To study the effect of Huangzhi oral liquid (HZOL) on I/R after 2 h and 4 h and determine its regulatory function on caspase-3 and protein networks. 70 SD male rats were randomly divided into seven groups and established myocardial I/R injury model by ligating the left anterior descending coronary artery. Myocardial infarction model was defined by TTC staining and color of the heart. The levels of CK-MB, CTnI, C-RPL, SOD, and MDA were tested at 2 h and 4 h after reperfusion. HE staining and ultramicrostructural were used to observe the pathological changes. The apoptotic index (AI) of cardiomyocyte was marked by TUNEL. The expression levels of caspase-3, p53, fas, Bcl-2, and Bax were tested by immunohistochemistry and western blot. HZOL corrected arrhythmia, improved the pathologic abnormalities, decreased CK-MB, CTnI, C-RPL, MDA, AI, caspase-3, p53, fas, and Bax, and increased SOD ans Bcl-2 with different times of myocardial reperfusion; this result was similar to the ISMOC (
The function of apoptosis in cardiovascular disease is gaining recognition. Necrosis is a myocardial cell injury caused by myocardial ischemia. Recent studies demonstrated that oxidative stress and ischemia/reperfusion (I/R) injury not only cause myocardial necrosis but also induce cardiomyocyte apoptosis. The degree of myocardial cell apoptosis is associated with the I/R time [
HZOL consists of leech, rhubarb, and Fructus arctii, which were mixed at a ratio of 5 : 3 : 3, with a concentration of 2.2 g/mL. HZOL was purchased from the Affiliated Zhongshan Hospital of Traditional Chinese Medicine for Guangzhou University of Chinese Medicine (Guangzhou, China). The quality of HZOL was controlled by thin layer chromatography analysis. The solution was stored in aliquots (10 mL/vase) at −20°C.
Male Wistar rats (SPF, no. 0099755, 200 g to 240 g) were purchased from the Experimental Animal Center of Southern Medical University, housed individually in clear plastic cages at a temperature- and humidity-controlled environment with a 12 h light/dark cycle (23 ± 1°C; 12 h light/dark cycle, light on at 7 a.m.) and given ad libitum access to rodent chow and water. Animals were handled in accordance with the Guidelines of Animal Care at Southern Medical University.
A total of 70 rats were randomly divided into seven groups, namely, the Model-2 h group (reperfusion for 2 h after 30 min of myocardial ischemia), Model-4 h group (reperfusion for 4 h after 30 min of myocardial ischemia), HZOL-2 h group (application of HZOL before Model-2 h), HZOL-4 h group (application of HZOL before Model-4 h), isosorbide mononitrate capsule- (ISMOC-) 2 h group (application of ISMOC before Model-2 h), ISMOC-4 h group (application of ISMOC before Model-4 h), and sham group (subjected to the same surgical procedure in the absence of left anterior descending (LAD) coronary artery). Rats in the HZOL-2 h, HZOL-4 h, ISMOC-2 h, and ISMOC-4 h groups were intragastrically administered with HZOL (2 g/kg/d) and ISMOC (3.6 mg/kg/d) at 1 h before surgery. This procedure was performed for seven consecutive days. Normal saline was used as control. Rats were anesthetized with pentobarbital sodium (80 mg/kg, Fluka), intubated, and ventilated artificially using a rodent ventilator (SAR-830, IITC, USA). Ischemia-reperfusion made mould method as mentioned in the paper [
The incidence of arrhythmias was determined and diagnosed based on the criteria of the Lambeth Conventions [
An arrhythmia score was used to evaluate the severity of arrhythmias by giving a grade to each animal as follows: 0 = no arrhythmias; 1 = less than 10 s of VT or other arrhythmias, no VF; 2 = 11 s to 30 s of VT or other arrhythmias, no VF; 3 = 31 s to 90 s of VT or other arrhythmias, no VF; 4 = 91 s to 180 s of VT or other arrhythmias, and/or less than 10 s of reversible VF; 5 = more than 180 s of VT or other arrhythmias, and/or more than 10 s of reversible VF; 6 = irreversible VF [
Experiments were terminated or excluded from the final data analysis if any of the following conditions occurred: absence of signs of successful coronary artery occlusion, severe arrhythmias prior to LAD occlusion and reperfusion, or severe atrioventricular block during the first 5 min of ischemia [
5 mL of blood was collected through the abdominal aorta of live rats to detect levels of serum creatine kinase mb isoenzyme (CK-MB), cardiac troponin I (CTnI), C reactive protein (CRP), superoxide dismutase (SOD), and malondialdehyde (MDA). The AAR of the left ventricle of rats in the sham, Model-2 h, Model-4 h, HZOL-2 h, HZOL-4 h, ISMOC-2 h, and ISMOC-4 h groups was removed and divided into four parts. One part was fixed in 10% formalin, another was fixed in 0.1 M phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde, and the other two parts were flash-frozen in liquid nitrogen and stored at −80°C for use.
Serum CK-MB, CTnI, CRP, SOD, and MDA determination. The serum biochemical parameters CK-MB, CTnI, and CRP, which closely reflect cardiac function [
Electron microscopy (TEM) examination part of the AAR of the left ventricle was fixed in 10% formalin, embedded in paraffin, cut into 4
We purchased a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit for paraffin sections from Nanjing KeyGen Biotech. Inc. (Nanjing, Jiangsu, China). Based on the manufacturer’s instructions, all the procedures were performed. Cells were defined as apoptotic if the entire nuclear area of the cell was positively labeled. The apoptotic cells and bodies were counted in five high-power fields. The apoptotic index (AI) was calculated as the percentage of positively stained cells using the following equation: AI = number of apoptotic cells/total number of nucleated cells [
Immunohistochemical staining for caspase-3, p-53, fas, Bcl-2, and Bax was performed using routine immunohistochemistry streptavidin peroxidase method. This method contained a rabbit polyclonal IgG antibody against caspase-3 (1 : 100; Cell Signaling Technology Inc., no. 9662, USA), p-53 (1 : 50; Bioworld Technology Inc., BS3736, Louis Park, USA), fas (1 : 100; Assay Designs, ADI-AAP-221D, USA), Bcl-2 (1 : 50; Bioworld Technology Inc., BS1511, Louis Park, USA) (Cell Signaling Technology, no. 2876, USA), and Bax (1 : 50; Bioworld Technology Inc., BS2538, Louis Park, USA) (Cell Signaling Technology, no. 2772, USA). Nuclear counterstaining was performed using hematoxylin. Five randomly selected fields from each section were examined at a magnification of ×200 and analyzed using Image-Pro Plus 6.0. The positive content (PC) was calculated using the following formula: PC = mean optical density × positive area [
Aliquots of heart tissue (50 mg) were homogenized in liquid nitrogen and dissolved in lysis buffer. Protein concentrations were determined by BCA protein quantitative assay. The protein lysates were loaded onto 10% SDS-polyacrylamide gel for separation, electrotransferred to PVDF membranes, and blocked in 5% nonfat milk in Tris-buffered saline, Membranes were incubated overnight using primary antibodies, (caspase-3 (Cell Signaling Technology Inc., no. 9662, USA) diluted to 1 : 1000, p-53 (Bioworld Technology Inc., BS3736, Louis Park, USA) diluted to 1 : 500, fas (Assay Designs, ADI-AAP-221D, USA) diluted to 1 : 500, Bcl-2 (Cell Signaling Technology, no. 2876, USA) diluted to 1 : 1000, and Bax (Cell Signaling Technology, no. 2772, USA) diluted to 1 : 1000) at 4°C. This step was followed by secondary antibodies, which were conjugated using horseradish peroxidase. We performed enhanced chemiluminescence (Merck-Millipore, Germany) detection. The images were captured and documented using a CCD system (image station 2000MM, Kodak, Rochester, NY, USA). Quantitative analysis of these images was performed using Molecular Imaging Software Version 4.0, which was provided by Kodak 2000MM System. The optical density was normalized against actin [
Each experiment was repeated at least three times. Data were represented in the form of means ± SD. Data were analyzed using SPSS statistical package (version 13.0, Armonk, NY, USA). Mean values were compared using one-way ANOVA, and multiple comparisons were performed. Data were analyzed using a homogeneity test for variance. If the variances were homogeneous, mean values were compared through ANOVA. The differences between two groups were analyzed based on least significant difference test. If the variances were not homogeneous, mean values were compared using Welch’s test. The differences between two groups were analyzed by Games-Howell. Statistical significance was set at
Naked eye observations showed that the heart color from the model group was paler than that of the sham group (Figure
Histological and electrocardiography (ECG) parameters. (a) Representative gross images of whole hearts. (b) Representative of 1% triphenyl tetrazolium chloride (TTC) staining results. (c) Representative of 2-lead electrocardiogram results. (d) Arrhythmia was scored according to 2-lead electrocardiogram. Model-2 h group (after 30 min of myocardial ischemia, reperfusion 2 h), Model-4 h group (reperfusion for 2 h after 30 min of myocardial ischemia), Model-4 h group (reperfusion for 4 h after 30 min of myocardial ischemia), HZOL-2 h group (application of HZOL before Model-2 h), HZOL-4 h group (application of HZOL before Model-4 h), isosorbide mononitrate capsule (ISMOC)-2 h group (application of ISMOC before Model-2 h), ISMOC-4 h group (application of ISMOC before Model-4 h), and sham group (subjected to the same surgical procedure in the absence of left anterior descending coronary artery).
The serum levels of CK-MB, CTnI, CRP, SOD, and MDA after treatment are shown in Figure
The serum levels of CK-MB, CTnI, CRP, SOD, and MDA after treatment. (a) CK-MB; (b) CTnI; (c) CRP; (d) SOD; (e) MDA; Model-2 h group (after 30 min of myocardial ischemia, reperfusion 2 h), Model-4 h group (reperfusion for 2 h after 30 min of myocardial ischemia), Model-4 h group (reperfusion for 4 h after 30 min of myocardial ischemia), HZOL-2 h group (application of HZOL before Model-2 h), HZOL-4 h group (application of HZOL before Model-4 h), isosorbide mononitrate capsule (ISMOC)-2 h group (application of ISMOC before Model-2 h), ISMOC-4 h group (application of ISMOC before Model-4 h), and sham group (subjected to the same surgical procedure in the absence of left anterior descending coronary artery).
Results of HE staining of AAR are shown in Figure
The effects of HZOL for I/R 2 h and I/R 4 h on the histological and ultrastructural changes in the myocardium (a) representative microscopic images of HE stain. (b) TEM images of ultrathin sections of myocardial tissue are changed. A: sham group; B: Model-2 h group; C: Model-4 h group; D: HZOL-2 h group; E: HZOL-4 h group; F: ISMOC-2 h group; G: ISMOC-4 h group.
HE
EMT
TUNEL staining suggested that more brown stained cells were found in the Model-2 h and Model-4 h groups than those in the sham group (
Effects of HZOL on apoptosis and expression of caspase-3. (a) TUNEL analysis of apoptosis in left ventricular AAR. (b) Immunohistochemical assay of caspase-3 expression. (c) Western blotting assay of caspase-3 expression. A: sham group; B: Model-2 h group; C: Model-4 h group; D: HZOL-2 h group; E: HZOL-4 h group; F: ISMOC-2 h group; G: ISMOC-4 h group;
Western blot showed that caspase-3 expression (
The expression levels of p53, fas, and Bcl-2/Bax are shown in Figure
Effects of HZOL on apoptosis proteins expression. (a) Immunohistochemical assay of p53 expression. (b) Immunohistochemical assay of Fas expression. (c) Immunohistochemical assay of Bcl-2 and Bax expression. (d) Western blotting assay of p53, Fas, Bcl-2, and Bax expression. A: sham group; B: Model-2 h group; C: Model-4 h group; D: HZOL-2 h group; E: HZOL-4 h group; F: ISMOC-2 h group; G: ISMOC-4 h group;
Our results indicate that the activation of HZOL at the beginning of reperfusion produced a protective effect in I/R injury and participated in a cascade of events that resulted in apoptosis. This study is the first to demonstrate the protective effect of HZOL inhibition against apoptosis caused by I/R injury.
The effects of HZOL prevented arrhythmias in rats by myocardial I/R for 2 and 4 h. Naked eye observations showed that the color of the heart in the model group was paler than that in the sham group. Almost all rats in the Model-2 h and Model-4 h groups experienced obvious ST-segment elevation, VT occurrence, and high VF frequency, which was the frequency of arrhythmias. By contrast, neither VT nor VF was observed in the sham group, and only a few ventricular premature beats appeared during the entire procedure. The serum results illustrate that CK-MB, CTnI, CRP, and MDA significantly increased, whereas SOD significantly decreased in the Model-2 h and Model-4 h groups (
The use of HZOL has been practiced for many years in the Affiliated Hospital of Zhongshan University of Traditional Chinese Medicine and is highly effective in relieving hypertension and hyperlipidemia [
Apoptosis typically proceeds through one of two signaling cascades, known as extrinsic and intrinsic pathways, both of which converge to activate the executioner caspase-3 [
The authors declare that there is no conflict of interests regarding the publication of this paper.
Xu Ran and Jian Xin Diao equally contributed to this work.