pocasa/) was used to predict protein binding sites, and AutoDock Vina 1.1.2 was used for docking with theogallin. The parameters related to IL-17RA were set to center_x = 57, center_y = 61.4, center_z = 36.3; the JUNB-related parameters were set to center_x = 12.3, center_y = 9.5, center_z = −29.2; The FRA1-related parameters were set to center_x = 31.7, center_y = −3.4, center_z = −8.5; the relevant parameters of MMP9 were set to center_x = −40.2, center_y = −31.3, center_z = 5.6. The search space for all four proteins was set at size_x: 37.5, size_y: 37.5, size_z: 37.5. Furthermore, the grid spacing was set to 0.375 Å, and the exhaustiveness was set to 10. The remaining parameters were set by default. Discovery Studio Visualizer 4.1 was utilized for identifying the hydrogen bonds, hydrophilic interactions, and coordination interactions between the residues at the IL-17RA, JUNB, FRA1, and MMP9 active sites and the theogallin structures.

2.13. Western Blotting

The border zones of the infarcted hearts were homogenized in RIPA lysis buffer containing protease inhibitors and a protease inhibitor cocktail. Protein concentrations were measured using a BCA protein assay kit. Equal amounts of protein (20 μg) were loaded onto SDS polyacrylamide gels (10%) for separation, and the proteins were transferred onto a nitrocellulose (NC) membrane. The membranes were blocked with 5% milk in TBS-T for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with relevant primary antibodies, followed by incubation with the appropriate HRP-conjugated secondary antibodies.

2.14. Statistical Analysis

Study data were analyzed by using GraphPad Prism 9.0 software. One-way or two-way analysis of variance (ANOVA), followed by Dunnett’s test, was used to compare multiple groups. Quantitative data were expressed as mean ± SD, and statistical charts were generated based on these statistical results. A p value of less than 0.05 was considered statistically significant.

3. Result

3.1. Theogallin Protected Myocardia Against I/R Injury in Isolated Normal Rat Hearts by Classic Langendorff Perfusion

There have been no previous reports on the cardiac-protective effects of theogallin. However, its precursor, gallic acid, has demonstrated protective effects in isolated rat hearts. To assess whether theogallin could protect against I/R injury, an ex vivo I/R model was first used with isolated rat hearts. The schematic of Langendorff’s perfusion setup is shown in Figure A. The results indicated that heart rates remained consistent across all groups, regardless of theogallin pretreatment, at every time point (Figure B). No significant differences were observed in LVDP (Figure C), ± dP/dt (Figure D,E), CF (Figure F), or RPP (Figure G) during the preischemic phase (time point: 0–20 min). Following 30 min of ischemia, there was a significant decline in hemodynamic parameters in the I/R group. In contrast, all hemodynamic parameters showed significant improvement in the theogallin groups compared with the I/R group (Figure C–G). Moreover, this recovery was more pronounced with higher doses of theogallin (200 ng/mL) compared to the lower dose (100 ng/mL). These findings suggest that theogallin pretreatment significantly alleviated I/R-induced contractile dysfunction in this ex vivo model.

3.2. Theogallin Pretreatment Ameliorated Myocardial Ischemia Reperfusion Injury in LAD Ligation Rats

To further investigate the cardioprotective effects of theogallin, we established an in vivo model of myocardial I/R injury by ligating the left anterior descending coronary artery in rats. The rats received theogallin at doses of either 5 or 20 mg/kg/day for 7 days prior to the LAD procedure. Cardiac function during myocardial I/R injury was assessed using echocardiography (Figure A–D). In the sham group, the EF, FS, LVIDs, and LVESV values were approximately 90.37 ± 0.90%, 56.16 ± 1.38%, 2.94 ± 0.28 mm, and 66.00 ± 16.73 μL, respectively, consistent with previous findings. Compared to the sham group, the I/R group showed significant reductions in EF (66.50 ± 3.67%) and FS (33.05 ± 3.23%), along with significant increases in LVIDs (5.31 ± 0.34 mm) and LVESV (364.00 ± 64.27 μL; Figure E–H).

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Theogallin pretreatment attenuated myocardial I/R injury in LAD ligation rats. (A-D) M-mode echocardiography of the long axis of the heart, (A) Sham group, (B) I/R group, (C) 5 mg TOG group, and (D) 20 mg TOG group, (E) Left ventricular ejection fraction (LVEF), (F) fractional shortening (FS), (G) left ventricular internal dimension systole (LVIDs) and, (H) left ventricular end-systolic volume (LVESV) were assessed by echocardiography. (I-L) Statistical results of myocardial enzyme in each group, (M) TTC-stained sections, (N) Statistical results of myocardial infarction size in each group. All data are presented as mean ± SD, n = 5. ** p < 0.01, *** p < 0.001 vs sham group, ## p < 0.01, ### p < 0.001 vs I/R group. I/R, ischemia/reperfusion; 5 mg TOG, 5 mg/kg/day theogallin; 20 mg TOG, 20 mg/kg/day theogallin.

However, pretreatment with 20 mg/kg/day theogallin significantly improved cardiac function, restoring EF and FS to 81.18 ± 3.68% and 44.72 ± 3.62%, respectively. Additionally, LVIDs and LVESV were reduced to 3.37 ± 0.56 mm and 114.00 ± 43.36 μL, respectively. The lower dose of theogallin (5 mg/kg/day) only reduced LVIDs and LVESV without significantly affecting EF or FS. These results indicate that I/R injury worsens myocardial contraction dysfunction, and theogallin pretreatment, particularly at a higher dose, offers significant protection against this dysfunction.

The release of cardiac enzymes, such as LDH, HBDH, CK, and CK-MB, serves as a crucial indicator of cardiac injury. To evaluate myocardial injury, we measured the serum activities of these enzymes. The results showed that, compared to the sham group, the I/R group exhibited significantly elevated levels of LDH, HBDH, CK, and CK-MB (Figure I–L), indicating that myocardial injury occurred following LAD ligation. However, pretreatment with theogallin at 20 mg/kg/day significantly reduced the abnormal activities of these enzymes, while the lower dose of 5 mg/kg/day did not produce significant effects. Additionally, the myocardial infarct size is another important indicator of I/R injury. As illustrated in Figure M,N, theogallin at a 20 mg/kg/day dosage significantly decreased the area of myocardial infarction caused by I/R injury, whereas the 5 mg/kg/day dosage had no effects. These findings further support the protective action of theogallin at 20 mg/kg/day against I/R-induced cardiac injury in rats.

3.3. Theogallin Inhibited Cardiomyocyte Necrosis and Fibrosis in LAD Ligation Rats

After I/R injury, the rat hearts showed signs of myocardial necrosis, infiltration of inflammatory cells, disordered arrangement of myocardial fibers, indistinct nuclei, and collagen fiber deposition. These changes were assessed by H&E and Masson staining, respectively. The histological analysis revealed that the I/R group exhibited significant myocardial damage. In contrast, pretreatment with theogallin at 20 mg/kg/day preserved the normal structure of myocardial fibers, reduced inflammatory cell infiltration, and lowered the degree of myocardial fibrosis (Figure I). These findings further indicate that theogallin effectively mitigates myocardial I/R injury in rats.

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Theogallin alleviated IR-induced myocardial histopathological damage and myocardial fibrosis in LAD ligation rats. Histopathological pictures of heart tissue sections were stained with H&E, and myocardial fibrosis pictures of heart tissue sections were stained with Masson. (A) and (E) Sham group, (B) and (F) I/R group, (C) and (G) 5 mg TOG group, and (D) and (H) 20 mg TOG group. (I) Statistical results of fibrosis area in each group. All data are presented as mean ± SD, n = 4. *** p < 0.001 vs sham group, ### p < 0.001 vs I/R group. I/R, ischemia/reperfusion; 5 mg TOG, 5 mg/kg/day theogallin; and 20 mg TOG, 20 mg/kg/day theogallin.

3.4. The Potential Action Mechanism of Theogallin on I/R Injury was Related to IL-17 Signaling Pathway by Bioinformatics Analysis

To identify the core genes associated with myocardial I/R injury, the GSE58486, GSE193997, and GSE214122 data sets were analyzed using the limma and GEOquery packages of the GEO2R web tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/). From the GSE58486 data set, we identified 1190 DEGs, comprising 661 upregulated and 529 downregulated genes (Figure A).The GSE193997 data set revealed 1375 DEGs, with 791 upregulated genes and 584 downregulated genes (Figure B). Finally, the GSE214122 data set included 1194 DEGs, consisting of 840 upregulated genes and 354 downregulated genes (Figure C). A subsequent search for DEGs common to all three data sets identified 255 shared DEGs (Figure D).

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Screening of key genes for myocardial I/R injury in the GEO database and analysis of GO and KEGG enrichment Volcano maps for the (A) data set of GSE58486, (B) data set of GSE193997, (C) data set of GSE214122, and (D) Venn diagrams of common targets in three myocardial ischemia reperfusion injury data sets. (E) Three data set common target protein interaction network. (F) Ranking of core targets for myocardial ischemia reperfusion injury. (G) Enrichment results of biological processes. (H) Enrichment results of KEGG pathways. (I) Multiple common targets were enriched in the IL-17 signaling pathway.

To further explore the biological characteristics of these DEGs, a PPI network was created by using the STRING database, which revealed 252 nodes and 908 edges (Figure E). We then utilized Cytoscape to identify hub genes, ultimately pinpointing ten key genes (Figure F): Mmp9, Itgam, Ptgs2, Il1b, Ccl2, Timp1, Cd44, Cxcl1, Serpine1, and Thbs1, with Mmp9 identified as the most critical target.

To further elucidate the main pathways enriched by these 255 DEGs, we conducted GO analyses. The results of the biological process (BP) from the GO enrichment analyses showed that the main pathways involving inflammatory response included regulating neutrophil chemotaxis, inflammatory response, and leukocyte migration (Figure G). In the subsequent KEGG analysis, more attention was paid to inflammation-related signaling pathways. The 255 DEGs were entered into the DAVID database for KEGG pathway enrichment. The signaling pathways related to inflammation in the top 5 KEGG signaling pathways (Figure H) primarily comprised the IL-17 signaling pathway and the TNF signaling pathway. The IL-17 signaling pathway was ultimately selected based on the correlation ranking. The top three common targets, MMP9, CXCL2, and CCL2, were all identified as downstreamed proteins of the IL-17 signaling pathway (Figure I). These findings suggest that the IL-17 signaling pathway may play a pivotal role in the pathogenesis of myocardial I/R injury.

3.5. Theogallin Protected Against I/R Injury via IL-17 Signaling Pathway in LAD Ligation Rats: Insights from Molecular Docking and Western Blot

Key proteins involved in the IL-17 signaling pathway are shown in Figure I. Among the 255 DEGs identified in this study, three proteins associated with the upstream of the IL-17 pathway were detected: IL-17RA, AP-1 (including JUNB and FRA1), and MMP9. Molecular docking analyses were conducted to evaluate the potential interaction between theogallin and these targets to explore the mechanism of its cardioprotective effects. Under optimized docking conditions, theogallin binding energies were −6.8 kcal/mol for IL-17RA (Figure A), −5.6 kcal/mol for JUNB (Figure B), −5.8 kcal/mol for FRA1 (Figure C), and −7.1 kcal/mol for MMP9 (Figure D), respectively. Specifically, theogallin showed a strong hydrogen bonding interaction with the following amino acid residues: IL-17RA: GLU506, 3.4 Å; VAL507, 3.7 Å; ASP512, 2.9 Å; ARG452, 2.3 Å; TRP584, 2.3 Å; and GLN578, 3.0 Å, forming a total of 7 hydrogen bonds. JUNB: THR283, 3.0 Å; ARG279, 3.1 Å; and ASN278, 2.5 Å of JUNB, forming a total of 6 hydrogen bonds. FRA1: GLN55, 2.0 Å; VAL54, 4.3 Å; ILE41, 2.0 Å; MET44, 2.9 Å9; and GLN51, 2.2 Å, forming a total of 6 hydrogen bonds. MMP9: GLU157, 3.8 Å; VAL170, 2.6 Å; ASN177, 2.6 Å; and SER107, 3.5 Å, forming a total of 5 hydrogen bonds. These docking results suggest that theogallin binds effectively with these four receptor proteins involved in the IL-17 signaling pathway.

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Molecular docking and Western blot experiments demonstrated that the protective effect of theogallin on myocardial ischemia reperfusion injury was related to the IL-17 signaling pathway. (A) Molecular docking of theogallin and IL-17RA, (B) molecular docking of theogallin and JUNB, (C) molecular docking of theogallin and FRA1, and (D) molecular docking of theogallin and MMP9. (E–H) Western blot results of the effects of theogallin on the expression of IL-17RA, JUNB, FRA1, and MMP9 in IR-treated rat hearts. All data are presented as mean ± SD, n = 3. ** p < 0.01, *** p < 0.001 vs sham group, # p < 0.05, ## p < 0.01 vs I/R group. IL-17, interleukin-17; IL-17RA, IL-17 receptor A; JUNB, transcription factor jun-B; FRA1, Fos-related antigen 1; MMP9, matrix metalloproteinase 9; I/R, ischemia/reperfusion; and TOG, 20 mg/kg/day theogallin.

To further validate the bioinformatics analysis and molecular docking findings, Western blotting was conducted to evaluate the expression of core proteins in rat heart tissue, including IL17-RA, JUNB, FRA1, and MMP9. The results showed that I/R significantly upregulated the expression of IL17-RA, JUNB, FRA1, and MMP9 (Figure E–H). However, pretreatment with 20 mg/kg/day theogallin notably reduced the expression levels of these proteins, indicating that theogallin can modulate the IL-17 signaling pathway to exert cardioprotective effects.

3.6. Theogallin Inhibited Myocardial Fibrosis by Modulating the TGF-β/SMAD Signaling Pathway

The upregulation of the IL-17 signaling pathway, as demonstrated in the study, leads to excessive activation of MMP9. This, in turn, enhances TGF-β1 bioactivity by cleaving latent TGF-β-binding protein-1. The TGF-β/SMAD pathway is well known for its association with cardiac fibrosis and its significant role in promoting fibrotic development. To further understand the effects of theogallin on this pathway, we analyzed the TGF-β/SMAD pathway in rat hearts (Figure A–C). Western blotting analysis revealed that I/R induced the upregulation of TGF-β1, p-SMAD2, and p-SMAD3. These upregulations were significantly reduced by pretreatment with theogallin. This finding indicates that theogallin suppresses the TGF-β/SMAD pathway, which is a downstream pathway of IL-17, contributing to its protective effects against I/R injury in LAD ligation rats. Therefore, inhibition of the TGF-β/SMAD pathway by theogallin plays a crucial role in mitigating cardiac fibrosis and I/R-induced myocardial damage.

6.

6

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Theogallin reduced myocardial fiber-associated protein levels induced by myocardial ischemia reperfusion injury in LAD ligation rat hearts. (A) TGF-β1 protein bands and statistical results, (B) SMAD2 phosphorylated protein and its total protein bands and their statistics, and (C) SMAD3 phosphorylated protein and its total protein bands and their statistics. All data are presented as mean ± SD, n = 3. ** p < 0.01, *** p < 0.001 vs sham group, # p < 0.05, ### p < 0.001 vs I/R group. I/R, ischemia/reperfusion; TOG, 20 mg/kg/day theogallin.

4. Discussion

Myocardial I/R injury is a pathological process in which myocardial tissue undergoes ischemia followed by reperfusion, exacerbating tissue damage and impairing cardiac function. This can result in irreversible harm, amplify myocardial infarction, and contribute to ventricular remodeling, which can further impair heart function and structure. Recent research highlights the protective role of tea polyphenols in cardiovascular diseases, including I/R injury, through their anti-inflammatory and antioxidant properties. , Gallic acid, a precursor of theogallin, has been reported to mitigate myocardial I/R injury by inhibiting inflammation, reducing oxidative stress, and preventing the opening of mitochondrial permeability transition pores. , Similarly, quinic acid has been shown to inhibit inflammation in cardiovascular disease. , Our study provides novel evidence that theogallin, a polyphenolic compound derived from both gallic acid and quinic acid in tea, may also protect the heart against I/R injury, potentially through its anti-inflammatory effects. To the best of our knowledge, this is the first report on the cardioprotective effects of theogallin.

In this study, we first utilized the Langendorff isolated heart perfusion technique to establish a myocardial I/R injury model in isolated rat hearts, aiming to determine whether theogallin exerts its protective effects through direct action on cardiomyocytes. Theogallin significantly improved key hemodynamic indexes, including LVDP, RPP, CF, and dP/dt. To further assess its efficacy in vivo, we employed an LAD ligation model in rats, which more closely mimics the complex pathophysiological environment of myocardial I/R injury. The results demonstrated that theogallin significantly inhibited the release of myocardial enzymes, improved myocardial contractility, and reduced the extent of myocardial infarction in LAD ligation rats. The protective effects observed in the Langendorff model are consistent with the in vivo outcomes, supporting the notion that theogallin’s direct myocardial actions contribute to its overall cardioprotective efficacy.

Understanding the molecular mechanisms underlying the cardioprotective effects of theogallin is crucial. Bioinformatics analysis identified the IL-17 signaling pathway as highly correlated with myocardial I/R injury. Molecular docking and Western blotting results revealed that theogallin binds closely to IL17-RA, JUNB, FRA1, and MMP9 targets, suggesting that its cardioprotective effects may be mediated through these proteins. These findings provide insights into the potential molecular mechanisms through which theogallin exerts its protective role against myocardial I/R injury.

The IL-17 signaling pathway plays a significant role in regulating inflammation, the immune response, and tissue repair. Research shows that IL-17A administration enhances neutrophil recruitment during I/R, while its inhibition reduces myocyte necrosis and apoptosis in rat hearts following I/R injury. IL-17A also negatively affects ventricular remodeling after a myocardial infarction, contributing to myocardial injury. IL-17A binds to the IL-17RA/IL-17RC complex, activating proinflammatory pathways and triggering inflammatory responses. AP-1, a dimeric transcription factor composed of proteins from the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra1, and Fra2) and activating transcription factor protein families, plays a crucial role in cellular processes such as differentiation, proliferation, apoptosis, and autophagy. Palomer et al. demonstrated that a deficiency in Fos proteins reduces cardiac inflammation by downregulating IL-6 and monocyte chemoattractant protein-1. Moreover, activation of c-Fos, Fosl1, and FosB has been linked to increased proinflammatory gene expression and macrophage recruitment after myocardial infarction. The administration of Fos/AP-1 inhibitors, such as T5224, effectively suppresses cardiac inflammation, decreases infarct size, and mitigates cardiac remodeling and heart failure. This study shows that theogallin modulates the expression of JUNB and FRA1, key proteins in the AP-1 complex, suggesting that theogallin’s cardioprotective effects may be mediated through its regulation of the IL-17 signaling pathway and suppression of AP-1 activity. These findings highlight the potential of theogallin as a therapeutic agent to reduce cardiac remodeling and dysfunction following I/R injury by targeting proinflammatory pathways.

The above bioinformatics analysis and experimental results led us to focus on the IL-17 signaling pathway. Studies have found that MMP9 plays a major mediating role in the pathogenesis of myocardial injury, inducing inflammation, apoptosis, and abnormalities in the ECM. MMPs, including MMP9, are zinc-dependent enzymes involved in ECM remodeling, and their elevated activity can lead to ECM damage and impaired cardiac contractility, as seen in conditions like myocardial infarction. Following I/R injury, cardiac cell death initiates an inflammatory phase, marked by the infiltration of neutrophils and monocytes to clear damaged tissue. , In the subsequent proliferative phase, myofibroblasts produce ECM proteins, which contribute to scar tissue formation. − Various cell typesincluding fibroblasts, cardiomyocytes, and immune cellsare capable of producing MMPs and tissue inhibitors of metalloproteinases. − The IL-17 signaling pathway plays a central role by recruiting neutrophils and macrophages to the injured myocardium, where these immune cells secrete MMP9. This enzyme contributes to myocardial I/R injury by breaking down ECM components and facilitating postinjury remodeling. Notably, MMP9 has been shown to activate TGF-β1 bioactivity by cleaving its latent binding protein, which is crucial for the development of myocardial fibrosis. , TGF-β1 is initially produced as a latent complex that binds to the ECM or is stored in the extracellular environment. By degrading ECM components, MMP9 exposes and releases latent TGF-β1, activating it. As an active and potent profibrotic cytokine, TGF-β1 drives cardiac fibrosis by promoting fibroblast activation, differentiation into myofibroblasts, and excessive ECM deposition. This MMP9-mediated activation of TGF-β1 amplifies profibrotic signaling. In addition, MMP9 also plays a role in promoting EMT and autophagy by activating the TGF-β/SMAD signaling pathway. In this process, TGF-β1 interacts with its receptors, leading to the phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4. This SMAD complex translocates to the nucleus to regulate gene expression, driving fibrosis and remodeling of heart tissue after injury.

In this study, activation of the IL-17 signaling pathway and its related proteins was observed following I/R injury. Theogallin, however, significantly inhibited these changes, suggesting its potential role in preventing myocardial fibrosis. By modulating the IL-17 pathway and downstream components like MMP9 and the TGF-β/SMAD axis, theogallin may offer protective effects against I/R-induced cardiac injury and fibrosis. These findings point to theogallin as a promising therapeutic agent for reducing cardiac remodeling and preserving heart function postinjury.

The NLRP3 inflammasome also plays a significant role in myocardial I/R injury. , The formation of the NLRP3 inflammasome in the myocardium during acute cardiac ischemia is likely to exacerbate the inflammatory response, thereby contributing to further tissue injury. Existing evidence indicates that the IL-17 signaling pathway can indirectly enhance the activation of the NLRP3 inflammasome by inducing reactive oxygen species (ROS) generation and releasing proinflammatory cytokines, thereby providing critical upstream signals for NLRP3 activation. Conversely, NLRP3-mediated release of IL-1β and IL-18 can promote Th17 cell differentiation, further amplifying the inflammatory effects of IL-17. Both IL-17 and NLRP3 contribute to neutrophil infiltration, proinflammatory cytokine release, and myocardial cell death through distinct yet interconnected pathways. Their synergistic interaction may establish a positive feedback loop of inflammation, exacerbating myocardial injury and fibrosis. This interplay between IL-17 and NLRP3 highlights a promising avenue for further investigation. Moreover, exploring whether theogallin, as a polyphenolic compound, can inhibit NLRP3 activation or mitigate oxidative stress represents a critical focus of our future research endeavors.

Theogallin is a unique compound found in tea. Initially, we planned to administer it via gavage to better mimic tea consumption. However, due to the current lack of large-scale production, the required oral dosage was impractically high. While intraperitoneal injection bypasses gastrointestinal digestion and absorption, a portion of the compound still reaches the liver through the portal vein, maintaining some pharmacokinetic similarity to oral administration. Although pharmacokinetic data for theogallin are currently unavailable, compounds with similar structures, such as chlorogenic acid, have shown moderate bioavailability, implying that theogallin may exhibit comparable absorption behavior. Furthermore, our unpublished in vitro digestion experiments demonstrated minimal theogallin loss during the gastrointestinal phase, while in vitro fermentation studies suggested that gut microbiota metabolize only about 20% of the compound. These findings indicate that the pharmacokinetic differences between gavage and intraperitoneal administration may be limited, supporting the feasibility of intraperitoneal administration as an alternative.

In addition, since the toxicity of theogallin has not been previously evaluated, we performed preliminary assessments, including a CCK-8 assay to measure the survival rate of H9c2 cells treated with theogallin and an in vitro cardiac perfusion study to examine whether theogallin affects hemodynamic parameters, thereby confirming that the selected concentration does not adversely impact cardiac function. However, these are only initial evaluations and are insufficient to fully establish its safety. A more comprehensive toxicity assessment, including systemic toxicity and long-term safety evaluation, will be conducted once a sufficient quantity of theogallin has been extracted. Oral gavage studies will also be conducted to directly compare the pharmacological effects of intraperitoneal and oral administration routes.

This study demonstrated the efficacy of theogallin in preventing myocardial I/R injury through both ex vivo and in vivo experiments. Theogallin appears to exert anti-inflammatory and antifibrotic effects by modulating the IL-17 signaling pathway and its core targets, which play a crucial role in the pathogenesis of myocardial injury. By inhibiting inflammatory responses and fibrotic processes, theogallin shows potential as a cardioprotective agent. These findings suggest that theogallin could serve as a novel therapeutic option for the clinical prevention and treatment of myocardial I/R injury.

F.Z.: data curation, software, writing original draft, writingreview and editing. H. C.: data curation, software. Z.L.: data curation, software. A.C.: data curation, software. T.Y.: formal analysis, methodology. H.G.: writingreview and editing. S.H.: writingreview and editing. Z.X.: Writingreview and editing. D. L.: funding acquisition, writingoriginal draft, writingreview and editing, supervision, conceptualization.

This work was supported by the earmarked fund for CARS (CARS-19).

The authors declare no competing financial interest.

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