Bioinformatics analysis
The GEO GSE62646 dataset contains data from 28 patients with ST-segment elevation myocardial infarction (STEMI) and 14 control patients without history of myocardial infarction. The blood was collected on the 1st day of myocardial infarction, after 4–6 days, and after 6 months. Peripheral blood mononuclear cells (PBMC) were isolated from the blood and gene expression analysis in the cells was performed with GPL6244 Affymetrix GeneChip® Human Gene 1.0 ST microarrays and GCS3000 TG system. The GEO GSE61145 dataset contains data from 10 non-disease controls, 7 patients with STEMI, 10 non-ST-elevation patents, and 9 patients with unstable angina. The PBMC were also isolated from their blood. It should be noted that PBMC were isolated from patients with STEMI both before and after percutaneous coronary intervention (PCI). The sample obtained before PCI was named as STEMI. The sample obtained after the recovery from STEMI via PCI was named as r_STEMI. Weighed Gene Co expression Network Analysis (WGCNA) is a bioinformatics algorithm for constructing co expression networks, and is used to identify modules associated with diseases and screen important pathogenic mechanisms or potential therapeutic targets. The data from the GSE62646 dataset were analyzed using the WGCNA package in R3.4.1 language (https://cran.r-project.org/web/packages/WGCNA/index.html) Version 1.61 Screening for Stable Modules Related to MI. The module partitioning screening threshold is set to: the module contains at least 50 mRNA, cutHeight = 0.99. The data from the GSE61145 dataset were analyzed using the R package pheatmap (Version: 1.0.8, https://cran.r-project.org/web/packages/pheatmap). The acquisition of differentially expressed genes was based on the screening criteria of adjust P < 0.05 and log2FC > 1. PCA analysis was performed on the samples using FactoMineR and Factoextra package. Intersection genes were taken from the differentially expressed genes of GSE62646 and GSE61145 to obtain MI/RI related differentially expressed genes, and gene ontology (GO) functional annotation analysis was performed using clusterProfiler R package [17].
Patients and health volunteers
From June 2022 to June 2023, 15 myocardial infarction (MI) patients and 15 healthy volunteers were recruited at the Liuzhou Municipal Liutie Central Hospital (Guangxi, China). For the recruited MI patients, those who were admitted with ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI) within the past 24 hours and underwent percutaneous coronary intervention (PCI) with stent implantation were included. These patients were excluded if they had cardiogenic shock, chronic kidney disease, autoimmune diseases, chronic inflammation, or cancer, among others. For the recruited 15 healthy volunteers, individuals aged 22 to 75 with no history of chronic diseases or chronic medication use were included. This study was approved by the ethics committee of the Liuzhou Municipal Liutie Central Hospital (Guangxi, China), and all patients signed informed consent forms. Blood samples were collected from these participants for the isolation of PBMC and serum for clinical research.
Cell culture and treatments
The AC16 and HL-1 myocardial cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were maintained in DMEM supplemented with 10% FBS (Biowest) at 37°C in an incubator with a humidified atmosphere containing 5% CO2. AC16 and HL-1 cells were subjected to various dosages of TNFSF14 recombinant protein (0, 10, 50, 250nM). The human TNFSF14 recombinant protein was purchased from Abnova Company (Shanghai, China), and the mouse TNFSF14 recombinant protein was purchased from AtaGenix Company (Wuhan, China). The impairment of AC16 and HL-1 cells caused by TNFSF14 was evaluated by cell viability, apoptosis, and cell cycle assays, as well as the levels of a few of cardiac damage-related markers. AC16 and HL-1 cells were also treated with traditional herbal extracts, including DOP (Pioneerherb, Nanchang, China), CA (MedChemExpress, Shanghai, China), Cur (Sigma-Aldrich, Shanghai, China), Res (MedChemExpress, Shanghai, China) and Val (GuideChem, Nanjing, China) to evaluate their protection against TNFSF14. Tunicamycin (TM, 2 µg/mL, MedChemExpress, Shanghai, China) and PNGase F (1000 U/mL, MedChemExpress, Shanghai, China) were added to suppress N-linked glycosylation on LTβR protein.
Cell Viability Assay
Cell viability was measured using the CCK8 kit according to the manufacturer's instructions. In brief, myocardial cells were seeded into a 96-well plate, and 10 µL of CCK-8 reagent was added to each well and incubated at room temperature for 3 hours. The absorbance was measured at 450 nm using a microplate reader (BioTek uQuant, USA).
Flow Cytometry for apoptosis and cell cycle tests
Cell apoptosis was evaluated using the V-FITC/PI apoptosis detection kit (Sigma-Aldrich, USA). According to the kit's instructions, cells were treated with 5 µL of FITC and 5 µL of PI. Subsequently, the cells were incubated for 10 minutes and apoptosis rate was measured by Attune NxT flow cytometry. Data analysis was performed using FlowJo 7.6 software (BD Biosciences, USA). For the cell cycle assay, cells in each experimental group were collected and washed using PBS. Then, the washed cells were resuspended using 500 µL PBS + 50 µg/mL PI (propidium iodide). Thereafter, 0.2%Triton X-100 and RNase A (100 µg/mL) were added to the cells and the cells were maintained in the dark at 4°C for 30 min. Finally, cells were analyzed using FlowJo 7.6 software to determine the percentage of cells in G2/M, S and G1 stages.
Immunofluorescence
Test cells from each group were fixed in 4% paraformaldehyde solution. After washing with PBS three times, the cells were blocked with 0.1% Triton solution in goat serum for 1 hour, followed by incubation with diluted anti-MGA antibody (1:100 dilution; Abcam, Shanghai, China) overnight at 4°C. Then, the cells were washed with PBS three times and incubated with IgG secondary antibody and DAPI at room temperature for 40 minutes. Finally, the cells were observed and photographed under a fluorescence microscope (Nikon, Japan).
Co-IP Assay
Cells or tissues from each experimental group were incubated in immunoprecipitation buffer (Thermo Fisher Scientific, USA) at 4℃ for 30 minutes to lyse the cells. The supernatant was collected for immunoprecipitation. According to the kit instructions, 30 µl of magnetic beads were added with LTβR and MAX primary antibodies antibodies and IgG (as a negative control). After mixing the antibodies with magnetic beads, the magnetic beads were washed and enriched, and mixed with the extracted cellular and tissue total protein samples. The mixture was inverted and incubated overnight at 4℃, followed by washing the magnetic beads with binding/washing buffer, centrifugation, and natural sedimentation. Subsequently, the supernatant was discarded, and the sediment was washed with TBS solution at a volume 20 times that of the magnetic beads. The protein precipitation was then examined by Western blotting.
Chromatin Immunoprecipitation (ChIP) assay
ChIP assay was performed using the ChIP Kit (CST, USA), following the manufacturer's instructions. In brief, cells from each experimental group were crosslinked with formaldehyde and sonicated to fragment chromatin into an average length of 200–1000 bp. Subsequently, immunoprecipitation was carried out using anti-c-Myc antibody (Abcam, Cambridge, UK) or IgG control. Finally, the precipitated DNA was amplified and analyzed by PCR.
RT-qPCR
Cells from each experimental group were collected and total RNA was extracted using the Trizol reagent kit (Invitrogen, USA). The RNA was reverse transcribed into cDNA, and then PCR was performed on the cDNA using the primers listed in Table 1. The qPCR thermal cycling parameters were as follows: pre-denaturation at 95℃ for 10 minutes, denaturation at 95℃ for 15 seconds, annealing at 60℃ for 35 seconds, extension at 72℃ for 10 seconds, with a total of 45 cycles. The relative expression level was calculated using the 2−ΔΔCt method.
Table 1
Gene | Species | Forward (5′-3′) | Reverse (5′-3′) |
TNFSF14 | Human | ATACAAGAGCGAAGGTCTCACG | CTGAGTCTCCCATAACAGCGG |
Mouse | ACTGCATCAACGTCTTGGAGA | TGGCTCCTGTAAGATGTGCTG |
HSPD1 | Human | ATGCTTCGGTTACCCACAGTC | AGCCCGAGTGAGATGAGGAG |
TMEM4 | Human | TCTGGTGGATGAACTAGAATGGG | TGTTTGGGGGAACAGTAACAGT |
UBR2 | Human | GTACCAGCATTTAGCCCACTATG | TGCAAGAATATGTAGGCTCTCCT |
SAP130 | Human | AACAGTTTCCTCGGTTAGGAGC | TGGGTTTATCAATCCAGCAGAAC |
NSFL1C | Human | CCCAGGAAGAAAAGTCCCAAC | CAGGGCTCTTGGTCACTCG |
SLC7A6 | Human | CCACCTACCATCTTGTCCCTA | GCATAGTTTCGGAGGACCTTTG |
MOGAT1 | Human | AAAGTGTGTCCTACATGGTAAGC | TGATCCTTCAGGGTTGTCAGTT |
SAMD4B | Human | CTCACTGGCGGACTGCAAT | GTAGCCTCATGTACTCCGACTT |
PDIA6 | Human | AGGAGGTCAGTATGGTGTTCAG | GAGGCGATCCTTCACGAGC |
TOLLIP | Human | TGGGCCGACTGAACATCAC | GTGGATGACCTTATTCCAGCG |
IL-29 | Human | CACATTGGCAGGTTCAAATCTCT | CCAGCGGACTCCTTTTTGG |
ETFA | Human | CAGAGGAACTGACACCATTGATT | AGGTTCTTTCCGAAGGCAGAT |
ING4 | Human | TGCAAGGAATTTGGTGACGAC | GCCGCCGAATGTGTTTGTC |
Cx43 | Human | GGTGACTGGAGCGCCTTAG | GCGCACATGAGAGATTGGGA |
Mouse | ACAAGGTCCAAGCCTACTCCA | CCGGGTTGTTGAGTGTTACAG |
MGA | Human | GTCATCGGGTCCGTCTTACAG | TGAGGCTACACGGGAGTCAT |
Mouse | GAGGAGCACCTACCTTCTTTGT | ACGGGCATCTCGATTAGTAACT |
c-Myc | Human | GGCTCCTGGCAAAAGGTCA | CTGCGTAGTTGTGCTGATGT |
Mouse | ATGCCCCTCAACGTGAACTTC | GTCGCAGATGAAATAGGGCTG |
GAPDH | Human | GGAGCGAGATCCCTCCAAAAT | GGCTGTTGTCATACTTCTCATGG |
Mouse | AGGTCGGTGTGAACGGATTTG | GGGGTCGTTGATGGCAACA |
Western blotting
Myocardial cells or tissue were placed in pre-chilled RIPA lysis buffer for lysis and total protein extraction, and the protein concentration was determined using the BCA assay kit (Sigma Aldrich, Cambridge, MA). Subsequently, the proteins were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was then incubated overnight at 4°C with primary antibodies against TNFSF14 (1:50 dilution), IL-29 (1:100 dilution), cTnT (1:100 dilution), cTnl (1:100 dilution), TNNI3K (1:100 dilution), Cx43 (1:50 dilution), GATA-4 (1:50 dilution), LTβR (1:20 dilution), MAX (1:20 dilution), MGA (1:50 dilution), c-myc (1:50 dilution), and β-Actin (1:200 dilution; Abcam, Shanghai, China). The secondary antibodies were incubated with the membrane at room temperature for 1 hour, and protein bands were visualized using an ECL assay kit.
IR Animal Model Establishment
Thirty C57BL/6 mice, aged 8 weeks and weighing between 22 and 26 g, were provided by the Experimental Animal Center of Guangxi Medical University. After one week of adaptive feeding, the animal experiments were conducted. This experiment was approved by the animal ethics committee of the Experimental Animal Center of Liuzhou Municipal Liutie Central Hospital affiliated to Guangxi Medical University. The mice were randomly divided into the sham group, IR group, and IR + Val group, with 10 rats in each group. Before modeling, mice in the IR + Val group were continuously fed with Val (5 mg/kg) for 7 days. For the IR model creation, the mice were intubated and connected to a small animal ventilator after intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg) anesthesia. Following the reference [14], thoracotomy was performed on the left chest to expose the heart between the 3rd and 4th ribs. A ligature was tied below the proximal end of the left anterior descending coronary artery to induce myocardial ischemia, which could be observed by the whitening of the anterior wall of the left ventricle. Thirty minutes later, the ligature was untied to restore arterial blood flow (i.e. reperfusion). The mice chest was sutured layer by layer and connected to an electrocardiograph, whereas the sham group underwent the same procedure without ligation. After 2 hours of reperfusion, myocardial tissue was collected for various indicators testing.
Statistical analysis
All results were analyzed using SPSS 20.0 and expressed as mean ± SEM. Differences between two or more groups were assessed using t-tests and one-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.