Autopsy cases were selected from the Forensic Judicial Expertise Centre of Guizhou Medical University from January 2017 to December 2019. The principles outlined in the Declaration of Helsinki and the Rules for Autopsy issued by the Ministry of Health of the People’s Republic of China were followed. It had obtained the informed consent from donor or next of kin of the deceased and was approved by the Ethics Review Committee of Guizhou Medical University (approval number: 201801). Sixty-eight coronary artery tissue specimens were included in the lesion group: there were 36 cases of sudden death of coronary heart disease caused by arrhythmia or myocardial infarction, accompanied by plaque rupture and intra-plaque haemorrhage (SCD group) and 32 cases of death caused by traffic accident, electric shock, etc., and coronary artery associated with atherosclerosis (CHD group). Twenty-two cases of acute death caused by traffic accident, electric shock, etc., and no coronary heart disease were used as the control group (Con group).
The following inclusion criteria were applied to the disease group. (1) The cadaver was cryopreserved within 6 h of death, and the autopsy was performed within 48 h of death. (2) The presence of coronary artery AS was confirmed by the naked eye and histological examination. Coronary artery intimal thickening, lumen narrowing, atherosclerotic plaque formation, atherosclerotic plaque rupture, intraplaque haemorrhage and aneurysm formation were observed in the thickened coronary artery intima, with or without any secondary lesions such as atherosclerotic plaque rupture, intraplaque hemorrhage, aneurysm formation, etc. (3) In CHD group, the deceased died quickly because of traffic accidents, electric shocks, etc. (4) In the SCD group, systematic autopsy and routine toxicological examination were performed within 24 h from the onset of coronary heart disease symptoms to death, excluding the possibility of poisoning or other diseases (Additional file 1).
The inclusion criteria of the control group was autopsy at the same time as the lesion group, no coronary artery disease as confirmed by the naked eye and histological examination. And the cause of death in control group was caused by traffic accidents, electric shocks, etc. The exclusion criteria were as follows: (1) tissue autolysis or unclear structure; (2) rheumatic heart disease, cardiomyopathy, myocarditis and other heart disease; and (3) multiple systemic inflammatory infections or multiple organ dysfunction.
According to the nano-exclusion standard, the left anterior descending coronary artery without any lesion was selected as the control group. The experimental group specimens were selected as the locations at which stenosis of the anterior descending coronary artery was the heaviest as observed by the naked eye, and the selected tissue was fixed with 4% formaldehyde. Part of the tissue was frozen at − 80 °C for protein extraction and analysis.
Observation of coronary artery structure
The coronary artery tissue in all cases were fixed with 4% paraformaldehyde for 72 h. The cross section of the blood vessel was embedded in paraffin, 4-μm sections were prepared and stained with conventional HE, and an EasyScan digital section scanning and application system (McAudi Industrial Group Co., Ltd., China) was used to observe the morphology and structure of the coronary artery. IPP 6.0 image analysis software was used to detect the related morphological indexes: (1) intimal and lesion thicknesses: From the free edge of the endocardial cavity surface to the vertical distance of the internal elastic membrane, the thickest and thinnest thickness of the intima was measured, and another straight line was perpendicular to the two test lines. The mean of the test line and the average thickness of the intima were calculated. (2) thickness of the fibrous cap: The fibrous cap on the surface of atherosclerotic lesions of coronary arteries was assessed in the experimental group. The thickness of the fibrous cap on both sides of the cap and at the thickest intimal was measured, and the average value was obtained. (3)Thickness of necrosis core: The vertical distance from the proximal rim to the proximal margin, the largest and smallest necrosis lesions in atherosclerosis were evaluated, and another test line was drawn perpendicular to the test line, with three lines in total. The mean value was the average thickness of the necrotic lesions, while necrosis was not evaluated. (4) Degree of lumen stenosis: The ratio of intimal area to the sum of intima and lumen area was assessed. The increase of intimal and lesion thicknesses and degree of lumen stenosis is related to the progression of atherosclerosis. The thinning of the fibrous cap and the enlargement of necrosis core are related to the plaque instability of atherosclerosis.
The expression and distribution of target proteins in coronary artery lesions were analysed by immunohistochemistry (IHC). There were 12 cases in each group. The coronary arteries were sectioned in paraffin and baked at 60 °C for 1 h and hydrated with concentration gradient after being dewaxed with xylene. Endogenous peroxidase was blocked with 3% H2O2, hyperbaric heat repair antigen was performed for 4 min, and the samples were blocked with goat serum and then incubated with antibodies against ATF3 (1:150, #ab254268, Abcam, UK), CD45 (1:200, #20103–1-AP, Proteintech, China), IL-1 β (1:150, #abs131179, Absin, China), TNF-α (1:100, #YM3478, Immunoway, China), MMP-9 (1:50, #10375-2-AP, Proteintech, China), and VCAM1 (1:200, #abs119895, Absin, China). PBS was selected as negative control and incubated with the sections overnight in wet box at 4 °C. On the next day, the primary antibody was washed away with PBS, and horseradish peroxidase-labelled sheep anti-rabbit IgG secondary antibody was added. After being incubated for 30 min at 37 °C, DAB was used for colour development, and the samples were stained with haematoxylin, cleared with xylene and sealed with neutral gum. The positive expression of the target protein was observed under a microscope, and was visualized as a brown colour. Three visual fields were selected and pictures were taken at 400 × magnification. The mean optical density (MOD) of the positive signal was measured by IPP6.0 software to evaluate the degree of target protein expression (MOD = positive expression/total measured area). The result is the average value of 3 repeated measurements.
Protein expression in the coronary artery was analysed by Western blotting. Vascular tissue was stored at − 80 °C, the homogenate was prepared, and the protein concentration was determined by ultraviolet spectrophotometry. SDS gel electrophoresis (10%) was performed, the proteins were transferred to a PVDF membrane, and the membrane was sealed with 5% skim milk for 2 h. According to the molecular size of the target protein (ATF3, 1:300; CD45, 1:1000; IL-1β, 1:1000; TNF-α, 1:1500; MMP-9, 1:600; VCAM1, 1:1000), the corresponding PVDF membranes were incubated overnight on a shaker at 4 °C. After the membranes were washed, HRP-conjugated goat ant-rabbit IgG (1:5000, Absin, China) was added and incubated at room temperature for 1 h. Chemiluminescent HRP substrate (Millipore, USA) was used for colour development on a chemiluminescence imaging system. β-actin was used as the internal reference, the grey value of the band was analysed and measured by ImageJ, and the mean value of three measurements was taken as the result.
The data were analysed by SPSS 22.0 statistical software, and normal distribution and homogeneity of variance were tested. The measurement data are expressed as the mean ± SD. The mean of the homogeneity of variance was compared by single factor analysis of variance (LSD), and data with uneven variances were analysed by the Games-Howell test. The chi-square test was used to compare the counting data between the two groups, and correlations were analysed by the Pearson correlation coefficient test. The difference was statistically significant at P < 0.05 and P < 0.01. The number of case experiments is indicated in each figure legend.