Patients
This study was approved by the Ethics Committee of West China Hospital (2019-332), and all patients signed informed consent. The study protocols were conducted in accordance with guidelines of WMA Declaration of Helsinki and CIOMS International Ethical Guidelines Biomedical Research Involving Human Subjects. All experimental protocols were approved by the the Ethics Committee of West China Hospital (2019-332). All samples were obtained from West China Hospital. Six scoring systems were included in the final analysis: gencini, syntax1, PCI syntax2 score, PCI 4-year mortality, CABG syntax2 score, CABG 4-year mortality. Peripheral blood samples were collected from 141 CHD patients (29 women and 112 men, aged 62.2 ± 11.9 years) and 46 healthy controls (8 women and 38 men, aged 61.24 ± 10.12 years) between June 2020 and August 2020. Patients were divided into 5 groups according to the coronary angiography score and the clinical manifestations: (1) Coronary atherosclerosis negative control group (n=46) including patients whose coronary artery examination showed no obvious stenosis; (2) Asymptomatic coronary heart disease group (N=17) including patients with coronary artery stenosis, but no clinical symptoms; (3) Stable angina pectoris group (n=20) including patients with coronary artery stenosis, clinical manifestations of patients with stable angina pectoris; (4) Unstable angina pectoris group (n= 69) including patients with coronary artery stenosis, clinical manifestations of unstable angina; (5) Acute myocardial infarction group (n=35) including patients with acute myocardial infarction. Patients with any one of the following diseases were excluded: tumor, severe infectious disease, and severe inflammation.
Preparation of peripheral blood mononuclear cells (PBMC) and plasma
Blood samples were collected from patients with coronary heart disease and healthy controls. 10 mL of peripheral blood were collected from each patient and put in an EDTA anticoagulation vacuum tube (BD, 9036857). 2ml of whole blood were transferred into 15ml centrifuge tube, centrifuge at 400×g for 5min. The plasma was stored at -80°C. The remaining whole blood was transferred to a centrifuge tube (Greiner bio-one, 163288) containing 3mL ficoll solution (Solarbio, P8610), and centrifuged at 800×g for 15 minutes at room temperature (minimum acceleration and deceleration). After centrifugation, the middle buffy coat layer was collected and washed for twice with PBS to obtain PBMC.
Flow cytometry for detecting Th cells
T cell subtypes in PBMCs were quantified by flow cytometry (BD, FACSAria SORP). CD4+IFN-γ+T cells were considered to be Th1 cells. CD4+ IFN-γ-GM-CSF+T cells were considered to be ThGM cells. CD4+IL-17A+ T cells were considered to be Th17 cells. 1×106 cells were transferred to a 1.5ml centrifuge tube containing 100ul FACS buffer (PBS solution containing 1% FBS). Anti-human CD4-PerCP-Cy5.5 flow cytometry antibody (BD, 552838) was added and incubated for 20min in the dark at room temperature. After washing with FACS buffer, 200ul of IC Fixation Buffer (invitrogen, 2235872) and Permeabilization Buffer (invitrogen, 1961659) were added into the tube, followed by incubating for 20 minutes at room temperature in the dark. After that, cells were resuspend using 100ul FACS buffer, add 5ul each of IFN-y-APC (BD, 554702), GM-CSF-PE (BD, 554507) and IL17A-BV421 (BD, 562933) into the tube and incubate for 30 minutes at room temperature. Then, resuspending cells with 300ul FACS buffer and detected using flow cytometry. Data were analyzed using Aria 2 software (BD, Inc.).
Luminex assay
levels of GM-CSF, IFN-γ, IL-10, IL-17A, IL-1β, IL-2, IL-4, IL- 23, IL-5, IL-6, IL-7, and TNF-α in plasma was detected using Luminex method. High throughput analysis was performed using the Milliplex Map Kit (Merck, HSTCMAG-28SK) according to the manufacturer's instructions. The operation method was shown in the instructions. Simply, setting up blank control wells (2), standard wells (14), QC wells (4) and sample wells (76) in the 96-well plate. Add 50ul of Serum Matrix to the blank well, 50ul of standards with different dilution concentrations into the standard wells, 50ul of quality control products into the QC wells, and 25ul of plasma and 25uL of Assay buffer into the sample wells. Add human monoclonal antibodies coupled with magnetic beads to 96 wells, 25uL per well and incubate with shaking for 16-18h at 4°C. Washing 3 times with wash buffer, and then add 50uL Detection Antibody to each well. After incubating for 1 h at room temperature, add 50uL Streptavidin-Phycoerythrin (R-PE) into each well. Then, incubating for 30min at room temperature, wash 3 times in wash buffer, adding 150uL of Sheath Fluid and run plate on LuminexR 200TM with Xponent software. Finally, calculating the amounts of cytokines based on the fluorescence intensity.
Statistical analysis
Using ANOVA for CHD clinical score studies. Using the student’s t test to assess between groups. The correlation was analyzed by Pearson correlation analysis. All statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 (SPSS, USA). P<0.05 was considered significant.