Berberine, an isoquinoline alkaloid originally isolated from the Chinese herb
Ischemic heart disease (IHD) is the leading cause of morbidity and mortality in the Western world; and, according to the World Health Organization, it will be the leading cause of death in the world [
Isoproterenol,
High mobility group box 1 (HMGB1), a 30 kDa nuclear protein involved in the structural organization of deoxyribonucleic acid (DNA), serves as a mediator of inflammation after being released by necrotic cells or upon cellular activation in various pathological conditions including diverse cardiovascular diseases and myocardial ischemia-reperfusion (I/R) injury [
Berberine, an isoquinoline alkaloid originally isolated from the Chinese herb
This study was approved by the Laboratory Animal Center of the Academy of the China Pharmaceutical University. 50 male healthy Sprague-Dawley rats weighing from 200 to 220 g were purchased from the Animal Experimental Animal Center of Jilin University. Animal experiment was carried out in accordance with the Guidelines for Animal Experiment of Jilin University. The animals were maintained in a temperature controlled room at 20.1–23.1°C and 40–50% humidity, a 12 h light/dark cycle, and free access to water. Berberine with purity of over 98% was purchased from National Institutes for Food and Drug Control (Beijing, China). Isoproterenol was purchased from Shanghai Hefeng Pharmaceutical Co. Ltd. (Shanghai, China). Sodium pentobarbital was purchased from Merck (Germany). Propranolol was purchased from Xi’an Li Jun Pharmaceutical Co. ltd. (Xi’an, China). CK-MB, LDH, T-SOD, MDA, TNF-
The rats were randomly assigned to five groups of 10 rats each: (1) control; (2) isoproterenol; (3) isoproterenol + propranolol (30 mg/kg, orally); (4) isoproterenol + berberine (30 mg/kg, orally); (5) isoproterenol + berberine (60 mg/kg, orally). Rats were pretreated for 14 days and then intoxicated with isoproterenol (isoproterenol, 85 mg/kg except for the control group) by subcutaneous injection on two consecutive days. Blood (3 mL) was collected from the abdominal aorta for serum enzyme assays. After treatment, hearts were excised, rinsed in ice-cold isotonic saline, blotted with filter paper, and homogenized in 0.05 M ice-cold phosphate buffer (pH 7.4) for biochemical assays.
Electrocardiograms (ECG) recorded ST-segment elevation and heart rate at 20 min after the final injection of isoproterenol or other drugs. ECGs were recorded under pentobarbital sodium anesthesia (22.5 mg/kg) using needle electrodes and a BL-420S Biological Function Experiment System purchased from Chengdu Thaimeng Technology Co. Ltd, (Chengdu, China).
After rats were sacrificed, the heart tissues were excised (excluding large blood vessels and connective tissue) and weighed after blotting with filter paper. The heart weight index (HWI) was computed as HWI = heart weight (HW)/bodyweight (BW).
CK-MB and LDH levels were measured by a rate assay using an RT-9600 Semiautomatic Biochemical Analyzer (ShenZhenLeiDu Life Science, LLC). TNF-
Immediately after the sacrifice of the rats, the hearts were removed and fixed in 10% formalin solution. The heart tissue was processed for sectioning and staining by standard histological methods. Sections (5 mm, Leica RM 2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and examined by light microscopy (Nikon, Tokyo, Japan) at 200x magnification.
The heart tissues were homogenized, washed with PBS, and incubated in lysis buffer in addition to a protease inhibitor cocktail (Sigma, St. Louis, MO) to obtain extracts of lung proteins. The samples were loaded to 10% SDS-PAGE gels and were electrotransferred to nitrocellulose. The blots were incubated with the appropriate concentration of specific antibody. After washing, the blots were incubated with horseradish peroxidase-conjugated second antibody. The membranes were stripped and reblotted with anti-
All values were expressed as the mean ± S.D. and analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test using SPSS version 13.0 software; a
Five minutes after isoproterenol administration, the ST-segment was elevated in the untreated model group but not in the groups treated with berberine. These results represented that the myocardial ischemia damage model has been established. Ten minutes after isoproterenol administration, ST-segment elevation was still seen in the untreated group. ST-segment elevation was reduced in the berberine groups compared with the untreated model rats. Heart rates tended to stabilize and approximate the rate observed in the propranolol treated group (Figure
Effect of berberine on ST-segment elevation. C (control); M (model); P (propranolol (30 mg/kg)); D (berberine (30 mg/kg)); E (berberine (60 mg/kg)). Values are expressed as means ± SDs. Compared with control, #
Significant increases in the myocardial injury marker enzymes, CK-MB, and LDH were observed in the untreated model rats compared with the control rats. Pretreatment with berberine decreased CK-MB and LDH levels compared with rats in the untreated model group in a dose-dependent manner. Compared with the control group, serum TNF-
Effects of berberine on CK-MB, LDH, TNF-
HWI were greater in the untreated model rats than in the control group rats. Pretreatment with berberine decreased the HWI compared with the untreated model group rats (Figure
Effect of berberine on heart weight indices. C (control); M (model); P (propranolol (30 mg/kg)); D (berberine (30 mg/kg)); E (berberine (60 mg/kg)). Values are expressed as means ± SDs. Compared with control, #
Light microscopy of tissue sections from control rat myocardium showed a normal myofibrillar structure with striations, branched appearance, and continuity with adjacent myofibrils. Tissue from the untreated model rats given isoproterenol revealed obvious myocardial cell swelling, degeneration, loss of transverse striations, and large numbers of infiltrating inflammatory cells. Tissues from rats pretreated with berberine showed normal, well preserved of cardiac muscle cell histology. Tissue sections from the propranolol group rats revealed approximately normal myofibrillar structure with clear transverse striations and presence of a few inflammatory cells (Figure
Effect of berberine on myocardial histology (×200). C (control); M (model); P (propranolol (30 mg/kg)); D (berberine (30 mg/kg)); E (berberine (60 mg/kg)).
The expressions of proteins of HMGB1, TLR4, Bax, Bcl-2, and TNF-
Effect of berberine on HMGB1-TLR4 axis signaling C (control); M (model); P (propranolol (30 mg/kg)); D (berberine (30 mg/kg)); E (berberine (60 mg/kg)). Values are expressed as means ± SDs. Compared with control, #
In this study, berberine reduced the ST-segment elevation induced by acute myocardial ischemia; alleviated myocardial ischemic injury; decreased HWI. Pretreatment with berberine also decreased CK-MB, LDH, TNF-
The acute myocardial ischemia induced by isoproterenol was confirmed by loss of integrity of myocardial membranes on histological examination, ST-segment elevation, and serum elevation of CK-MB and LDH enzymes. Berberine alleviated myocardial histological injury, reduced heart rate and ST-segment elevation, and decreased CK-MB and LDH enzyme in a dose-dependent fashion.
Isoproterenol is a synthetic
As a previous study [
Inflammation has been recognized as a major driving force in the ischemic process, and increasing evidence has shown that enhanced levels of inflammatory markers are related to ischemia [
The levels of SOD and MDA activity are among the principal pathophysiological parameters in evaluating free radical metabolism. SOD activity reflects the cellular capability of scavenging/quenching free radicals [
High mobility group box 1 protein (HMGB1), a highly conserved nuclear protein that could regulate gene transcription and maintain the nucleosome structure, could be passively released from necrotic cell or apoptotic cell or actively secreted by innate immune cells (such as macrophages and monocytes) [
Several toll-like receptors (TLRs), TLR4, play important roles in mediating myocardial inflammatory and injurious responses to I/R [
Inflammation is involved in many cardiovascular diseases, such as myocardial infarction, atherosclerosis, and cardiomyopathy [
Cardiomyocyte apoptosis is involved in various heart diseases, such as myocardial hypertrophy, heart failure, and myocardial ischemia [
In conclusion, this study demonstrated that berberine had cardioprotective effects against acute ischemic myocardial injury induced by isoproterenol in rats.
The authors declare that there is no conflict of interests regarding the publication of this paper.