Participants and Data Collection Patients

A total of 115 patients with STEMI who underwent emergency PCI at Nanjing Drum Tower Hospital from October 2021 to December 2022 were selected for the study. Blood samples were collected for routine blood tests before PCI, and cardiac magnetic resonance (CMR) and echocardiography were performed within 1 week after PCI. Inclusion criteria of patients were: (1) consistent with the diagnostic criteria for acute STEMI [2], (2) primary PCI within 12 h after onset, (3) age ≤ 85 years old, and (4) informed consent was signed. This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital. The approval number was 2021-531-02.

CMR Assessment

All patients underwent CMR examination within 1 week after PCI using the Philips 3.0T Ingenia magnetic resonance imaging system. The patients were positioned in a supine position, and the breath-holding sequence was scanned over 12 cardiac cycles with a breath-holding time of 12–17 s. Gadolinium was then administered via a peripheral vein for first-pass perfusion scans, and myocardial delayed gradient echo inversion recovery sequence enhancement scans were performed 10 min after injection (TR: 6 ms, TE: 3 ms, FA: 25°, TI: 260–350 ms, shot duration: 100–125 ms, voxel: 1.6 mm × 1.9 mm × 8 mm). CMR images were analyzed by experienced radiologists. The area of IS was shown as hyperintensity in images, and MVO was described as hypointense areas in the area of IS in late-gadolinium enhancement (LGE) CMR images. The representative CMR-LGE images are shown in Supplementary Fig. 1. Both IS and MVO were expressed as their percentage of left ventricle (LV).

Animal Experiments and Ethics Statement

All animal experiments were approved by the Institutional Ethics Committee of Nanjing Drum Hospital (2021AE02005) and followed the guidelines outlined in the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male Sprague−Dawley (SD) rats (n = 12, weighing 220−250 g, 7−8 weeks) and C57BL/6 mice (n = 50, weighing 20−25 g, 7−8 weeks) were purchased from the Center for Model Animals, Nanjing University. Animals were placed in 22 °C and 65–70% humidity chambers for 12 h light–dark cycles and fed a standard laboratory diet with free access to food and water. In our study, the rat experiment was only used to observe the MVO area since MVO, which stands for microvascular obstruction, is defined as a very small area that is not easily observable. The rat heart volume is larger than that of mice, making it more convenient to observe the MVO size in hearts. The other animal experiments in this study were all conducted on mice. Rats or mice were divided into three groups—sham group, I/R+ PBS group, and I/R + colchicine group. At the end of the study, all animals were given 1.5% isoflurane inhalation anesthesia followed by euthanasia by cervical dislocation.

Rats or mice were used to establish the myocardial I/R model. They were anesthetized by inhalation of isoflurane (1.5–2%) and ventilated with room air using a ventilator. A thoracotomy was performed at the fourth intercostal space to expose the heart and left anterior descending coronary artery (LAD). The rats’ LAD was ligated with a 4–0 silk suture and the mice’s LAD was ligated with a 7–0 silk thread and released after 45 min to restore blood perfusion [15]. For colchicine treatment, rats or mice were administered colchicine once (intraperitoneal injections, 0.1 mg/kg) (C3915, Sigma-Aldrich, USA) immediately after reperfusion. The dose of colchicine was determined according to a previously published study [16]. The artery was separated without ligation in the sham operation group.

Assessment of Microvascular Obstruction (MVO) Size

After 24 h of reperfusion, the rats (n = 4 in sham, I/R+PBS, I/R+ colchicine group separately) were placed under general anesthesia with isoflurane, and the limbs were immobilized in a supine position on an animal surgical plate. The thoracic cavity was reopened along the original incision to expose the heart, and the rat’s aorta was carefully separated to expose it; 1 mL thioflavin S (4% solution, Sigma Aldrich) was injected via the aorta to delineate the no-reflow zone. The heart was quickly excised and cut crosswise into 1 mm-thick slices by heart slice mold (1 mm spacing). Slices were exposed to UV light (302 nm) using a UV transilluminator for digital imaging. Light blue indicates myocardial uptake after infusion of thioflavin-S into the area at risk, whereas dark blue indicates lack of perfusion which was delineated as areas of MVO by Image J software [17]. Percentage MVO was expressed as a percentage of left ventricular (LV) [18].

Flow Cytometry Analysis Blood and Bone Marrow Neutrophils

Mice (n = 4 each group) were euthanized at 12 h after reperfusion and their ocular venous blood was collected in EDTA tubes, and erythrocytes were lysed using red blood cell (RBC) lysis buffer (Invitrogen, USA). They were washed, rinsed, and resuspended in phosphate-buffered saline (PBS) or FACS buffer for fluorescent antibodies (anti-mouse PE-labeled antibody against Ly6G (1:50, 12-9668-80, Invitrogen, USA) and FITC-labeled antibody against CD45 (1:50, 11-0451-81, Invitrogen, USA)) staining. The femur and tibia of each mouse were isolated and washed, and their ends were cut to expose the marrow cavity. The BM was washed directly with cold PBS using a 40 μm nylon mesh strainer. After the supernatant was aspirated, the RBCs were lysed in the RBC lysis buffer. Cells were then resuspended in FACS buffer and subjected to fluorescent antibody (anti-mouse PE-labeled antibody against Ly6G (1:50, 12-9668-80, Invitrogen, USA) and a FITC-labeled antibody against CD45 (1:50, 11-0451-81, Invitrogen, USA)) staining, washed, and resuspended in FACS buffer for flow cytometry analysis.

Heart and Spleen Neutrophils

The hearts (n = 4 each group) were collected at 24 h after reperfusion and spleen tissues (n = 4 each group) were collected at 12 h after reperfusion after the euthanization of the mice. A single-cell suspension of the tissues was obtained by using a gentle MACSTM Dissociator (Miltenyi Biotec). Then, the samples were stained with the antibodies (anti-mouse PE-labeled antibody against Ly6G (1:50, 12-9668-80, Invitrogen, USA) and FITC-labeled antibody against CD45 (1:50, 11-0451-81, Invitrogen, USA)), washed, and resuspended in FACS buffer for flow cytometry analysis.

Flow cytometry was carried out on an FACS Aria flow cytometer (BD Bioscience, USA), and data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

In Vivo Proliferation Assay (EdU)

For proliferation studies, the I/R+ ABR-215757 treated mice group was built. The S100A8/A9 inhibitor (ABR-215757) was administered orally once (10 mg/kg) 4 h before LAD ligation, and the treatment was continued (10 mg·kg−1·d−1) in drinking water until termination mice [19]. Mice (n = 4 in sham, I/R+PBS, I/R+ colchicine, I/R+ S100A8/A9 inhibitor group separately) were injected with 0.2 mg of 5-ethyny l-2’-deoxyuridine (EdU) for 12–14 h via intraperitoneal injection before euthanization. In preparation for flow cytometry, after 12 h of reperfusion, cell populations were immunostained CD45+Ly6G+ and the incorporation of EdU was quantified using Click-iTTM Plus EdU flow cytometry assay kit (Molecular Probes, Eugene OR) according to the manufacturer’s instructions. Proliferation was quantified and expressed as a percentage of EdU+ cells [19].

Cell Culture

Primary neutrophils were collected from bone marrow as previously described [20]. Briefly, bone marrow cells were collected from the femurs and tibias of 4-week-old mice by flushing with RPMI 1640 medium containing 10 U/mL heparin, 50 U/mL penicillin, and 50 g/mL streptomycin. Then, cells were treated with RBC lysis buffer and filtered through a 70 μm mesh. The cell pellet was resuspended in 200 μL buffer per 5×107 cells. Primary neutrophils were sorted from the cells by magnetic beads according to the manufacturer’s instructions (130-097-658, Miltenyi Biotec, Germany). After washing with PBS, primary neutrophils were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 g/mL streptomycin, followed by inoculation in a humidified atmosphere of 5% CO2 and 95% O2 at 37 °C [21]. To verify whether colchicine inhibits the S100A8/S100A9 signaling pathway in neutrophils, recombinant mouse S100A8/S100A9 (2 µg/ml, R&D Systems, MN, USA) was added to the culture medium in primary neutrophils for 8–12 h with or without colchicine (10 μmol/L, C3915, Sigma-Aldrich, USA) pretreatment [19, 21]. NLRP3 expression was measured using western blotting.

Preparation of Hypoxia/Reoxygenation (H/R) Cell Model and Measurement of Intracellular ROS

The H9c2 cardiomyocytes (Rat cardiomyocytes; American Typer Culture Collection, Manassas, USA) were maintained in DMEM supplemented with 10% FBS and 50 U/mL penicillin, and 50 g/mL streptomycin. The culture conditions contained 95% O2 and 5% CO2 at 37 °C. Cells in the control group were cultured under normal conditions. The H/R group cells were placed in a three-gas incubator (1% O2, 94% N2, and 5% CO2) under hypoxia for 12 h. Then, the cells were cultured with normal DMEM in a carbon dioxide incubator (5% CO2 and 95% O2) for 6 h for reoxygenation. The H/R group was treated with colchicine (5 nmol/L) or PBS and exposed to H/R, simultaneously [22].

Myocardial oxidative stress was measured using a reactive oxygen species assay kit (ROS Assay Kit) (S0033S, Beyotime, China) [16]. After 6 h of reoxygenation, the medium of H9c2 cells was changed to a serum-free medium containing DCFH-DA (10 μM). After incubation at 37 °C for 30 min, images were taken by fluorescence microscope (Leica Thunder, Germany) [16]. ROS were green (excitation wavelength 488 nm, emission wavelength 535 nm) in cells [22].

Enzyme-linked immunosorbent Assays (ELISA)

Peripheral blood samples were collected at 6, 12, 24, and 48 h of reperfusion after myocardial I/R for ELISA detection. EDTA anticoagulant blood samples (n = 4 each group) were collected and incubated at room temperature on a shaker for 30 min. Platelet-depleted plasma was collected after centrifugation at 2400 g for 20 min at 4 °C, and interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and S100A8/A9 protein levels were measured using commercial ELISA kits according to the manufacturer’s instructions (Sbjbio-Z/E, Nanjing, China).

Immunostaining Fluorescence

After 24 h reperfusion of the myocardial I/R model, fresh heart tissue (n = 4 each group) was embedded in an optimal cutting temperature compound (OCT) at –80 °C for 2h, cut into 6mm sections, and stored at –80 °C. Sections were washed with PBS, blocked with goat serum for 1h, and incubated overnight at 4 °C with primary antibodies, marker of Nets: Ly6G+(1:200, ab25377, Abcam, UK) MPO+(1:100, 14569, CST, USA) CitH4+(1:100, 07–596, Sigma-Aldrich, USA), three co-localized, markers of cardiomyocyte: α-Actinin (Sarcomeric) (α-Actinin) (1:200, A7811, Sigma-Aldrich, USA). After washing three times with PBS, the cells were incubated with secondary antibodies (Alexa Flour 488, 1:500, 4412, CST, Alexa Fluor 555,1:500, 4413, CST, and Alexa Flour 647 1:500, 4414, CST, USA) or (Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H+L), 1:400, 711-165-150, Jackson ImmunoResearch, USA) for 2 h at room temperature in the dark. Nuclei were counterstained with DAPI. Samples were sealed with 50% glycerol. Images were captured using a Leica imaging system.

TUNEL Assay

TUNEL assay was used to detect cardiomyocyte apoptosis in myocardial tissue. The sections were examined with a TUNEL assay kit (Beyotime Biotechnology, Shanghai, China). Images of the infarct area were acquired in each section using an OLYMPUS microscope (Japan). Image J software was used to count the number of TUNEL-positive cells in each of the total number of myocardial nuclei, and to calculate the percentage of apoptotic cardiomyocytes [23].

Immunohistochemistry

After the mice (n = 3 each group) were euthanized after 24 h reperfusion, the femur, tibia, and fibula were harvested for histological studies. Immunohistochemical staining was used to detect the expression of IL-1RI, IL-1RII, and IL-1Ra in the BM. Paraffin sections were deparaffinized and rehydrated, then deposited and stored at 4 °C overnight before being incubated with primary antibodies IL-1RI (1:50, sc-393998, Santa Cruz, USA), IL-1RII (1:50, sc-376247, Inc), IL-1RII (1:50, sc-376247, Santa Cruz, USA), and IL-1Ra (1:50, sc-374084, Santa Cruz, USA). The next day, the corresponding secondary antibody was applied at 20 to 25 °C for about 50 min, followed by the prepared 3,3’-diaminobenzidine (DAB) substrate and hematoxylin. The results were evaluated by two qualified pathologists. Motic Images Plus version 2.0 system (Motic China Group Co, Xiamen, China) was used for image acquisition and the ImageJ analysis system was quantified.

Western Blotting

Cultured cells or heart tissues in mice euthanized after 24 h reperfusion were homogenized in a lysis buffer containing a mixture of protease and phosphatidase inhibitors. The homogenate was denatured in a loading buffer at 95 °C for 10 min; 30 ug of protein was loaded onto SDS-PAGE gels and transferred to PVDF membranes. Subsequently, cell membranes were blocked with TBST containing BSA (5%), followed by incubation with specific antibodies (caspase-3(1:1000, 14220, CST, USA), cleaved caspase-3 (1:1000, 9664, CST, USA), Tubulin (1:1000, 11224-1-AP, Proteintech, USA), NLRP3(1:1000, 15101, CST, USA), and GAPDH (1:1000, 5174, CST, USA)) overnight at 4 °C. Finally, cells were incubated with horseradish peroxidase-labeled anti-rabbit immunoglobulin G. The signals were detected with an electrochemiluminescence (ECL) system and quantified by scanning densitometry with an ImageJ analysis system.

Bioinformatics Analysis

The datasets were collected from the Gene Expression Omnibus (GEO) database. The initial work was to identify differentially expressed genes (DEGs) and then find common DEGs for Colchicine, NETs, MVO, and I/R. Gene Ontology (GO) analysis of selected differential genes was performed using the online tool DAVID (https://david.ncifcrf.gov/), and the Kyoto Encyclopedia of Genomes (KEGG) pathway enrichment analysis of differential genes was performed using the KOBAS online analysis database (http://kobas.cbi.pku.edu.cn/). The results were presented as bar graphs and bubble plots, respectively. Then the STRING online database protein–protein interaction (PPI) network was used to analyze the association of DEGs and to identify the interacting molecules of colchicine in myocardial I/R injury.

Statistical Analysis

The Clinical Research Section used the Kolmogorov–Smirnov test to test for normality. Continuous normally distributed data are represented as mean ± standard deviation (SD) and are compared using the unpaired Student t-test. Nonnormal distribution variables are reported as median (interquartile range) and compared using the Mann–Whitney U test. Categorical variables are expressed as frequency and percentage and are appropriately studied by either chi-square or Fisher’s exact test. The univariate analysis was divided into two groups: extensive and no or mild MVO groups; we selected 2.6% as the segmentation value, divided patients into two groups, included all baseline variables of P <0.05 in univariate analysis into the logistic regression model, calculated their odds ratio and 95% confidence interval, and P-values <0.05 were considered statistically significant. All statistical analyses were performed using the IBM SPSS software package (IBM SPSS Statistics 25).

Basic Experimental Section

All values are expressed as mean ±SD or median, with interquartile ranges as appropriate. For data with normal distribution and homogeneity of variance, the unpaired t-test was used to compare the two groups. For the comparison of multiple samples at the same time point, the one-way ANOVA was used to confirm the existence of differences between groups, and then Tukey’s test was used to compare the means of each group. For the comparison of multiple samples at multiple time points, two-way ANOVA was used to confirm the existence of differences between groups, and Bonferroni’s test was used to compare the means of each group. The difference in P-value < 0.05 was considered statistically significant. Statistical analysis was carried out using GraphPad Prism 9.0.