Abstract
Background
Nicorandil, a vasodilator, exhibits potential in improving myocardial blood supply, but its protective mechanism against myocardial ischemia-reperfusion (I/R) injury remains unclear. This study aims to explore the cardioprotective effects of nicorandil on myocardial I/R injury and elucidate its molecular mechanisms, providing a theoretical basis for clinical treatment.
Methods
Myocardial I/R injury models were established using SPF-grade male Sprague-Dawley (SD) rats, randomly divided into control, I/R, and I/R+Nicorandil groups (n = 6 per group). The I/R+Nicorandil group, nicorandil (5 mg/kg) was injected intraperitoneally every day from 3 days before the establishment of myocardial I/R model. Myocardial injury was assessed through TTC staining, hematoxylin-eosin (HE) staining, and multi-omics analyses (transcriptome sequencing, proteomics, and metabolomics) to investigate alterations at genetic, protein, and metabolite levels.
Results
Biochemical and histological analyses revealed that nicorandil significantly reduced serum levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK), while decreasing myocardial infarction area and pathological damage. Transcriptome sequencing identified differentially expressed genes (DEGs) enriched in TNF and chemokine signaling pathways between control vs. I/R and I/R vs. I/R+Nicorandil groups. Proteomics analysis screened differentially expressed proteins (DEPs) primarily involved in glycolysis and protein glycosylation. Metabolomics detected two key differential metabolites in positive ion mode. Multi-omics integrative analysis identified CYP4F5 as a pivotal protein node, showing significant correlations with multiple metabolites. Western blot results demonstrated that nicorandil modulated the expression of PPAR-α, NF-κB, cGAS, and STING pathway proteins in myocardial I/R injury.
Conclusion
Nicorandil may alleviate myocardial I/R injury by regulating CYP4F5-associated metabolites and signaling pathways.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13019-026-04325-0.
Keywords: Nicorandil, Myocardial ischemia-reperfusion injury, CYP4F5, Transcriptome, Metabolome, Proteome
Introduction
Myocardial ischemia-reperfusion (I/R) injury is an unavoidable pathological process in clinical settings such as reperfusion therapy for acute myocardial infarction and cardiac surgery. It is characterized by oxidative stress, inflammatory responses, and apoptosis following the restoration of blood flow after ischemia, which exacerbates myocardial cell damage [1]. In the United States, the overall prevalence of acute myocardial infarction among adults aged ≥ 20 years is 3.0%. In China, the mortality rates of acute myocardial infarction in urban and rural areas are approximately 58.69 per 100,000 and 74.72 per 100,000 population, respectively, with up to 60% of patients developing myocardial I/R injury [2, 3]. This process not only reduces the benefits of reperfusion therapy and hinders clinical efficacy but also imposes economic burdens on healthcare systems and families, severely threatening patients’ lives and health [4].
Nicorandil, a drug combining potassium channel-opening and nitrate-like effects, exerts a dual regulatory mechanism by releasing nitric oxide (NO) to effectively improve blood perfusion in ischemic regions. It is widely used for the treatment of angina [5–7]. However, its mechanism in treating myocardial I/R injury remains unclear. Cytochrome P450 (CYP450), previously implicated in the metabolism and detoxification of numerous drugs, metabolizes approximately 90% of U.S. Food and Drug Administration (FDA)-approved drugs and aids in early detection of potential drug-drug interactions [8]. Among CYP450 isoforms, the CYP4F subfamily participates in the metabolism of various endogenous and exogenous substances and may serve as a potential therapeutic target for myocardial I/R injury [9]. Whether nicorandil exerts cardioprotective effects by regulating CYP4F and its downstream metabolic pathways has not been reported. In this study, we established a rat model of myocardial I/R injury and employed multi-omics techniques (transcriptomics, proteomics, and metabolomics) to analyze the effects of nicorandil intervention on myocardial gene/protein expression and metabolite profiles. This investigation aims to explore the critical role of nicorandil in alleviating myocardial I/R injury, providing a novel theoretical foundation for its clinical application in treating this condition.
Materials and methods
Establishment of myocardial I/R rat model
The rats were provided by the Animal Experiment Center of Xinjiang Medical University. The experimental protocol was approved by the Animal Ethics Committee of Xinjiang medical university (IACUC-JT-20241119-07) and complied with ethical guidelines for animal experiments. SPF male SD rats (250–300 g) were anesthetized with 2% isoflurane and fixed on their backs. After sterilizing their chests, a 1 cm incision was made between the left 3rd and 4th ribs of the sternum. The pectoralis major muscle and intercostal muscles were bluntly separated along the rib space, and the thoracic cavity was gently retracted using a rib spreader to expose the heart, which was then carefully lifted out of the thoracic cavity with sterile forceps. The left anterior descending coronary artery (LAD) was found 2–3 mm below the intersection of the left atrial inferior margin and the pulmonary artery cone, and the left anterior descending coronary artery was ligated with 6 − 0 silk thread (the control group only threaded). Electrocardiogram was observed within 5 min after ligation, and ST segment elevation ≥ 0.25mV was considered as ischemic success. The rats woke up in 2–3 min minutes, and were reperfusion after 45 min of ischemia. After 24 h, the electrocardiogram was recorded, and the ST segment dropped by 1/2, which was considered as successful reperfusion [10, 11]. The experiment was divided into control group(sham group), model group (I/R group) and nicorandil intervention group (I/R+Nicorandil group), with 6 rats in each group. In the I/R+Nicorandil group, nicorandil (5 mg/kg) was injected intraperitoneally for 3 days from 3 days before the establishment of myocardial I/R model, and the solvent was physiological saline with a volume of 1mL/kg. The control group only performed thoracotomy, threading under LAD, but not ligation, and then closed the chest cavity. The other operations were the same as those in the model group.
ELISA assay
Levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK), leukotriene B4, and 20-hydroxyeicosatetraenoic acid (20-HETE) were measured using ELISA kits (Nanjing Jiancheng Bioengineering Institute). Standards and samples were added to 96-well plates and incubated at 37 °C for 2 h. After discarding the liquid, plates were washed three times with wash buffer. Detection antibody working solution was added and incubated at 37 °C for 1 h. Following another wash, enzyme conjugate working solution was added and incubated at 37 °C for 60 min. After thorough washing, substrate solution was added for 30-minute chromogenic reaction at 37 °C in the dark. The reaction was terminated with stop solution, and absorbance was measured at 450 nm using a microplate reader. Sample concentrations were calculated based on standard curves.
TTC staining
Hearts were harvested post-experiment and rinsed with phosphate-buffered saline (PBS). Cardiac tissue was sectioned into 2-mm-thick slices and incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37 °C in the dark for 20 min. Viable myocardium stained red, while infarcted areas remained pale. Sections were fixed in 4% paraformaldehyde, and infarction area (percentage of left ventricular area) was quantified using ImageJ software.
HE staining
Myocardial tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through graded ethanol series (100%, 95%, 90%, 80%, and 70%), and embedded in paraffin. Serial 5-µm-thick sections were mounted on slides. After deparaffinization and rehydration, sections were stained with hematoxylin for 5 min, rinsed, counterstained with eosin for 3 min, dehydrated, and cleared in xylene. Morphological changes in cardiomyocytes were observed under a light microscope.
Transcriptome sequencing and data analysis
Total RNA was extracted from myocardial tissue samples using TRIzol reagent (Thermo Fisher Scientific). RNA purity and concentration were measured using a NanoDrop 2000 spectrophotometer, with RNA integrity number (RIN) > 7.0. RNA libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and sequenced on an Illumina HiSeq platform with paired-end 150-bp reads. Raw sequencing data were quality-filtered using FastQC software to obtain high-quality clean reads [12]. Differential gene expression analysis between groups was performed using the DESeq2 package in R, with thresholds set at |log2FC| ≥1 and P < 0.05 to identify significantly differentially expressed genes (DEGs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the clusterProfiler package [13].
Proteomic analysis
Label-free proteomic analysis was performed on selected samples from each group. Myocardial tissues were homogenized in ice-cold RIPA lysis buffer supplemented with protease/phosphatase inhibitor cocktail (1:100, Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were centrifuged at 16,000 × g for 20 min at 4 °C, and supernatants were collected for protein quantification via BCA assay. Equal protein amounts (50 µg) were subjected to filter-aided sample preparation (FASP) digestion: reduction with 100 mM dithiothreitol (DTT) at 56 °C for 1 h, alkylation with 50 mM iodoacetamide (IAA) in the dark for 45 min, and overnight digestion with trypsin (enzyme: substrate = 1:50) at 37 °C. Digested peptides were desalted using C18 columns (Thermo Scientific) and vacuum-dried.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using a Vanquish Neo UHPLC system coupled to an Orbitrap Astral mass spectrometer. Peptides were separated on a µPAC Neo column (Thermo Scientific) via an 8-minute gradient of 4%–99% acetonitrile (0.1% formic acid). Data were acquired in data-independent acquisition (DIA) mode with a scan range of 380–980 m/z, MS1 resolution of 240,000, MS2 resolution of 80,000, and 25% collision energy (HCD). Raw data were processed using DIA-NN v1.8 against the UniProt Rattus norvegicus proteome database. Differentially expressed proteins (DEPs) were screened with thresholds of |log2FC| ≥0.5 and P < 0.05, followed by GO and KEGG enrichment analyses.
Metabolomic analysis
Myocardial tissue samples were homogenized in pre-cooled 80% methanol (pH-adjusted with 0.1% formic acid) for metabolomic analysis. Homogenates were centrifuged at 15,000 × g for 10 min at 4 °C to remove debris, and supernatants were collected. Quality control (QC) samples were prepared by pooling equal aliquots from all tissue samples. Untargeted metabolomic analysis was conducted using a high-resolution UHPLC-MS/MS system. Metabolites of varying polarities were separated on a C18 reversed-phase column via gradient elution and detected in both positive and negative ion modes.
Raw MS data were preprocessed using Compound Discoverer 3.3 software for denoising, calibration, peak extraction, and identification. Metabolite peaks were characterized based on mass-to-charge ratio (m/z) and retention time (RT). Peak area quantification and metabolite identification were achieved by matching against high-resolution MS/MS spectral databases. Metabolites with a coefficient of variation (CV) > 30% in QC samples were excluded. Relative quantification was normalized to total peak area.
Principal component analysis (PCA) was performed on metabolite peaks from all samples. Partial least squares-discriminant analysis (PLS-DA) was used to compare intergroup metabolite levels. Metabolites with variable importance in projection (VIP) > 1 and P < 0.05 were defined as differentially expressed metabolites. KEGG pathway enrichment analysis was conducted for identified differential metabolites.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissues and cells using TRIzol reagent. cDNA was synthesized using a reverse transcription kit (Takara Bio Inc.). RT-qPCR was performed on a real-time PCR system with SYBR Green PCR Master Mix under the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. β-actin served as the internal reference gene, and relative expression levels of CYP4F5 mRNA were calculated using the 2−ΔΔCt method. Primer sequences are listed in Table 1.
Table 1.
Primer sequences
| gene | Primer sequences(5′-3′) |
|---|---|
| CYP4F5 | F: 5′-CTTGCCTGGACTTACTCATTCTGTG-3′ |
| R: 5′-CATGCCTTCCTCATTGCTCTGG-3′ | |
| β-actin | F: 5′-AGAGCTATGAGCTGCCTGAC-3′ |
| R: 5′-AGCACTGTGTTGGCGTACAG-3′ |
Western blot
Myocardial tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors, followed by centrifugation at 12,000 ×g for 15 min at 4 °C. Supernatants were collected for protein quantification using a BCA assay kit (Beyotime Biotechnology). Protein samples were mixed with 5× SDS loading buffer, denatured at 100 °C for 5 min, and separated by SDS-PAGE (10%–12% separating gel, 5% stacking gel) at 80 V initially, then adjusted to 120 V after entering the separating gel. Proteins were transferred to a 0.45 μm PVDF membrane at 300 mA, blocked with 5% skim milk for 1.5 h, and incubated overnight at 4 °C with primary antibodies (PPAR-α, NF-κB, cGAS, STING, CYP4F5, and β-actin; 1:1000 dilution). After washing three times with TBST (10 min each), membranes were incubated with HRP-conjugated secondary antibodies (1:5000 dilution) at room temperature for 1 h. Signals were visualized using ECL reagent (Merck Millipore), captured with a gel imaging system, and analyzed using ImageJ software. Band intensities were normalized to β-actin for relative quantification.
Statistical analysis
All statistical analyses were performed using R (v4.2) and GraphPad Prism (v10.0). Data are expressed as mean ± standard deviation (SD). Normality and homogeneity of variance were routinely tested before statistical analysis to ensure the rationality of subsequent statistical methods. Comparisons between two independent groups were analyzed using unpaired Student’s t-test. Multiple groups were compared via one-way analysis of variance (ANOVA), followed by Tukey’s post hoc multiple comparison test for pairwise comparisons. For multi-omics differential analysis, the Wilcoxon rank-sum test was used for non-normal distributed data, and false discovery rate (FDR) correction was applied to eliminate false positive results. A P < 0.05 was considered statistically significant.
Results
Nicorandil attenuates myocardial I/R injury
ELISA analysis of myocardial tissues across groups revealed significantly elevated levels of LDH, AST, and CK in the I/R group compared to the Control group. These enzyme levels were markedly reduced in the I/R+Nicorandil group versus the I/R group (Fig. 1A). TTC staining demonstrated extensive pale infarct areas in the I/R group, whereas the I/R+Nicorandil group exhibited significantly reduced infarct volume (Fig. 1B). HE staining further confirmed these findings: the Control group displayed orderly myocardial cell alignment with intact structure, the I/R group showed disrupted cell architecture and interstitial widening, and the I/R+Nicorandil group exhibited mitigated structural damage with improved cellular organization (Fig. 1C).
Fig. 1.
Biochemical and histopathological assessments in myocardial I/R injury rats. (A) Serum levels of LDH, AST, and CK in myocardial I/R injury rats measured by ELISA. (B) Myocardial infarction area observed by TTC staining. (C) Pathological morphology of myocardial tissue evaluated by HE staining
Transcriptomic profiling of myocardial I/R injury
A total of 1,051 differentially expressed genes (DEGs) were identified between Control and I/R groups, with 243 downregulated and 808 upregulated genes (Fig. 2A). Comparison of I/R and I/R+Nicorandil groups revealed 315 DEGs (128 downregulated, 187 upregulated; Fig. 2B). Intersection analysis identified 101 overlapping DEGs (Fig. 2C). Gene Ontology (GO) enrichment highlighted these DEGs in biological processes such as cell cycle and division (Fig. 3A). KEGG pathway analysis indicated enrichment in TNF signaling, chemokine signaling, and IL-17 signaling pathways (Fig. 3B). To further explore the functional pathway changes at the transcriptomic level, we performed Gene Set Enrichment Analysis (GSEA) on the transcriptome data (Fig. 4). For the Control vs. I/R comparison, Hallmark pathway analysis revealed enrichment of inflammatory-related pathways (including TNF signaling via NF-κB, inflammatory response, IL6-JAK-STAT3 signaling) and metabolic pathways (oxidative phosphorylation) in the I/R group (Fig. 4A). GO-based GSEA further confirmed the upregulation of biological processes related to inflammatory response, cytokine production, and mitochondrial proton motive force-driven ATP synthesis (Fig. 4B). In contrast, for the I/R vs. I/R+Nicorandil comparison, GSEA showed that nicorandil treatment downregulated cell cycle-related pathways and inflammatory pathways in the intervention group (Fig. 4C, D), consistent with the results of differential gene enrichment analysis.
Fig. 2.
Identification of differentially expressed genes (DEGs). (A) Volcano plot of DEGs between Control and I/R groups. (B) Volcano plot of DEGs between I/R and I/R+Nicorandil groups. (C) Venn diagram showing overlapping DEGs between the two comparisons
Fig. 3.
GO and KEGG analyses of DEGs. (A) GO functional enrichment analysis, including biological processes (BP), molecular functions (MF), and cellular components (CC). (B) KEGG pathway enrichment analysis
Fig. 4.
Gene Set Enrichment Analysis (GSEA) of transcriptomic data. (A, B) Hallmark and GO-based GSEA plots comparing the Control and I/R groups. (C, D) Hallmark and GO-based GSEA plots comparing the I/R and I/R+Nicorandil groups
Nicorandil alters protein expression in myocardial I/R injury
Proteomic analysis identified 243 differentially expressed proteins (DEPs) between Control and I/R groups (43 downregulated, 201 upregulated; Fig. 5A). The I/R vs. I/R+Nicorandil comparison yielded 167 DEPs (105 downregulated, 62 upregulated; Fig. 5B), with 25 overlapping DEPs (Fig. 5C). GO analysis associated these DEPs with glycolytic processes, protein N-linked glycosylation, and oxidation-reduction (Fig. 6A). KEGG pathways included fructose/mannose metabolism, N-glycan biosynthesis, and the pentose phosphate pathway (Fig. 6B). Consistent with the transcriptomic analysis, we also performed GSEA on the proteomic data to characterize functional pathway changes at the protein level (Fig. 7). For the Control vs. I/R comparison, Hallmark pathway analysis showed enrichment of MYC target pathways, interferon response pathways, and coagulation-related pathways in the I/R group (Fig. 7A). GO-based GSEA further revealed the upregulation of biological processes such as RNA processing, ribonucleoprotein complex biogenesis, and mitochondrial metabolic processes (Fig. 7B). In the I/R vs. I/R+Nicorandil comparison, nicorandil treatment downregulated pathways related to extracellular space organization, secretory vesicle function, and phagocytosis (Fig. 7C, D), reflecting the regulatory effect of nicorandil on protein expression and functional pathways during myocardial I/R injury.
Fig. 5.
Identification of differentially expressed proteins (DEPs). (A) Volcano plot of DEPs between Control and I/R groups. (B) Volcano plot of DEPs between I/R and I/R+Nicorandil groups. (C) Venn diagram showing overlapping DEPs between the two comparisons
Fig. 6.
GO and KEGG analyses of DEPs. (A) GO functional enrichment analysis. (B) KEGG pathway enrichment analysis
Fig. 7.
Gene Set Enrichment Analysis (GSEA) of proteomic data. (A, B) Hallmark and GO-based GSEA plots comparing the Control and I/R groups. (C, D) Hallmark and GO-based GSEA plots comparing the I/R and I/R+Nicorandil groups
Nicorandil modulates key protein CYP4F5 expression
Integrated transcriptomic and proteomic analyses identified CYP4F5 as the sole overlapping protein with differential expression at both mRNA and protein levels (Fig. 8A). CYP4F5 mRNA and protein levels were significantly elevated in the I/R group versus Control, but reduced in the I/R+Nicorandil group (Fig. 8B). RT-qPCR validated these trends, confirming upregulated CYP4F5 in I/R and downregulation after nicorandil treatment (Fig. 8C).
Fig. 8.
Multi-omics analysis of CYP4F5 in myocardial I/R injury rats. (A) Overlap analysis of DEGs and DEPs. (B) Expression levels of CYP4F5 at transcriptomic and proteomic levels. *P < 0.05, **P < 0.01. (C) RT-qPCR validation of CYP4F5 mRNA expression. *P < 0.05 vs. Control; #P < 0.05 vs. I/R group
Metabolomic alterations in myocardial I/R injury
Metabolomic profiling identified 721 metabolites in positive ion mode (5 upregulated, 15 downregulated in I/R vs. Control; Fig. 9A) and 625 metabolites in negative ion mode (1 upregulated, 26 downregulated; Fig. 9B). Comparing I/R+Nicorandil and I/R groups revealed 721 metabolites in positive mode (8 upregulated, 18 downregulated; Fig. 9C) and 625 in negative mode (8 upregulated, 18 downregulated; Fig. 9D). Intersection analysis identified two shared metabolites in positive ion mode (Fig. 9E), with no overlaps in negative mode (Fig. 7F).
Fig. 9.
Analysis of differential metabolites. (A) Volcano plot of differential metabolites (positive ion mode) between I/R and Control groups. (B) Volcano plot of differential metabolites (negative ion mode) between I/R and Control groups. (C) Volcano plot of differential metabolites (positive ion mode) between I/R+Nicorandil and I/R groups. (D) Volcano plot of differential metabolites (negative ion mode) between I/R+Nicorandil and I/R groups. (E) Venn diagram of overlapping metabolites (positive ion mode) from I/R vs. Control and I/R+Nicorandil vs. I/R comparisons. (F) Venn diagram of overlapping metabolites (negative ion mode) from I/R vs. Control and I/R+Nicorandil vs. I/R comparisons
CYP4F5-associated metabolite regulation
Correlation analysis demonstrated negative associations between CYP4F5 and 20-carboxy-leukotriene B4, and positive correlations with 11-trans-leukotriene C4 (Fig. 10A). Metabolite levels varied across groups: 12-epi-leukotriene B4 and 20-carboxy-leukotriene B4 were lower in I/R versus Control and I/R+Nicorandil groups, while 11-trans-leukotriene C4 was elevated in I/R (Fig. 10B). ELISA confirmed elevated leukotriene B4 and 20-HETE in I/R, both reduced by nicorandil (Fig. 11).
Fig. 10.
Correlation analysis between CYP4F5 and metabolites. (A) Heatmap showing correlations between CYP4F5 mRNA/protein expression and metabolite levels (red: positive correlation; blue: negative correlation). (B) Box plots of four metabolite levels across groups. *P < 0.05, **P < 0.01
Fig. 11.
ELISA quantification of Leukotriene B4 and 20-HETE levels. *P < 0.05 vs. Control; #P < 0.05 vs. I/R group
Nicorandil restores CYP4F5-linked signaling pathways
Western blot analysis (Fig. 12) revealed significantly reduced PPAR-α and elevated NF-κB, cGAS, and STING protein levels in the I/R group versus Control. Nicorandil treatment reversed these effects, restoring PPAR-α expression and suppressing NF-κB, cGAS, and STING.
Fig. 12.
Western blot analysis of CYP4F5-associated signaling pathway proteins. *P < 0.05 vs. Control; #P < 0.05 vs. I/R group
Discussion
Nicorandil has been shown to effectively prevent reperfusion injury in myocardial infarction patients undergoing percutaneous coronary intervention (PCI) and is recognized as a multi-target cardioprotective agent [14, 15]. This study further investigates the protective mechanisms of nicorandil against myocardial I/R injury through multi-omics profiling, revealing three key findings: (1) Nicorandil reduces serum levels of myocardial injury biomarkers (LDH, AST, CK) and mitigates pathological changes in myocardial tissue. (2) Integrated transcriptomic and proteomic analyses identified CYP4F5 as a critical protein associated with myocardial I/R injury and regulated by nicorandil. (3) Metabolomic analysis detected two differential metabolites related to myocardial I/R injury, including four subtypes: 20-Carboxy-Leukotriene B4, 12-Epileukotriene B4, 12-epi-Leukotriene B4, and 11-trans-Leukotriene C4.
In terms of biochemical assessments, LDH, AST, and CK are key biomarkers for evaluating myocardial injury in clinical and preclinical studies [16]. Liu [17] demonstrated that during myocardial I/R injury, energy metabolism dysregulation and impaired membrane integrity in cardiomyocytes lead to massive release of intracellular LDH, AST, and CK into the bloodstream, with serum levels positively correlating with the extent of cardiomyocyte necrosis. In this study, the I/R+Nicorandil group exhibited significantly lower serum LDH, AST, and CK levels compared to the I/R group, indicating that nicorandil effectively reduces cardiomyocyte necrosis and leakage of cellular contents. TTC staining confirmed that nicorandil significantly reduced myocardial infarct size in I/R-injured rats, effectively limiting the scope of cardiomyocyte death. HE staining revealed reduced myocardial cell swelling, improved cellular alignment, and decreased inflammatory cell infiltration in the I/R+Nicorandil group, further validating nicorandil’s protective effects on myocardial structure [18].
Notably, in this study, CYP4F5 expression was significantly upregulated at both mRNA and protein levels in the I/R group, while nicorandil intervention suppressed its expression. Metabolomic analysis revealed significant correlations between CYP4F5 expression levels and multiple arachidonic acid metabolites (20-carboxy-leukotriene B4, 11-trans-leukotriene C4), suggesting that nicorandil may attenuate myocardial injury by inhibiting CYP4F5 and blocking abnormal activation of arachidonic acid metabolism at the metabolite level. As a key member of the CYP450 superfamily, the CYP4F subfamily is widely recognized to mediate the biotransformation and metabolic clearance of numerous cardiovascular clinical drugs. Although nicorandil is primarily metabolized by hepatic glucuronidation, local myocardial CYP4F5 upregulation during I/R injury may alter the microenvironmental metabolic capacity of the ischemic myocardium. Nicorandil-mediated CYP4F5 suppression may optimize local drug metabolism homeostasis and potentially affect the metabolic profile of commonly combined medications, including statins, antiplatelet agents, and anti-inflammatory drugs. ELISA further confirmed that nicorandil significantly reduced serum and myocardial levels of 20-HETE, demonstrating its inhibitory effect on CYP4F5 downstream metabolites. During I/R, 20-HETE—produced via CYP4F subfamily enzyme catalysis—is widely recognized as a key mediator of reperfusion injury, promoting vasoconstriction, reactive oxygen species (ROS) generation, inflammatory responses, and apoptosis [19]. Multiple studies have identified 20-HETE as a “pro-injury factor” in cardiovascular diseases [20–22]. Osama [23] demonstrated that 20-HETE activates voltage-gated calcium channels on vascular smooth muscle cells, enhancing calcium influx and inducing vasoconstriction, thereby reducing post-reperfusion myocardial blood flow and exacerbating hypoperfusion in ischemic regions. Additionally, 20-HETE induces ROS production, activates inflammatory pathways such as NF-κB, and triggers the release of TNF-α and IL-6, leading to inflammatory damage and cardiomyocyte apoptosis [24].
On the other hand, CYP450, a novel system regulating Ca2 + channels and contractility in mammalian cardiac cells, encompasses over 60 distinct P450 isoforms [25]. CYP450 is also implicated in signaling pathways associated with cancer, neurodegenerative diseases, and cardiovascular injury [26]. PPAR-α, a member of the nuclear receptor superfamily, exerts anti-inflammatory, antioxidant, and lipid metabolism-regulating functions in the cardiovascular system [27, 28]. Prior studies confirmed that PPAR-α activation in myocardial I/R injury models competitively inhibits NF-κB binding to DNA response elements, suppresses transcription of inflammatory cytokines (e.g., TNF-α, IL-6), and upregulates antioxidant enzymes (e.g., superoxide dismutase, glutathione peroxidase), thereby alleviating oxidative stress [29]. The cGAS/STING pathway, a DNA damage-sensing system, is activated during myocardial I/R due to cytosolic DNA accumulation. cGAS binds to DNA and catalyzes the production of the secondary messenger 2’3’-cGAMP, activating STING and triggering IRF3- and NF-κB-dependent inflammatory responses and pyroptosis [30]. Mechanistically, I/R-induced inflammatory activation identified in transcriptomic analysis (TNF and chemokine signaling) serves as the upstream driver to promote CYP4F5 transcription and expression. Abnormally elevated CYP4F5 accelerates the synthesis of downstream arachidonic acid metabolites represented by 20-HETE, which further triggers multi-dimensional pathological disorders. On the one hand, excessive 20-HETE inhibits PPAR-α activity, resulting in impaired energy metabolism and abnormal glycolysis and protein glycosylation observed in proteomic profiling. On the other hand, 20-HETE accumulation exacerbates oxidative stress, further activating NF-κB-mediated inflammatory cascade and cGAS-STING innate immune pathway, thus forming a vicious cycle of metabolism-inflammation-immunity imbalance. As the core regulatory target of nicorandil, downregulated CYP4F5 reduces the production of pro-damage metabolites such as 20-HETE, thereby restoring PPAR-α-controlled energy homeostasis, relieving protein glycosylation disorders, and simultaneously inhibiting the overactivation of NF-κB and cGAS-STING pathways. This unified regulatory network centered on CYP4F5 ultimately explains the consistent protective effects of nicorandil across transcriptomic, proteomic, and metabolomic layers. This study found that nicorandil upregulated PPAR-α expression while suppressing NF-κB and cGAS/STING pathway proteins in myocardial I/R rats. Combined with CYP4F5 expression changes, these findings suggest a synergistic regulatory network mediated by nicorandil: (1) PPAR-α activation may directly bind to the CYP4F5 gene promoter or indirectly inhibit upstream regulators to downregulate CYP4F5 transcription, reducing pro-inflammatory metabolites like 20-HETE; (2) inhibition of NF-κB and cGAS/STING pathways lowers inflammatory cytokine levels, mitigating CYP4F5-mediated oxidative stress. This dual “metabolic-inflammatory” inhibitory mechanism underscores nicorandil’s cardioprotective efficacy.
This study has several limitations. First, the conclusions are primarily based on rat models, and their applicability to humans requires further validation in large animal models or clinical samples. Second, the current study lacks direct functional validation experiments targeting CYP4F5, including specific inhibitors, gene knockdown, overexpression or gene knockout models. Therefore, we cannot fully confirm that CYP4F5 downregulation is the necessary and sufficient causal event for the cardioprotective effect of nicorandil. Targeted loss-of-function and gain-of-function experiments will be designed in future research to verify the direct causal relationship between CYP4F5 and myocardial I/R injury. Third, our current label-free proteomic analysis mainly focused on the screening of differentially expressed total proteins, without targeted enrichment and quantitative detection of other critical post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination. Therefore, whether nicorandil exerts regulatory effects on these PTMs cannot be fully clarified in the present study. Additionally, the analysis was conducted at the whole-tissue level, lacking single-cell resolution to dissect heterogeneous changes in CYP4F5 and associated pathways across distinct cell types. This limits a comprehensive understanding of the complex pathological mechanisms. Future studies will focus on clarifying these molecular mechanisms and exploring nicorandil’s potential for clinical translation.
Conclusion
This study demonstrates that nicorandil exerts cardioprotective effects against myocardial I/R injury by modulating CYP4F5 and its associated metabolic and signaling pathways. Nicorandil significantly suppresses CYP4F5 upregulation at transcriptional and translational levels, reduces the production of its downstream metabolite 20-HETE, and regulates PPAR-α, NF-κB, and cGAS-STING signaling, thereby enhancing myocardial protection. These findings provide novel strategies for the clinical application of nicorandil in treating myocardial I/R injury.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
Not applicable.
Author contributions
Conception and design: Huanxin Zhang. Data collection, analysis and processing: Jianlong Hao. Paper writing: Zhenrong Ge . Paper revision: Longfei Sun and Shubin Jiang. Final approval of manuscript: All authors.
Funding
This study was supported by the General Project of Natural Science Foundation of Xinjiang Uygur Autonomous Region (2024D01C123). Key project of hospital-level project of traditional Chinese medicine hospital affiliated to Xinjiang Medical University (ZYY20232ZD1). Noncommunicable Chronic Diseases-National Science and Technology Major Project(2024ZD0528300).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
This study was approved by the Animal Ethics Committee of Xinjiang medical university (Approval No. IACUC-JT-20241119-07).
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
No datasets were generated or analysed during the current study.