The animal experimental center at Guizhou Medical University (Guiyang, China) was responsible for raising mice under controlled conditions, namely a constant temperature of 22–24°C, a relative humidity of 55–60% and a 12-h light/dark cycle. The experimental procedures were approved by the animal experiment ethics committee of Guizhou Medical University (approval no. 2100328). To determine the effect of a HFD on LCN2 expression, male C57BL/6J WT mice (Cyagen Biosciences Inc.; weight, 18–20 g; age, 6 weeks) were fed either a HFD (60% kcal from fat; D12492 formula; Research Diets, Inc.) or normal chow diet for 24 weeks, and then blood and liver samples were collected to detect LCN2 expression. These mice (n=5/group) were given free access to food/water. To investigate the effect of LCN2 gene knockout on MI/R injury in HFD-fed mice, male WT and LCN2^−/− mice (Cyagen Biosciences Inc.; weight, 18–20 g; age, 6 weeks) were fed a HFD for 24 weeks, and then underwent MI/R injury or sham surgery. The mice were divided into the following four groups: WT + HFD + sham, WT + HFD + I/R, LCN2^−/− + HFD + sham and LCN2^−/− + HFD + I/R. The hearts (n=5/group) collected from the four groups underwent hematoxylin and eosin (H&E) staining. The degree of MI in the hearts of the four groups was assessed through TTC staining (n=5/group). Immunohistochemistry was performed to assess the expression of CD42b in the hearts collected from the four groups (n=5/group). In addition, immunohistochemistry was employed to detect the recruitment of Ly6G⁺, CD3⁺ and B220⁺ cells in the myocardial tissues collected from the mice in the WT + HFD + I/R and LCN2^−/− +HFD + I/R groups (n=5/group).

Cyagen Biosciences, Inc. provided C57BL/6J mice with a background that lacked the LCN2 gene (LCN2^−/−). Mouse tail genomic DNA was extracted using lysis buffer (cat. no. GD01-02; Bioland Scientific LLC) from mouse tail tissue, which was mixed with DNA stabilization buffer. PCR was then conducted using the 2X Es Taq Master Mix (cat. no. CW0690M; Jiangsu CoWin Biotech Co., Ltd.), as follows: 35 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The following PCR primers were utilized: LCN2, forward (F) 5′-CAACTCAGAACTTGATCCCTGCC-3′ and reverse (R) 5′-TTTCCCTAAGTCCCGTTCAATCC-3′. Agarose gel electrophoresis (2%) was used to determine the size of the PCR products and the gels were stained with nucleic acid dye SuperRed/GelRed (cat. no. BS354A; Biosharp Life Sciences). For WT mice, a PCR product of 1,287 bp was generated. By contrast, a PCR product of 586 bp was generated for LCN2^−/− mice.

Male mice were anesthetized by intraperitoneal injection of Avertin (1.25%, 250 mg/kg) (cat. no. BR4108423; Bioleaper). After anesthesia, the animals naturally laid down; their head, limbs, tail and whiskers did not respond to touch, their heartbeat and breathing were uniform in a supine position, and their muscles were relaxed. These signs indicated full anesthesia and that the subsequent experimental operations could be carried out. The tongue was pulled out with tweezers and endotracheal intubation was performed through the glottis. A 1.5-cm incision was made at the intersection of the skin between the left midclavicular line and the fourth intercostal space in mice, and hemostatic forceps were used to stretch the fourth intercostal space to expose the heart. The left anterior descending (LAD) coronary artery was identified, and a 1.5-mm suture was made at the lower margin of the left auricle through the LAD coronary artery and tied into a knot. In addition, the sham operation group underwent the same procedure with the suture passing under the LAD branch, but without binding.

Mice were intraperitoneally injected with 1% pentobarbital sodium (150 mg/kg) and were sacrificed by cervical dislocation. Death was verified by the absence of a heartbeat and dilated pupils. Liver and heart samples were then obtained and fixed with 4% paraformaldehyde (cat. no. P0099; 100 ml; Beyotime Institute of Biotechnology) for 24 h at room temperature. The liver and heart tissues were then dehydrated, embedded in paraffin and sliced into 5-µm sections. These tissue sections were placed onto slides and subjected to dewaxing before staining with the H&E staining kit (cat. no. C0105M; Beyotime Institute of Biotechnology). The staining procedure was carried out according to the manufacturer's instructions at room temperature, with hematoxylin staining performed for 6 min and eosin staining for 1 min. The degree of MI was assessed through TTC staining (cat. no. T8170; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Images of the tissue sections were captured using an optical microscope (80i; Nikon Corporation).

The heart tissues of mice collected from different treatment groups were fixed with 4% paraformaldehyde solution for 24 h and dehydrated in an ascending series of alcohol at room temperature. After permeabilization with xylene for 30 min, heart tissues were then embedded in paraffin and sliced into 5-µm sections. These tissue sections were attached to slides and dewaxed with xylene. For antigen retrieval, they were placed in dyeing tanks containing citrate buffer, and were boiled in a pressure cooker for 15 min. To inhibit the activity of endogenous peroxidase, a 3% solution of hydrogen peroxide was applied for 10 min at room temperature. The heart tissue sections were then blocked with goat serum (cat. no. SAP-9100; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at 24°C for 30 min, and were subsequently incubated with primary antibodies [anti-CD42b antibody (1:200; cat. no. ab183345; Abcam), anti-CD3 antibody (1:200; cat. no. ab16669; Abcam), anti-B220 antibody (1:200; cat. no. ab10558; Abcam) and anti-Ly6G antibody (1:200; cat. no. ab238132; Abcam)] at 4°C for 12 h, followed by incubation with a horseradish peroxidase (HRP)-IgG secondary antibody (1:50; cat. no. A0208; Beyotime Institute of Biotechnology) at 24°C for 60 min. The expression of specific proteins in the heart tissues was visualized using DAB staining. Images of the tissue sections were captured using an optical microscope (80i; Nikon Corporation). Three visual field images of the infarct area were obtained from each histochemical section. The DAB-positive cell number in each image was manually counted by ImageJ (version 1.8.0; National Institutes of Health), and the mean value was defined as the positive cell number in the section.

All blood samples used in the present study were obtained from healthy volunteers (age, 18–26 years) who provided written informed consent. Blood samples were collected from 18 volunteers, including 10 men and 8 women, with an mean age of 24 years, between March 2021 and March 2023. The Human Ethics Committee of Guizhou Medical University approved the experimental procedures [(approval no. 2021 (35)]. The collected blood was stored in a VACUETTE^® vessel and mixed with 0.109 M sodium citrate at a ratio of 9:1. Subsequently, centrifugation at 120 × g for 20 min at 25°C was conducted to obtain the washed platelets for further experiments.

After mice were intraperitoneally injected with 1% pentobarbital sodium (150 mg/kg), the blood serum was obtained by centrifuging blood samples (150 µl) collected from the tail veins of mice at 3,000 × g for 15 min at room temperature. The serum LCN2 levels were determined using a mouse ELISA kit (cat. no. ab199083; Abcam) according to the manufacturer's instructions.

Platelets were exposed to LCN2 (2 µg/ml; cat. no. 1757-LC-050; R&D Systems, Inc.) for 5 min at 37°C, and were then stimulated with thrombin (0.04 U/ml; cat. no. T6884; Sigma-Aldrich; Merck KGaA), collagen (1 µg/ml; cat. no. P/N 385; Chrono-Log Corporation) or adenosine diphosphate (ADP; 2.5 µM; cat. no. A2754; Sigma-Aldrich; Merck KGaA) for 5 min at 37°C. An equivalent volume of normal saline was used as a vehicle. Aggregation of platelets was then assessed using an aggregator (Model 700; Chrono-Log Corporation), while constant stirring was maintained at a rate of 1,200 rpm for 5 min. The expression levels of P-selectin were analyzed by performing flow cytometric analysis. Platelets were stimulated with or without LCN2 (2 µg/ml; cat. no. 1757-LC-050; R&D Systems, Inc.) for 5 min at 37°C, and were then exposed to thrombin for 5 min at 37°C; untreated platelets were used as a control. After incubating with FITC-conjugated P-selectin antibodies (0.2 µg/ml; cat. no. ab33279; Abcam) for 20 min at 37°C, the expression of P-selectin on the platelet surface was detected by flow cytometry (CytoFLEX S; Beckman Coulter, Inc.). Flow cytometry data were analyzed using CytExpert software (version 2.3.1.22; Beckman Coulter, Inc.).

To investigate the adhesion of platelets, cellular coverslips were categorized into three distinct groups: The first group was coated with 1% bovine serum albumin (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at 20°C and subsequently incubated with non-treated platelets (2×10⁷). The remaining two groups were coated with 5 µg/ml collagen for 14 h at 4°C, and incubated with 2 µg/ml LCN2-stimulated platelets (2×10⁷) and unstimulated platelets (2×10⁷) at 37°C. After a 45-min incubation, the platelets were stained with iFluor^™ 680-phalloidin (1 µg/ml; cat. no. 40788ES75; Shanghai Yeasen Biotechnology Co., Ltd.) for 25 min at 37°C, and the adhesion of platelets was observed using a fluorescence microscope.

Platelets were incubated with or without LCN2 (2 µg/ml) for 5 min at 37°C, and subsequently stimulated with thrombin (0.4 U/ml) at 37°C. The process of clot retraction was monitored and recorded at intervals of 0, 20, 40 and 60 min after thrombin stimulation by capturing images of platelet clots.

Liver tissues were collected for extraction of total RNA using a DNA/RNA co-extraction kit (cat. no. DP422; Tiangen Biotech Co., Ltd.). Subsequently, cDNA synthesis was carried out using the FastKing gDNA Dispelling RT SuperMix (cat. no. KR118; Tiangen Biotech Co., Ltd.) according to the manufacturer's instruction. qPCR was performed according to the protocol specified by the manufacturer of FastFire qPCR PreMix (SYBR Green) (cat. no. FP207; Tiangen Biotech Co., Ltd.). The qPCR program consisted of an initial phase of pre-denaturation at 95°C for 1 min, followed by 40 cycles at 95°C for 5 sec, 55°C for 10 sec and 72°C for 15 sec. For the expression analysis of the target genes, GAPDH was employed as the internal reference for normalization. The primer sequences were as follows, LCN2, F 5′-AAGGCAGCTTTACGATGT-3′, R 5′-TGGTTGTAGTCCGTGGTG-3′; GAPDH, F 5′-GCACAGTCAAGGCCGAGAAT-3′ and R 5′-GCCTTCTCCATGGTGGTGAA-3′. Data were normalized to GAPDH levels as a reference. Subsequently, the gene expression levels were calculated using the 2^−ΔΔCq method (21).

The data were analyzed using GraphPad Prism v5.01 (Dotmatics), and the results are presented as the mean ± SEM derived from ≥3 independent experiments. Differences between two groups were assessed using unpaired Student's t-test, while differences among multiple groups were determined by one-way ANOVA and Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Notably, LCN2 has been shown to be highly expressed in adipose tissue, and to serve a role in both obesity and obesity-related diseases (22,23). Considering that obesity is an important risk factor for CAD and MI/R injury, a mouse model of obesity was established in the present study through the use of a HFD. The results revealed a significant increase in the weight of the mice (Fig. 1A), as well as the accumulation of large lipid droplets in their liver tissues (Fig. 1B) following administration of a HFD. Additionally, a significant upregulation of LCN2 levels was observed in both the liver (Fig. 1C) and serum (Fig. 1D) of mice fed a HFD.

In cardiovascular disorders, the formation of atherosclerotic plaques and microthrombi are the consequences of platelet aggregation, adhesion and activation (24). These processes also contribute to the worsening of MI/R injury. Previous research has indicated that endogenous proteins possessing pro-inflammatory properties can enhance platelet hyperresponsiveness, and exacerbate atherosclerosis and MI/R injury by promoting platelet aggregation (25,26). The present study investigated the effects of LCN2 on platelet function. When platelet agonists (thrombin, collagen and ADP) were applied, incubation with LCN2 promoted platelet aggregation (Fig. 2A). Additionally, compared with the untreated platelets, a higher number of platelets adhered to collagen-coated coverslips following treatment with LCN2 (Fig. 2B). Furthermore, it was observed that LCN2-treated platelets had more pronounced clot retraction (Fig. 2C) and higher expression of P-selectin (Fig. 2D) upon stimulation with thrombin.

Obesity serves an important role in the development of cardiovascular diseases. One of the main contributors to MI/R injury in obese individuals is the increase in endogenous bioactive molecules, which stimulate platelet activation (25). The gene phenotypes of WT mice and LCN2^−/− mice were determined by PCR (Fig. 3A). For WT mice, a PCR product of 1,287 bp was generated, whereas a PCR product of 586 bp was generated for LCN2^−/− mice. The PCR product of 586 bp in LCN2^−/− mice indicated that LCN2 was knocked out, whereas the PCR product of 1,287 bp in WT mice indicated that LCN2 was not knocked out. These mice were then subjected to MI/R injury or sham operation after being fed a HFD. Subsequently, cardiac pathology was observed by H&E staining (Fig. 3B). After sham operation, the myocardia of WT mice and LCN2^−/− mice were closely arranged, with normal structure and no obvious inflammatory cell infiltration. WT mice and LCN2^−/− mice with MI/R injury exhibited notable histological features of myocardial tissue injury and inflammatory cell infiltration. In the case of MI/R injury, there was less structural damage to the myocardium and relatively less inflammatory cell infiltration in LCN2^−/− mice compared with that in the WT mice (Fig. 3B). TTC staining showed no obvious ischemic features in the myocardia after sham operation in WT mice and LCN2^−/− mice. After MI/R injury, the myocardial infarct size of LCN2^−/− mice was smaller than that of WT mice (Fig. 3C).

During MI/R injury, platelets tend to form microthrombi within the microvessels of the heart and worsen myocardial damage by releasing certain bioactive molecules (24). Thus, the current study aimed to investigate whether the absence of LCN2 affects the accumulation of platelets induced by I/R injury in myocardial tissue. As shown in Fig. 4A, the number of CD42b⁺ platelets in myocardial tissues was lower in LCN2^−/− mice compared with that in WT mice following reperfusion post-ischemia. These findings suggested that LCN2 induced by HFD may enhance platelet accumulation in myocardial tissue during MI/R injury. Notably, activated platelets form complexes with leukocytes and adhere to microvessels within the heart, thereby exacerbating MI/R injury. Suppression of platelet activation can reduce the recruitment of inflammatory cells in myocardial tissue, ultimately mitigating MI/R injury (24,25). In the present study, it was observed that LCN2 gene knockout decreased the accumulation of Ly6G⁺ cells within the region of myocardial injury induced by I/R, while having no significant impact on the recruitment of CD3⁺ or B220⁺ cells (Fig. 4B). These results indicated that LCN2 knockout might alleviate MI/R injury by inhibiting platelet and Ly6G⁺ cell recruitment.