Tanshinone-IIA Mediated Attenuation of Post-Infarction Myocardial Fibrosis via IL-33/ST-2 Signaling Changes in Rat Epicardial Adipose Tissue

Summary

This study demonstrates that Tanshinone IIA (Tan IIA) mitigates post-myocardial infarction fibrosis, likely via IL-33/ST2 modulation, as shown in animal models and molecular docking, and validated by ELISA and Western blot.

Abstract

Myocardial infarction (MI), a severe form of coronary artery disease, often results in myocardial fibrosis, a key contributor to cardiac dysfunction and heart failure. Epicardial adipose tissue (EAT) is increasingly implicated in cardiovascular pathology through cytokine-mediated modulation of cardiac function. Tanshinone IIA (TanIIA), a lipophilic compound derived from Salvia miltiorrhiza, exhibits anti-inflammatory, antioxidant, and anti-fibrotic properties. This study aimed to evaluate the therapeutic potential of TanIIA in attenuating post-MI myocardial fibrosis and to explore its underlying mechanisms using an integrative approach combining in vivo experiments and in silico analyses. A murine MI model was established, and molecular docking was employed to assess TanIIA binding to key components of the interleukin-33/growth stimulation expressed gene 2 (IL-33/ST2) signaling pathway. Our results showed that TanIIA treatment significantly enhanced cardiac function, reduced histological injury, and mitigated myocardial fibrosis, alongside decreased serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I (hs-cTnI). TanIIA also attenuated inflammatory responses and oxidative stress in the serum, myocardium, and EAT. Mechanistically, TanIIA downregulated IL-33 and ST2 expression, suppressed the myeloid differentiation factor 88 (MyD88)/nuclear factor kappa-B (NF-κB) pathway, and reduced transforming growth factor-β1 (TGF-β1) levels. In silico analyses further revealed that TanIIA exhibited strong binding affinities for IL-33, ST2, MyD88, and NF-κB p65, with the most stable interaction predicted for ST2. Collectively, these findings indicate that TanIIA alleviates MI-induced myocardial fibrosis by modulating EAT-associated inflammation and oxidative stress, potentially via multi-target inhibition of the IL-33/ST2 axis. These results support TanIIA as a promising therapeutic candidate for the management of post-infarction myocardial fibrosis.

Introduction

Myocardial infarction, pathologically defined as cardiomyocyte death due to prolonged ischemia, remains a life-threatening cardiovascular event¹. Despite significant advances in reperfusion strategies and pharmacotherapy, MI continues to be a leading cause of morbidity and mortality worldwide^2,3,4,5,6. A key pathophysiological consequence of MI is myocardial fibrosis, the excessive deposition of extracellular matrix proteins, which is a major determinant of subsequent adverse clinical outcomes⁷. This fibrotic scarring, while essential for preventing ventricular wall rupture, often becomes a maladaptive repair process initiated by complex inflammatory and intercellular signaling pathways^5,7,8,9,10. By replacing functional myocardium with non-contractile scar tissue, maladaptive fibrosis leads to increased ventricular stiffness, progressive cardiac dysfunction, and ultimately, heart failure^9,11. Therefore, investigating novel pharmacological treatments that specifically target and attenuate post-infarction fibrosis is a critical therapeutic goal for improving long-term patient morbidity and mortality.

Salvia miltiorrhiza (Danshen), a traditional Chinese medicine, and its various preparations are widely employed for the management of cardiovascular diseases. Tanshinone IIA, its principal lipophilic constituent, is credited with multiple cardioprotective properties, including anti-atherosclerotic, anti-myocardial ischemic, and antioxidant effects^12,13,14,15. Mechanistically, TanIIA exerts its anti-inflammatory and anti-fibrotic effects by modulating diverse signaling pathways^{13,14,15,16,17}. Notably, TanIIA has been demonstrated to directly target adipocytes, suppressing their inflammatory responses and thereby ameliorating cellular dysfunction¹⁸. However, whether these protective effects of TanIIA extend to the epicardial adipose tissue remains to be elucidated.

Epicardial adipose tissue, a unique visceral adipose depot, is strongly implicated in the pathogenesis of coronary artery disease (CAD), atrial fibrillation (AF), and heart failure. Anatomically located between the myocardium and the visceral layer of epicardium without an intervening fascial layer, EAT facilitates direct paracrine communication with the adjacent cardiac structures^19,20,21,22. In its physiological state, EAT is cardioprotective, providing metabolic support and releasing anti-inflammatory and antioxidant adipokines^{20,21,23,24,25,26}. Conversely, during pathological conditions, EAT becomes a source of pro-inflammatory, pro-oxidative, and pro-fibrotic mediators that drive adverse cardiac remodeling^27,28,29,30. Consequently, the hypothesis was formulated that TanIIA may inhibit myocardial fibrosis by attenuating inflammatory responses and oxidative stress in epicardial fat, potentially via a novel signaling pathway.

Interleukin-33, a member of the IL-1 family of cytokines³¹, is recognized for its potent anti-hypertrophic and anti-fibrotic properties in the cardiovascular system²⁵. Its biological effects are mediated through a distinct receptor complex, ST2, which exists in two isoforms: a transmembrane receptor (ST2L) that initiates signaling, and a soluble decoy receptor (sST2) that sequesters IL-33³². Clinically, serum sST2 levels are acutely elevated following myocardial infarction (MI) and serve as a robust prognostic marker for adverse outcomes in heart failure and myocarditis^33,34,35,36. The canonical IL-33/ST2L signaling cascade involves the recruitment of the adaptor protein MyD88 and subsequent activation of TRAF6, leading to the phosphorylation and degradation of the NF-κB inhibitor (IκB) and the consequent nuclear translocation of NF-κB^37,38. This pathway is intricately linked with other key inflammatory cytokines; for instance, IL-33/MyD88 signaling can promote the secretion of the anti-inflammatory cytokine IL-10³⁹, while the pro-inflammatory cytokine TNF-α can, in turn, amplify the inflammatory response by activating NF-κB to induce IL-33 expression^40,41. Importantly, both IL-33 and ST2 are expressed in adipose tissue, where they are thought to exert local anti-inflammatory effects^31,42,43. However, whether the cardioprotective effects of TanIIA involve the modulation of the IL-33/ST2 axis remains unexplored.

Therefore, the present study was designed to test the central hypothesis that TanIIA prevents myocardial fibrosis by mitigating inflammation and oxidative stress arising from epicardial adipose tissue via modulation of the IL-33/ST2 pathway. Specifically, we hypothesized that this therapeutic action is driven by the direct, multi-target engagement of TanIIA with key proteins in this signaling cascade. To validate this, we adopted a dual approach. First, a rat model of myocardial infarction was utilized to confirm the in vivo efficacy of TanIIA in attenuating adverse cardiac remodeling. Second, to provide a direct molecular basis for these observations, computational docking was employed to characterize the potential binding of TanIIA to four key nodes of the pathway—IL-33, ST2, MyD88, and NF-κB p65. By investigating the mechanism from the tissue level down to specific molecular binding events, this study aims to establish a novel mechanistic framework for the cardioprotective effects of TanIIA.

Protocol

All experimental procedures and animal welfare practices were conducted in strict accordance with the Ethical Regulations on the Care and Use of Laboratory Animals of the Ethical Animal Committee of the Jiangxi University of Chinese Medicine (No. JZLLSC2023-0702), China.

Molecular docking simulations
An in silico molecular docking approach was employed to explore direct interactions between TanIIA and IL-33, ST2L, MyD88, and NF-κB p65. The 3D coordinates of TanIIA were obtained from the PubChem database, and the initial SDF file was subsequently converted to a PDB format. The crystal structure of IL-33 (PDB ID: 4KC3), ST2L (PDB ID: 4KC3), MyD88 (PDB ID: 7L6W), and NF-κB p65 (PDB ID: 1NFI) was sourced from the protein data bank. Initial processing of the receptor was performed in PyMOL (v2.3.4), where water molecules and original ligands were removed, followed by further preparation in AutoDockTools for hydrogen addition and charge assignment. Both molecules were ultimately converted to the PDBQT format. AutoDock Vina (v1.1.2) was utilized to perform the docking simulation within a defined grid box centered on the active site. The optimal binding pose was selected based on the lowest binding energy, and detailed interactions were identified using the PLIP web tool. The final docked conformation was visualized using PyMOL (v2.3.4).

Animals
Male Sprague-Dawley (SD) rats, SPF class, aged 6 weeks and weighing 200 ± 20g (certificate No. SCXK(Su)2023-0009). The rats were maintained under standardized conditions, including a 12 h light/dark cycle and a temperature of 22 °C ± 2 °C. The animals were permitted unlimited access to both food and water during the experiment.

Experimental protocol
A rat model of myocardial infarction was established by ligating the left anterior descending (LAD) coronary artery. Sham-operated rats underwent the same surgical procedures without LAD ligation. Briefly, rats were anesthetized via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg), followed by endotracheal intubation and ventilation. A left thoracotomy was performed to expose the heart. The LAD was then ligated with a 4-0 non-absorbable suture. Successful MI induction was confirmed by ST-segment elevation on a continuous electrocardiogram and visible blanching of the left ventricular anterior wall. Post-surgery, rats received cefoxitin sodium (200 mg/kg/d, i.p.) for three days. Twenty-four hours after surgery, the surviving rats were randomly assigned to six groups (n = 10 per group):

Sham group: received intraperitoneal injections of saline (1 mL·kg^-1·day^-1).

MI group: received intraperitoneal injections of saline (1 mL·kg^-1·day^-1).

TanIIA-L group (low-dose): received intraperitoneal injections of TanIIA (7.5 mg·kg^-1·day^-1).

TanIIA-M group (medium-dose): received intraperitoneal injections of TanIIA (15 mg·kg^-1·day^-1).

TanIIA-H group (high-dose): received intraperitoneal injections of TanIIA (30 mg·kg^-1·day^-1).

XST group (positive control): received oral gavage of valsartan (30 mg·kg^-1·day^-1).

All treatments were administered daily for 28 consecutive days. The doses of TanIIA and XST were selected based on previous studies and our preliminary unpublished data^44,45.

Electrocardiogram (ECG) and echocardiography
After 28 days of treatment, cardiac function was evaluated by transthoracic echocardiography. Under 2% isoflurane anesthesia (motion mode), M-mode images were acquired to determine left ventricular end-diastolic/systolic diameters (LVIDd/s). Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were subsequently calculated from an average of three consecutive cardiac cycles. Additionally, electrocardiograms were recorded pre-surgery, 15 min post-ligation, and at the experimental endpoint to monitor cardiac electrical activity.

Sampling and 2,3,5-triphenyltetrazolium chloride (TTC) staining
Blood from the abdominal aorta was centrifuged at 1370 × g for 15 min to isolate serum, which was stored at -80 °C. The heart and epicardial adipose tissue were rapidly excised and weighed. For molecular analyses, EAT and infarct border zone tissues were snap-frozen in liquid nitrogen and stored at -80 °C. Other tissue samples were fixed in 4% paraformaldehyde for histological examination. Additionally, selected hearts were frozen at -20 °C, sectioned, and stained with TTC to assess infarct size. Materials and animal tissues are used and handled in compliance with laboratory safety regulations.

Histological examination
For histopathological evaluation, paraffin sections were subjected to hematoxylin and eosin (H&E) staining to observe myocardial morphology. Masson's trichrome staining was utilized to assess the extent of myocardial fibrosis and the size of epicardial adipocytes, with the collagen volume fraction (CVF) being quantified. Additionally, the ratio of type I to type III collagen in the non-infarcted area was determined by picrosirius red (PSR) staining. All sections were digitally scanned for quantitative analysis.

Immunohistochemistry
Paraffin-embedded myocardial sections were deparaffinized and rehydrated. Antigen retrieval was performed using a citrate buffer (pH 6.0). Sections were blocked with 3% BSA, then incubated overnight at 4 °C with primary antibodies against collagen I, collagen III, IL-33, ST2, MyD88, NF-κB p65, and TGF-β1. Subsequently, sections were incubated with an HRP-labeled secondary antibody for 1 h at room temperature. Staining was visualized with DAB, and nuclei were counterstained with hematoxylin. Images were captured using a digital scanner and analyzed with image analysis software to quantify the integrated optical density (IOD) of positive staining.

Enzyme-linked immunosorbent assay
The concentrations of hs-cTnI, NT-proBNP, IL-33, and ST2 were quantified in serum samples. Additionally, IL-10 and TNF-α levels were measured in serum, myocardial homogenates, and epicardial adipose tissue lysates. All assays were performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocols.

Western blotting
Total protein was extracted from tissue homogenates, and concentrations were determined by bicinchoninic acid (BCA) assay. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with primary antibodies against IL-10, tumor necrosis factor-α (TNF-α), IL-33, ST2, NF-κB p65, TGF-β1, and GAPDH (1:1000). Subsequently, membranes were incubated with an HRP-conjugated secondary antibody (1:5000) and visualized using an ECL reagent. Band intensities were quantified with software, and protein levels were normalized to GAPDH as an internal control.

Statistical analysis
All statistical analyses were performed using medical statistics software. Data is presented as the mean ± standard error of the mean. For comparisons between two independent groups, a t-test was employed. In the case of multiple groups, either a one-way or a two-way ANOVA followed by a Tukey post hoc test was applied. For all analyses, a P < 0.05 was considered statistically significant.

Results

Effects of TanIIA on cardiac function in rats with myocardial infarction (Figure 1)
Health status and body weight changes: Rats in the MI group exhibited poor general condition, with abdominal distension, while rats in the TanIIA groups showed improved health status, increased food and water intake, and increased activity. After 4 weeks, a significant increase in body weight was observed among the rats in the TanIIA group in comparison t...

Discussion

The present study investigates the effects of TanIIA on myocardial fibrosis after myocardial infarction, along with the underlying mechanisms involved. The primary findings are as follows: (1) TanIIA attenuates adverse cardiac remodelling and improves cardiac function by inhibiting cardiac fibrosis. (2) TanIIA has been found to inhibit myocardial fibrosis by attenuating inflammatory responses and oxidative stress in epicardial fat. (3) TanIIA mediates antifibrotic effects through the IL-33/ST2 signalling pathway.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCA Protein Quantification Kit	Thermo Fisher Corporation	PICPI23223
CAT kit	Shanghai Enzyme Link Biotechnology	202211202
Collagen I antibody	Wuhan Servicebio Biotechnology Co., Ltd	GB114197
Collagen III antibody	Shenyang Wanleibio Biotechnology Co., Ltd	WL03186
GAPDH antibody	Cell Signaling Technology Corporation	#5174
GraphPad Prism	GraphPad Software Corporation	version 9.0
GSH kit	Shanghai Enzyme Link Biotechnology	202211131
H&E staining solution	Beijing Solarbio Technology Co., Ltd	H8070;G1100
HRP-labeled goat anti-rabbit IgG antibody	Beijing Solarbio Technology Co., Ltd	SE134
Hs-cTnI  kit	Shanghai Yuanjie Biotechnology Co., Ltd	20220808
IL-10 antibody	Shenyang Wanleibio Biotechnology Co., Ltd	WL03088
IL-10  kit	Shanghai Yuanjie Biotechnology Co., Ltd	202211172
IL-33  kit	Shanghai Yuanjie Biotechnology Co., Ltd	202211192
IL-33 antibody	Shenyang Wanleibio Biotechnology Co., Ltd	WL04438
ImageJ software	National Institutes of Health	version Fiji
Image-Pro	Media Cybernetics, USA	version 6.0
Male Sprague-Dawley (SD) rats	Jiangsu Jicui Yaokang Biotechnology Co., Ltd	SCXK(Su)2023-0009
Masson staining kit	Wuhan Bolf Biotechnology Co., Ltd	20221218
MDA kit	Shanghai Enzyme Link Biotechnology	202211171
MyD88 antibody	Shenyang Wanleibio Biotechnology Co., Ltd	WL02494
NT-proBNP  kit	Shanghai Yuanjie Biotechnology Co., Ltd	20220805
Nuclear factor kappa-B(NF-κB)(p65) antibody	Abmart Corporation	TP56372
Open Babel	Open-source cheminformatics toolkit project	version 2.3.2
Polyvinylidene fluoride (PVDF) membranes	Merck Millipore	R9M2784
PSR solution	Beijing Solarbio Technology Co., Ltd	G1472
SOD kit	Shanghai Enzyme Link Biotechnology	202211201
ST2 antibody	Proteintech Group Corporation	11920-1-AP
ST2 kit	Shanghai Yuanjie Biotechnology Co., Ltd	202211183
Statistical Product and Service Solutions (SPSS)	IBM Corporation	version 26.0
Sulfotanshinone Sodium Injection	Shanghai Pharmaceutical First Biochemical Pharmaceutical Co., Ltd	2012303
Super sensitive ECL luminescence reagent	Dalian Meilun Biotechnology Co., Ltd	MA0186
TGF-β1 antibody	ABclonal Corporation	A2124
Titanvin Valsartan Capsule	Hunan Qianjin Xiangjiang Pharmaceutical Co., Ltd	211120
TNF-α  kit	Shanghai Yuanjie Biotechnology Co., Ltd	202211202
TNF-α antibody	Shenyang Wanleibio Biotechnology Co., Ltd	WL01581
TTC solution	Fuzhou Phygene Biotechnology Co., Ltd	20210620
Ultra-high resolution small animal ultrasound imaging system	VEVOcompany	Vevo 2100