Jia-jia Xu1†
Min-wei Chen1†
Long-zhen Lai1Di Xiao1Shu-yan Jiang1Jun-xin Lin1
Zheng-hao Zhang2*
Zhong-gui Shan1*- 1Department of Cardiovascular Surgery, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China
- 2Department of Hematology, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, China
Introduction: Acute myocardial infarction (MI) is a leading global cause of morbidity and mortality, where inflammatory response and programmed cell death (PCD) are critical in disease progression. β-sitosterol (β-SITO), a phytosterol with known cardioprotective effects, has been implicated in cardiovascular diseases, but its specific role and mechanisms in MI remain underexplored.
Methods: This study employed both in vivo and in vitro models. Male C57BL/6J mice with MI were used to evaluate the effects of β-SITO treatment. Cardiac function was assessed via echocardiography, infarct size and fibrosis were analyzed histologically. In vitro, cardiomyocyte viability under hypoxia and TGF-β-induced cardiac fibroblast activation were examined. Mechanistic insights were gained through transcriptomic profiling, molecular docking studies, and validation by Western blotting.
Results: β-SITO treatment significantly reduced myocardial infarct size, alleviated cardiac fibrosis, and improved cardiac function in MI mice. In vitro, it enhanced cardiomyocyte viability under hypoxia and inhibited TGF-β-induced fibroblast activation. Transcriptomic analysis revealed that β-SITO modulated pathways related to immune-inflammatory responses, NF-κB, and necroptosis signaling. Molecular docking confirmed its strong binding affinity to key components of these pathways. Western blotting validated the inhibition of NF-κB activation and necroptosis in both hypoxic cardiomyocytes and MI mouse heart tissue.
Conclusion: β-SITO demonstrates significant therapeutic potential for improving post-MI recovery. Its cardioprotective effects are likely mediated through the modulation of NF-κB and necroptosis signaling pathways, highlighting it as a promising candidate for MI treatment.
1 Introduction
As a prevalent cardiovascular disorder, acute myocardial infarction (MI) remains a leading global health burden, accounting for significant morbidity and mortality worldwide (Zhao et al., 2019; Roth et al., 2020). In general, MI triggers a pathological cascade characterized by coronary artery occlusion, leading to acute myocardial ischemia and hypoxia (Chapman et al., 2025). Dysregulated immune modulation is a key driver of post-MI inflammation. During the acute phase of MI, pro-inflammatory immune cells infiltrate the infarcted myocardium, initiating a robust inflammatory cascade (Ma et al., 2025). Prolonged inflammation not only impairs cardiac repair but also exacerbates cardiomyocyte apoptosis, necrosis, and fibrotic remodeling, ultimately contributing to adverse ventricular remodeling and progressive cardiac dysfunction (Wang et al., 2025a). Programmed cell death (PCD) is a hallmark pathological feature of MI, significantly influencing disease progression (Galluzzi et al., 2018). Given its critical role, therapeutic strategies targeting anti-inflammatory pathways and inhibition of cardiomyocyte death are essential to mitigate post-MI injury (Yang et al., 2022a; Wang et al., 2025b).
Emerging evidence highlights the central involvement of RIPK3-dependent necroptosis and NF-κB signaling in post-MI pathophysiology (Chai et al., 2023; Zhang et al., 2025a; Maslov et al., 2022). Necroptosis, a regulated form of necrosis mediated by RIPK1/RIPK3 and MLKL, is increasingly recognized as a major contributor to ischemic injury (Luedde et al., 2014). Necroptosis not only induces membrane rupture and cell lysis but also facilitates the release of danger-associated molecular patterns (DAMPs), thereby creating a feed-forward loop that intensifies inflammatory responses following MI (Zhang et al., 2019). Previous studies have reported substantial activation of RIPK3 and MLKL in ischemic myocardium, implicating necroptosis as a therapeutic target for attenuating myocardial injury and adverse left ventricular remodeling (Wu et al., 2025a). The NF-κB signaling pathway is another central regulator of MI pathophysiology (Shan et al., 2024). As a master transcriptional controller of inflammatory mediators, NF-κB activation occurs rapidly after ischemic insult and contributes to the production of cytokines, chemokines, and adhesion molecules that propagate inflammatory cell recruitment (Zhuang et al., 2023; Zhang et al., 2025b). Importantly, recent evidence indicates extensive crosstalk between NF-κB signaling and necroptosis. NF-κB can modulate the expression of RIP kinases, while RIPK1/RIPK3 activation may further stimulate NF-κB-dependent inflammatory cascades, jointly aggravating myocardial injury (Zhang et al., 2023; Alghibiwi et al., 2025). Despite these insights, the integrated roles of NF-κB and RIPK3-dependent necroptosis in MI remain incompletely understood, and therapeutic approaches targeting both pathways simultaneously are still lacking.
Pharmacological modulation of these pathways may offer promising therapeutic avenues to attenuate excessive inflammation and improve cardiac outcomes. To date, reperfusion therapy remains the cornerstone of MI management, with coronary recanalization demonstrating significant prognostic benefits (Wei et al., 2025). However, the concomitant ischemia/reperfusion (I/R) injury paradoxically exacerbates myocardial damage, resulting in impaired cardiac function and increased risk of severe complications (Yan et al., 2025). While contemporary therapeutic strategies have improved acute outcomes, they remain insufficient to prevent subsequent ventricular remodeling and heart failure progression (Zhu et al., 2025). This critical limitation underscores the urgent need to develop novel treatment modalities that can fundamentally reverse post-AMI heart failure.
Accumulating clinical studies have validated the historical application of Traditional Chinese Medicine in cardiovascular therapeutics, demonstrating measurable treatment benefits (Lu et al., 2019; Sun et al., 2024). β-sitosterol (abreviated as β-SITO in this study), a prominent member of the phytosterol family, is widely distributed in numerous medicinal plant species, including Aconiti Lateralis Radix Praeparata (Yang et al., 2022b), Trema orientalis (Mekarunothai et al., 2024), bitter melon (Kim et al., 2025), Angelica sinensis (Chen et al., 2025a), Curcuma longa L. (Wang et al., 2025c), S. surattense (Yang et al., 2025), and soybean (Yang et al., 2024). Previous studies have reported that β-SITO has various biological actions, including anti-inflammation (Wu et al., 2025b), hepatoprotection (Zhang et al., 2024; Wang et al., 2025d), neuroprotection (Dolrahman and Thong-Asa, 2024), cardioprotective properties (Jaiswal et al., 2024; Adhimoolam et al., 2024), anti-diabetes (Liu et al., 2024), anti-oxidation (Gupta et al., 2011), and anti-tumor effects (Wang et al., 2024a; Chen et al., 2024; Wang et al., 2025e). A study by Wang et al proven that β-SITO triggers ovarian cancer apoptosis via ASS1-mediated Nrf2 degradation and PTEN/PI3K/AKT-dependent ROS generation (Wang et al., 2024b). Additionally, β-SITO demonstrated significant anti-diabetic effects in rat models of type 2 diabetes induced by high-fat diet and sucrose. Their study revealed that β-SITO can exert reno-protective effects by maintaining cellular homeostasis, balancing apoptosis, and reinforcing antioxidant activity. Mechanistically, β-SITO targets the TGF-β1/Nrf2/SIRT1/p53 pathways, suggesting its therapeutic utility in diabetic nephropathy and related metabolic disorders (Jayaraman et al., 2025). It is worth noting that some studies have confirmed that β-SITO can effectively prevent and treat cardiovascular diseases. Jiang and colleagues indicated that β-SITO attenuates atherosclerosis by upregulating catalase, which suppresses the PI3K/Akt/mTOR pathway, thereby reducing lipid accumulation and VSMC phenotypic switching (Jiang et al., 2024). Moreover, β-SITO exhibits cardioprotective effects in a monocrotaline-induced rat model of pulmonary hypertension. Studies indicated that β-SITO ameliorates pulmonary arterial hypertension by modulating vascular smooth muscle cell phenotype switching and suppressing DNA damage-mediated cGAS/STING signaling (Li et al., 2024). Nevertheless, the precise effects of β-SITO on myocardial infarction pathogenesis and recovery remain poorly characterized. In this research, through integrated in vitro and in vivo approaches, we explored β-SITO’s therapeutic potential against myocardial infarction and deciphered its mechanistic basis.
2 Materials and methods
2.1 Materials
β-sitosterol (C29H50O) with over 96% purity was obtained from Sigma-Aldrich (S9889, soluble in ethanol, sonication-assisted dissolution, United States). Hematoxylin Eosin (H&E) stain kit (G1005), Masson’s Trichrome stain kit (G1006), wheat germ agglutinin (WGA) staining (GDP1020) and TTC staining (G1017) were purchased from Service-bio (Wuhan, China).
2.2 Construction of MI model and treatment protocol
Male C57BL/6J mice of 8–12 weeks were used in the experiments. The mice were randomly allocated to four groups: Sham + Vehicle, MI + Vehicle, MI + β-SITO (10 mg/kg), MI + β-SITO (50 mg/kg). After acclimation, the MI models were established in mouses. Briefly, Mice were anesthetized with isoflurane, and the surgical site was aseptically prepared by depilating the cervical region extending to the mid-sternal area using electric clippers. A thoracotomy was performed through the 3rd or 4th intercostal space using ophthalmic curved forceps under a stereomicroscope. The left anterior descending coronary artery (LAD) was identified and ligated approximately 1.5 mm distal to the inferior edge of the left auricle. After ligation, residual air within the thoracic cavity was evacuated, followed by layered closure of the incision. Sham-operated mice underwent the same procedure, including LAD exposure and threading, but without ligation. After that, the mice were orally fed with 200 mL β-SITO for 28 consecutive days. Following euthanasia, tissue specimens were harvested for subsequent analysis.
2.3 Echocardiography determination
Briefly, cardiac function was evaluated using the VEVO2100 high-resolution ultrasound system (Visual-Sonics, Canada). Under continuous 1%–2% isoflurane anesthesia, murine chest hair was removed with depilatory cream. M-mode echocardiography was performed to obtain left ventricular functional parameters.
2.4 Histopathological analysis
Cardiac tissues were fixed, paraffin-embedded, and sectioned (5 μm) for histological analysis. Myocardial architecture, fibrosis, and hypertrophy were evaluated by H&E, Masson’s trichrome, and WGA staining, respectively. Digital whole-slide images were acquired using a Leica Aperio Versa 200 scanning system (Germany), with cardiomyocyte cross-sectional area (WGA) and collagen deposition (Masson) quantified using ImageJ software.
2.5 TTC staining
Myocardial infarct size was measured by TTC staining. In short, the mice were anesthesia with 5% isoflurane and then sacrificed, hearts were excised and sectioned transversely into 1 mm slices using a precision matrix. For infarct quantification, slices were incubated in 1% TTC (Solarbio, China) for 10 min, with the reaction terminated by 4% paraformaldehyde. The myocardial infarct area was imaged under a stereomicroscope (Zeiss Stemi 508, Germany) and calculated by ImageJ.
2.6 Cell isolation and culture
Primary neonatal mouse cardiomyocytes (NMCMs) and cardiac fibroblasts (NMCFs) were obtained as previously described (Yang et al., 2023). In brief, NMCMs and NMCFs were isolated from 1 to 3 days-old C57BL/6 mice. Following surface disinfection with 75% ethanol, hearts were aseptically excised and transferred to a laminar flow hood. Cardiac tissues were enzymatically dissociated using 0.25% trypsin (Service-bio) and type II collagenase (Thermo-Fisher Scientific) in sequential digestions.
Cell suspensions were centrifuged (1,500 × g, 5 min) and resuspended in complete high-glucose DMEM (Thermo-Fisher Scientific) supplemented with 10% FBS (Thermo-Fisher Scientific) and 1% penicillin/streptomycin. NMCMs and NMCFs were separated by differential adhesion (1–2 h at 37 °C). The adherent population (NMCFs) and non-adherent supernatant (NMCMs) were maintained in complete medium under standard culture conditions (37 °C, 5% CO2). For experimental treatments, cells were exposed to: 10 ng/mL TGF-β (Sigma-Aldrich), β-SITO (10 μM or 40 μM), Hypoxic conditions (5% CO2, 95% N2 at 37 °C).
2.7 Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-well plates (1 × 104 cells/well) and cultured for 24 h prior to treatment with hypoxia (5% CO2/95% N2), TGF-β (10 ng/mL), and/or β-SITO at serial concentrations for 24 h. Following treatment, 10 μL CCK-8 reagent was added per well, incubated for 3 h, and absorbance was measured at 450 nm using a microplate reader.
2.8 Lactate dehydrogenase (LDH) release assay
NMCMs were plated in 96-well plates and treated with TGF-β (10 ng/mL) and/or β-SITO (10 or 40 μM) as indicated. Following treatment, culture supernatants were collected and analyzed for LDH release using a commercial cytotoxicity detection kit (C0017, Beyotime Institute of Biotechnology) according to the manufacturer’s instructions.
2.9 Transwell migration assay
Cell migration was assessed using Transwell chambers (8 μm pore size; Millipore, Billerica, MA). Transfected cells (2 × 104) in serum-free medium were seeded in the upper chamber, while the lower chamber contained DMEM supplemented with 10% FBS as a chemoattractant. Following 24 h incubation, migrated cells on the membrane underside were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified by counting ten random fields at ×200 magnification using an inverted light microscope (Olympus IX73).
2.10 Immunofluorescence (IF)
For immunofluorescence analysis, NMCFs grown on coverslips were treated with β-SITO/TGF-β for 24 h. After fixation (4% paraformaldehyde, 30 min) and permeabilization (0.1% Triton X-100, 15 min), nonspecific sites were blocked with 2% BSA. Primary antibody incubation (anti-α-SMA, ab7817, abcam) was performed overnight at 4 °C, followed by fluorescent secondary antibody (1 h, RT). Nuclear staining with DAPI (C0065, Solarbio) preceded imaging on an Olympus IX73 system.
2.11 Transcriptomics analysis
The transcriptomics analysis was performed by Hangzhou Guangke Ande Biotechnology Co., LTD. In short, total RNA was extracted from NMCMs using standard extraction methods. Then, RNA quality assessment was performed using a Nanodrop ND-2000 spectrophotometer and Agilent 2,100 Bioanalyzer. Sequencing libraries were prepared using the ABclonal mRNA-seq Library Preparation Kit following manufacturer’s specifications, with library quantification and size distribution verification conducted on an Agilent 4,150 TapeStation system. High-throughput sequencing was performed on an Illumina Nova-seq 6,000 platform (Applied Protein Technology), generating 150 bp paired-end reads. Raw data processing and differential expression analysis were executed using DESeq2, with significantly differentially expressed genes (DEGs) defined as those exhibiting >2-fold change at adjusted p-value <0.05. Lastly, the enrichment analyses of differential genes were implemented following the procedures as previously described by Gao et al. (2025).
2.12 Molecular docking
The structures of NLRP3, RIPK3, and MLKL were obtained from the PDB database with respective ID 7LFH, 6OKO, and 4BTF. The models for P65 and SRC3 were generated using AlphaFold3 (Abramson et al., 2024). And then prepared structurally using the Protein Preparation Wizard module of Schrödinger (Release 2021–2) (Napolitano and Ballabio, 2016). All operating parameters were set using the software default settings unless otherwise specified. The protein structure preparation process includes removing water molecules from the native structure, adding hydrogen atoms, removing crystalline solvent molecules, completing missing residues and loops using Prime (Jacobson et al., 2002; Jacobson et al., 2004), optimizing the hydrogen bond networks, and energy minimization of the structure using the OPLS4 force field (Lu et al., 2021). The compound β-Sitosterol used for docking was prepared using the LigPrep (LigPrep, Schrödinger, LLC, New York, NY, 2021) tool with default parameters. Maestro (Maestro, Schrödinger, LLC, New York, NY, 2021) and PyMol (The PyMOL Molecular Graphics System, Version 2.3 Schrödinger, LLC) were used for structural visualization and docking result profiling.
2.13 Quantitative reverse Transcription-PCR
In brief, total RNA isolation from samples was performed with TRIzol reagent (Invitrogen), followed by cDNA synthesis using a reverse transcription system (Transcriptor First Strand cDNA Synthesis Kit, Thermo-Fisher). Quantitative PCR analysis was conducted with SYBR Green Master Mix (Thermo-Fisher) on a Step-One-Plus Real-Time PCR System (Applied Biosystems). Gene-specific primer sequences are detailed in Supplementary Table S1.
2.14 Protein extraction and Western blotting
Protein extraction and immunoblotting were conducted as follows: Cardiac tissues and NMCMs were homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein quantification was performed using a BCA assay. Equal protein aliquots were electrophoresed on 8%–12% gradient SDS-polyacrylamide gels and subsequently transferred to PVDF membranes. After blocking with 5% non-fat dried milk in TBST for 1 h at room temperature, membranes were probed with specific primary antibodies targeting: GAPDH (60004-1-Ig, Protein-tech), NF-κB p65 (8242S, CST), phosphorylated p65 (3033S, CST), NLRP3 (ab214185, Abcam), RIPK3 (ab56164, Abcam), phospho-RIPK3 (ab195117, Abcam), MLKL (ab184718, Abcam), phospho-MLKL (ab196436, Abcam), and IL-1β (12242S, CST). Following overnight incubation at 4 °C, membranes were exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein-antibody complexes were visualized using the ECL detection system and quantified by densitometry.
2.15 Statistical analysis
All quantitative data are presented as mean ± standard deviation (SD). Statistical comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons (GraphPad Prism 8.0.1; GraphPad Software, San Diego, CA). A two-tailed p-value < 0.05 was considered statistically significant.
3 Results
3.1 β-SITO improves myocardial injury and cardiac function in MI mouses
The molecular structure of β-SITO is presented in Figure 1A. To investigate its cardioprotective potential, we established a myocardial infarction (MI) model in male C57BL/6J mice through permanent coronary artery ligation. Postoperative animals received daily β-SITO treatment (10 or 50 mg/kg, oral gavage) for 28 days (Figure 1B), with physiological and histological endpoints evaluated. At first, the TTC staining was performed on the tissue slices to measure myocardial infarct size (Figures 1C,D), and the results showed that β-SITO effectively decreased infarct areas in MI mice compared to the Vehicle group. Furthermore, H&E and WGA staining revealed that MI mice developed significant cellular edema and cardiomyocyte hypertrophy, which were attenuated by β-SITO treatment (Figures 1E–G). Besides, HW/TL ratios were significantly reduced in the β-SITO treated group compared to MI controls (Figure 1H). Meanwhile, echocardiography was used to determine cardiac function in various mice groups. As described in Figures 1I–M, our results manifested that a significant decrease in LVEF, LVFS and cardiac output after MI induction, when compared with those in the sham group. By contrast, treatment with β-SITO remarkably enhanced left ventricular function. Consistent with these above results, RT-qPCR results further proven that the mRNA expression of heart failure-related proteins (ANP and BNP) were obviously reduced in the β-SITO treated group when compared with the MI group (Figures 1N,O). From all above results, we revealed that β-SITO effectively improved myocardial injury and cardiac function in vivo.
Figure 1. β-SITO improves MI-induced myocardial injury and cardiac function in vivo. (A) The chemical structural formula of β-SITO. (B) Schematic representation of the murine MI experimental protocol. (C,D) Myocardial infarct size was measured by TTC staining in MI + Vehicle and MI + β-SITO (50 mg/kg) group. Quantitation of myocardial infarct area were calculated by ImageJ (n = 5). (E) Representative images of HE staining of the myocardial tissues. Scale bar = 1,000 or 100 μm. (F,G) Representative WGA-stained left ventricular sections across experimental groups. Scale bar = 100 μm. Quantitative analysis of cardiomyocyte cross-sectional area (n = 6). (H) Ratio of heart weight (HW) to tibia length (TL) in different groups (n = 6). (I) Representative echocardiographic images across experimental groups. (J–M) Quantitative analysis of cardiac function parameters, including heart rate (HR), left ventricular ejection fraction (LVEF), fractional shortening (LVFS), Cardiac output (n = 6). (N,O) The myocardial mRNA expression of ANP and BNP was quantified by real-time quantitative PCR (RT-qPCR), with GAPDH serving as the endogenous control (n = 6). **P < 0.01, ***P < 0.001; ns means no significance.
3.2 β-SITO ameliorates cardiac fibrosis in MI mouses
To assess the effect of β-SITO on MI-triggered cardiac fibrosis, collagen deposition was quantified using Masson staining. As shown in Figures 2A,B, β-SITO treatment considerably reduced cardiac fibrosis in the left ventricle compared to the MI group. Additionally, the mRNA expression of COL-1, COL-3, Galectin-3 and α-SMA in MI mouses was higher than that in the sham group. This trend was reversed to varying degrees following β-SITO treatment (Figures 2C–F). Collectively, these findings indicated that β-SITO can inhibit cardiac fibrosis in MI mouses.
Figure 2. β-SITO ameliorates cardiac fibrosis in MI mouses. (A,B) Masson trichrome staining (left) and quantification of the scar area (right) at 28 days after MI (n = 6, scale bar = 1,000 or 100 μm). (C–F) The myocardial mRNA expression of COL-1, COL-3, Galectin-3 and α-SMA was quantified by RT-qPCR, with GAPDH serving as the endogenous control (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001; ns means no significance.
3.3 β-SITO alleviates HO-induced cardiomyocytes damage in vitro
Next, NMCMs were used to evaluate β-SITO’s effects on cardiomyocyte injury. As depicted in Figures 3A,B, CCK-8 assay demonstrated that Low-concentration β-SITO exhibited negligible cytotoxicity in NMCMs. Concurrently, β-SITO treatment significantly enhanced NMCM viability under hypoxic conditions (Figure 3C). Moreover, as illustrated in Figure 3D, the experimental results demonstrated that β-SITO dose-dependently inhibited LDH release in NMCMs. Altogether, these above results certified that β-SITO significantly alleviated HO-induced cardiomyocytes damage in vitro.
Figure 3. β-SITO alleviates HO-induced cardiomyocytes damage in vitro. (A) Representative images of NMCMs after 24 h β-SITO exposure (0, 80, 160, and 320 μM). (B) NMCM viability following β-SITO treatment (0, 10, 20, 40, 80, 160, and 320 μM, 24 h) was quantified via CCK-8 assay (n = 3). (C) NMCMs were exposed to β-SITO (0, 10, 20, 40, and 80 μM, 24 h) under normoxic or hypoxic conditions, with cell viability assessed using CCK-8 assay (n = 3). (D) LDH release was measured in NMCMs following 24 h β-SITO (0, 10, and 40 μM) treatment under normoxic or hypoxic conditions (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001; ns means no significance.
3.4 β-SITO ameliorates the activation of NMCFs induced by TGF-β in vitro
TGF-β serves as a pivotal regulator of fibroblast-to-myofibroblast transition, driving enhanced proliferative capacity, collagen deposition, and migratory activity. For that reason, we next estimated the effects of β-SITO on the migratory and proliferation capacities of NMCFs stimulated by TGF-β. First of all, the results of cell viability assay showed that low-concentration of β-SITO had no effect on the viability of NMCFs (Figure 4A). Subsequently, we demonstrated that β-SITO suppressed TGF-β-stimulated NMCF proliferation (Figure 4B). What’s more, as presented in Figures 4C,D, β-SITO treatment evidently reduced the mRNA expression of COL-1 and COL-3 compared to the TGF-β-challenged group alone. At the same time, the experimental results suggested that β-SITO reduced the migration of NMCFs induced by TGF-β (Figure 4E). Lastly, immunofluorescence analysis revealed that β-SITO partially attenuated TGF-β-mediated α-SMA enhancement in NMCFs (Figure 4F). Collectively, these findings indicated that β-SITO ameliorated the activation of NMCFs induced by TGF-β in vitro.
Figure 4. β-SITO alleviates the activation of NMCFs induced by TGF-β in vitro. (A) NMCFs viability following β-SITO treatment (0, 10, 20, 40, 80, 160, and 320 μM, 24 h) was quantified via CCK-8 assay (n = 3). (B) NMCFs were exposed to β-SITO (0, 10, 20, 40, 80 and 160 μM, 24 h) in the presence of TGF-β (10 ng/mL) or saline, with cell viability assessed using CCK-8 assay (n = 3). (C,D) The mRNA expression of COL-1 and COL-3 was quantified by RT-qPCR in NMCFs with or without β-SITO treatment, with GAPDH serving as the endogenous control (n = 4). (E) Representative map of cell migration (scale bar = 100 μm). (F) NMCFs were exposed to β-SITO (10 and 40 μM, 24 h) in the presence of TGF-β (10 ng/mL) or saline. DAPI (blue) and α-SMA (red) staining were performed in NMCFs. Immunofluorescence photographs were obtained using an inverted fluorescence microscope (scale bar = 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001; ns means no significance.
3.5 Transcriptomic insights and molecular docking accounting for the efficacy of β-SITO in the prevention of MI
To elucidate β-SITO’s cardioprotective mechanisms, we performed transcriptomic profiling of hypoxic NMCMs treated with either vehicle (V) or β-SITO (T). Initially, Principal component analysis (PCA) revealed significant separation between V and T groups (Figure 5A). Moreover, GO enrichment analysis of DEGs displayed that β-SITO treatment significantly regulated many biological processes associated with immune response and inflammation, such as regulation of innate immune response, leukocyte proliferation and regulation of inflammatory response (Figure 5B). Meanwhile, KEGG pathway enrichment analysis results of GSEA showed that several signaling pathways, such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and TNF signaling pathway (Figure 5C). GSEA analysis (Reactome) indicated differential gene enrichment in signaling pathways TNFR2 non-canonical NF-kB pathway, signaling by Interleukins and Cytokine Signaling in Immune system (Figure 5D). Additionally, as described in Figure 5E, the correlation heat map of GSEA analysis revealed that β-SITO treatment effectively reduced the expression of proteins in pathways related to immunity, inflammation, and programmed death, including Nlrp3, IL-1β, IL-6, Cxcl1, Zbp1, Ripk3, Mlkl, Gsdmd, Jak2, Stat1 and Myd88. Of these, as a well-established mediator of programmed cell death, TNF-α triggers RIPK3-dependent necroptosis via TNF-R1. In addition, LPS can activate the ZBP1-RIPK3 axis through TLR4 to induce the same process (Jiao et al., 2020). Considering these findings, we suggested that β-SITO could exerts cardioprotective effects against MI by modulating immune-inflammatory responses and programmed cell death pathways.
Figure 5. Transcriptomics analysis of β-SITO treatment on MI. (A) PCA of transcriptomic profiles in HO + Vehicle (V) and HO + β-SITO group (T). (B) Bubble chart of GO enrichment analysis of DEGs between V and T groups. (C) The ridge plot of KEGG enrichment of GSEA analysis results across experimental groups. (D) Ridge plot displaying the results of GSEA analysis using the Reactome Database. (E) The correlation heat map of GSEA analysis results in V and T group. n = 3.
On basis of the results of RNA-Seq analysis, the NF-κB and necroptosis signaling pathways may be modulated by β-SITO in MI. Subsequently, molecular docking analysis evaluated β-SITO’s binding affinities for specific molecular targets. NF-κB p65, SRC3, NLRP3, RIPK3, and MLKL were selected as target receptors for β-SITO binding prediction, with molecular docking results visualized in Figure 6. The molecular docking scores (kcal/mol) was used to determine the binding strength. Values less than −5 kcal/mol indicate strong binding affinity, and values less than 0 kcal/mol usually indicate spontaneous binding. As shown in Table 1, the binding energies of β-SITO with all target proteins, which ranged from −9.397 to −6.837 kcal/mol, were less than −6.0 kcal/mol, suggesting strong binding affinity. These results demonstrate β-SITO’s high-affinity binding to key targets in NF-κB and necroptosis signaling pathways, while concurrently validating the accuracy of the transcriptome screening.
Figure 6. Docking solutions were visualized as both 3D structural models and 2D interaction diagrams. (A) P65. (B). SRC3. (C). NLRP3. (D). RIPK3. (E). MLKL.
Table 1. Docking score.
3.6 β-SITO represses activation of NF-κB and necroptosis signaling pathways
To validate the key signaling pathways mediating β-SITO’s cardioprotective effects in MI, we analyzed the protein expression of NLRP3 inflammasome components (NLRP3, IL-1β), NF-κB signaling markers (p-P65/P65), and necroptosis executors (p-RIPK3/RIPK3, p-MLKL/MLKL) by Western blotting. As presented in Figures 7A–F, the results suggested that the expression levels of NLRP3, p-P65, p-RIPK3, p-MLKL and IL-1β in HO-induced NMCMs were elevated obviously, but this phenomenon could be reversed by β-SITO administration in a dose-dependent manner. Consistent with in vitro findings, β-SITO treatment significantly downregulated myocardial expression of NLRP3, p-P65, p-RIPK3, p-MLKL and IL-1β in MI mice (Figures 7G–L). According to these results, we suggested that β-SITO could ameliorate MI probably via modulating NF-κB and necroptosis signaling pathways.
Figure 7. β-SITO represses activation of NF-κB and necroptosis signaling pathways. (A) The protein expression levels of NLRP3, p-P65, P65, p-RIPK3, RIPK3, p-MLKL, MLKL and IL-1β were analyzed by immunoblotting in NMCMs. (B–F) Protein expression levels were quantified using ImageJ software (n = 3). GAPDH was used as a loading control. (G). The protein expression levels of NLRP3, p-P65, P65, p-RIPK3, RIPK3, p-MLKL, MLKL and IL-1β were analyzed by immunoblotting in mice. (H–L) Protein expression levels were quantified using ImageJ software (n = 6). GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001; ns means no significance.
4 Discussion
In the present study, we comprehensively investigated the cardioprotective effects and underlying mechanisms of β-SITO in MI using both in vivo and in vitro models. Our findings demonstrate that β-SITO significantly improves cardiac function, mitigates myocardial fibrosis, reduces cardiomyocyte injury under hypoxic conditions, and inhibits fibroblast activation. Mechanistically, transcriptome sequencing combined with molecular docking and protein validation assays revealed that β-SITO exerts its beneficial effects primarily through the regulation of inflammatory and necroptotic signaling pathways, particularly via modulation of NF-κB, NLRP3 inflammasome, and RIPK3/MLKL-mediated necroptosis (Figure 8).
Figure 8. Mechanistic schematic depicting the cardioprotective pathways mediated by β-SITO in MI.
MI is characterized by ischemic cardiomyocyte death, followed by an inflammatory response and fibrotic remodeling, ultimately leading to progressive cardiac dysfunction and heart failure (Li et al., 2025a). Despite the widespread use of reperfusion therapy and pharmacologic agents such as β-blockers and ACE inhibitors (Chen et al., 2025b), the long-term prognosis for patients with MI remains unsatisfactory, highlighting the need for novel therapeutic approaches targeting the underlying pathophysiology. β-SITO, a plant-derived phytosterol known for its anti-inflammatory and anti-oxidant properties, has emerged as a potential candidate with multifaceted bioactivities (Tang et al., 2024; Jiang et al., 2025). However, its role in cardiovascular disease, particularly in post-MI remodeling, has not been thoroughly elucidated.
Our in vivo data provide compelling evidence that β-SITO administration significantly improves left ventricular function post-MI, as evidenced by increased LVEF, LVFS, and cardiac output, alongside reduced infarct size and HW/TL ratios. These functional improvements were accompanied by decreased myocardial expression of hypertrophy markers ANP and BNP, suggesting attenuation of pathological remodeling. Histological analyses further revealed reduced interstitial fibrosis and lower expression of fibrotic markers, including COL-1, COL-3, Galectin-3 and α-SMA, indicating that β-SITO mitigates myocardial fibrosis.
In vitro, we demonstrated that β-SITO confers direct cytoprotective effects on NMCMs subjected to hypoxic stress, significantly improving cell viability and reducing cardiomyocytes damage. These results suggest that β-SITO may preserve cardiomyocyte integrity in the ischemic myocardium, thereby preventing the loss of functional myocardial tissue. Additionally, in NMCFs, β-SITO inhibited TGF-β–induced fibroblast proliferation, migration, and myofibroblast differentiation. These findings demonstrate a dual mechanism by which β-SITO acts—protecting cardiomyocytes and preventing excessive fibroblast activation, thereby limiting both cell loss and fibrotic scar expansion.
To investigate the cardioprotective mechanisms of β-SITO, we conducted RNA-seq analysis of HO-induced NMCMs treated with either vehicle or β-SITO. Functional enrichment of DEGs indicated significant involvement of inflammation- and cell death-related pathways, including NF-κB signaling, cytokine-cytokine receptor interaction, and necroptosis. Notably, KEGG and GO analyses highlighted the involvement of genes such as NLRP3, RIPK3, MLKL, and IL-1β. Moreover, the interaction between β-SITO and the identified targets was further supported by molecular docking analysis, which showed favorable binding affinities between β-SITO and NLRP3, RIPK3, MLKL, and P65. These in silico findings provide a mechanistic rationale for the observed effects and suggest that β-SITO may function as a direct modulator of key inflammatory and cell death signaling pathways.
The NLRP3 inflammasome plays a critical role in sterile inflammation following MI by promoting the maturation and release of pro-inflammatory cytokines, notably IL-1β, thereby exacerbating cardiomyocyte injury and fibrosis (Cui et al., 2025). In this study, β-SITO treatment significantly suppressed NLRP3 and IL-1β expression in both in vivo and in vitro models, suggesting that inhibition of inflammasome activation is a central mechanism of its cardioprotective effect. Additionally, β-SITO reduced the phosphorylation of NF-κB p65, key mediators of canonical NF-κB activation, further confirming its anti-inflammatory potential. These findings are consistent with previous reports of β-SITO’s anti-inflammatory activity in other disease contexts, such as metabolic and neuroinflammatory disorders (Li X. et al., 2025; Dolrahman and Thong-Asa, 2024).
Necroptosis, a regulated form of necrotic cell death mediated by the RIPK3-MLKL axis, has recently emerged as a major contributor to cardiomyocyte death during ischemia-reperfusion injury, MI and heart failure (Qin et al., 2024). In our study, β-SITO markedly reduced p-RIPK3 and p-MLKL expression, indicating inhibition of necroptotic signaling. This finding is particularly significant given that necroptosis not only contributes to myocardial cell loss but also promotes inflammation through the release of damage-associated molecular patterns (DAMPs). By suppressing this pathway, β-SITO likely exerts both cytoprotective and anti-inflammatory effects, creating a more favorable environment for myocardial healing.
Collectively, our study provides robust evidence that β-SITO mitigates myocardial injury and fibrosis following MI through a multifaceted mechanism involving suppression of inflammation and necroptosis. These effects are mediated, at least in part, by direct inhibition of the NLRP3 inflammasome and RIPK3/MLKL pathway, as well as modulation of NF-κB signaling. Importantly, these molecular effects translate into significant improvements in cardiac structure and function, highlighting the translational potential of β-SITO as a therapeutic agent.
5 Limitation
Despite these promising findings, several limitations warrant consideration. First, while we demonstrated the efficacy of β-SITO in a murine model of MI, further studies in large animal models are needed to validate its therapeutic potential and determine optimal dosing strategies. Second, although molecular docking provides insight into potential binding interactions, confirmatory studies such as surface plasmon resonance or co-immunoprecipitation are needed to validate direct target engagement. Lastly, the bioavailability and pharmacokinetics of β-SITO in the context of cardiac injury remain to be fully characterized.
6 Conclusion
Our findings demonstrate that β-SITO is a promising cardioprotective agent capable of mitigating myocardial injury and remodeling through the suppression of inflammation and necroptosis. These results pave the way for future studies aimed at translating β-SITO into a viable therapeutic strategy for patients with MI and potentially other forms of cardiovascular disease.
Data availability statement
The original contributions presented in the study are publicly available. This data can be found here: NCBI GEO repository, accession number GSE314346.
Ethics statement
The animal study was approved by the Ethics Committee of Xiamen university (approval no. XMULAC20200137, Xiamen, China). The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
J-jX: Conceptualization, Data curation, Formal Analysis, Methodology, Writing – original draft. M-wC: Data curation, Methodology, Software, Validation, Visualization, Writing – review and editing. L-zL: Data curation, Investigation, Software, Validation, Writing – review and editing. DX: Investigation, Methodology, Writing – review and editing. S-yJ: Investigation, Methodology, Writing – review and editing. J-xL: Methodology, Writing – review and editing. Z-hZ: Formal Analysis, Funding acquisition, Resources, Supervision, Writing – review and editing. Z-gS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Natural Science Foundation of China (82070291), Natural Science Foundation of Xinjiang Uygur Autonomous Region (2024D01C103) and Tianchi Talent Program of Xinjiang Uygur Autonomous Region.
Acknowledgements
The authors would like to thank Ph. D Wenbin Hong (The First Affiliated Hospital of Xiamen University, Xiamen, China) for supporting this study.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2025.1719074/full#supplementary-material
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Keywords: cardioprotection, myocardial infarction, necroptosis, NF-κB signaling, β-sitosterol
Citation: Xu J-j, Chen M-w, Lai L-z, Xiao D, Jiang S-y, Lin J-x, Zhang Z-h and Shan Z-g (2026) β-sitosterol ameliorates myocardial infarction injury via modulating the NF-κB and necroptosis signaling pathways. Front. Pharmacol. 16:1719074. doi: 10.3389/fphar.2025.1719074
Received: 05 October 2025; Accepted: 04 December 2025;
Published: 06 January 2026.
Edited by:
Mahmoud El-Mas, Alexandria University, EgyptReviewed by:
Jingquan Zhong, Shandong University, ChinaJia He, Jiangxi University of Traditional Chinese Medicine, China
Copyright © 2026 Xu, Chen, Lai, Xiao, Jiang, Lin, Zhang and Shan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zheng-hao Zhang, NDA1ODQ0ODU4QHFxLmNvbQ==; Zhong-gui Shan, c3pnZG9jdG9yQHhtdS5lZHUuY24=
†These authors have contributed equally to this work