A severe loss of contractile function is the main characteristic of end-stage heart failure. Pathological analyses of the myocardium from patients with end-stage heart failure indicate a loss of cardiomyocytes as well as fibrosis and cardiomyocyte hypertrophy [
Furthermore, recent evidence from both animal and human studies suggests that increased production of inflammatory cytokines and cardiac cell oxidative stress are associated with a poor prognosis following MI and may play a critical role in the pathogenesis and progression of heart failure. Indeed, myocardial cell death is reported to trigger an acute inflammatory reaction that leads to chemokine and cytokine production and the recruitment of macrophages that results in leukocyte infiltration of the infract area [
In the present study, we assessed the therapeutic effects of (
In one of our previous studies, we demonstrated that (
The rats used in the current study were supplied by the experimental animal center of Fudan University. Adult male Sprague-Dawley rats weighing 230-250 g (8-10 w) were used for the study. The animals were housed with a 12 h light-12 h dark cycle and had free access to standard pellet food and water throughout the experiment. All of the experiments were approved by the local ethics committee of Fudan University.
For the experiments, the adult male Sprague-Dawley rats were randomly divided into five groups: a sham operation group, MI group, and MI with drug treatment groups (15, 30, and 60 mg/kg). The drug was dissolved in DMSO (dimethyl sulfoxide). The drug was injected intraperitoneally 6 weeks after surgery once daily. The sham operation group and the MI group were injected only with DMSO according to animal weight. Prior to the induction of MI, the animals were intubated and artificially ventilated with a rodent ventilator (DHX-150, China) under anesthesia with 7% chloral hydrate (60 mg/kg i.p.). The left anterior descending coronary artery was ligated with a 5-0 suture, 1-2 mm below the left atrial appendage [
Left ventricular pressures were measured via a saline-filled cannula that was inserted through the right carotid artery and connected to a pressure transducer. The cannula was inserted into the left ventricle to monitor left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as to measure the maximum rate of the rise of the left ventricular pressure rising rate (
Six weeks after surgery, the whole blood of the rats was centrifuged (3000 r/min, 10 min, 4°C) to obtain serum. The antioxidant enzyme activities of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) were measured in the serum. MDA, CAT, SOD, GSH, GSH-Px, and GST levels were measured by using a commercially available kit according to the manufacturer’s instructions (Jiancheng Institute of Biotechnology, Nanjing, China).
MDA activity was measured by adding 1 ml of the standard agent to the standard tubes. Next, 1 ml of absolute ethyl alcohol was added to the blank tubes. Then, 1 ml of the sample was added to the sample tubes and control tubes. The tubes were shaken for a long period. Then, 3 ml of reagent 2 was added to all the tubes, and then, 1 ml of reagent 3 was added to the standard tubes, blank tubes, and sample tubes. Next, 1 ml of 50% glacial acetic acid was added to the control tubes. The tubes were mixed and covered with plastic wrap. A small hole was made in the plastic wrap. The tubes were incubated at 95°C for 40 minutes and cooled in flow water. The tubes were centrifuged at 3000 to 4000 rpm for 10 minutes. The absorbance of the supernatant was measured using a plate reader at 532 nm.
CAT activity was measured by first preparing positive control tubes and sample tubes. Then, 0.1 ml of serum was added to the sample tubes, and 1.0 ml of reagent 1 and 0.1 ml of reagent 2, both kept at 37°C, were added to each tube. They were incubated on a shaker at 37°C for precisely 1 min. After that, 1.0 ml of reagent 3 and 0.1 ml of reagent 4 were added to all tubes. Then, 0.1 ml of the serum was added to the positive control tubes. Finally, the absorbance was read at 405 nm using a plate reader.
To measure GSH activity, the preparation of the sample was as follows: 0.5 ml of the sample and the buffer of reagent 1 were mixed and centrifuged at 3000 to 4000 rpm for 10 minutes. Then, 1 ml of the sample supernatant was added to sample tubes, while 1.0 ml of reagent 1 and 1.0 ml of standard 20
SOD activity was measured as follows: 0.5 ml of the sample and the buffer of 1.0 ml reagent 1 were mixed; meanwhile, 0.5 ml of water and the buffer of 1.0 ml reagent 1 were mixed as the positive control. Then, 0.1 ml of reagents 2, 3, and 4 was added to the sample tubes and the positive control tube. Then, all of the tubes were incubated at 37°C for precisely 40 minutes. 2.0 ml of color reagents were added to each tube. Finally, all the tubes were placed at room temperature for 15 minutes. The absorbance was read at 550 nm using a plate reader.
GSH-Px activity was measured by first adding 0.2 ml of aqueous 1 mmol/l GSH to the control tubes and the sample tubes. Then, 0.1 ml of the sample was added to the sample tubes. All of the tubes and reagent 1 were kept at 37°C for 5 minutes. Then, 0.1 ml of reagent 1 was added to each tube. All of the tubes were incubated at 37°C for precisely 5 minutes. Two milliliters of reagent 2 were added to every tube, and 0.1 ml of the sample was added to the control tubes. The tubes were centrifuged at 3000 to 4000 rpm for 10 minutes. One milliliter of the supernatant from the control tubes and the sample tubes was transferred to new tubes labeled the same as the old tubes. Simultaneously, 1 ml of the GSH standard buffer was added to the blank tubes and 1 ml of the aqueous 20
GST activity was measured by first adding 0.3 ml of aqueous 1 mmol/l GSH to the control tubes and the sample tubes. Then, 0.1 ml of the sample was added to the sample tubes. The tubes were mixed and incubated at 37°C for 30 minutes. Then, 2 ml of reagent 2 was added to all the tubes. Next, 0.1 ml of the sample was added to the control tubes. The tubes were centrifuged at 3000 to 4000 rpm for 10 minutes. One milliliter of the supernatant from the control tubes and the sample tubes was transferred to new tubes labeled the same as the old tubes. Then, 2 ml of the GSH buffer and 2 ml of aqueous 20
Total RNA was isolated from cardiac myocytes by using TRIzol (Invitrogen, Carlsbad, CA) as previously described. The RNA concentration was determined by measuring the absorbance at 260 nm. Reverse transcription (RT) was conducted by using a PrimeScript™ 1st Strand cDNA Synthesis Kit according to the manufacturer’s recommended protocol. The primers for RT-PCR were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd, China. The primers used for amplification were synthesized as follows: TNF-
Frozen LV specimens were dispersed mechanically in the lysis buffer. The lysate was centrifuged at 10,000 r/min for 10 min at 4°C, and the supernatant was collected. The protein concentrations were quantified using an enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, China). After the protein concentrations were determined, 30-50
The data are presented as
The synthetic route of (
Table
Cardiac parameter changes following (
Variable | Sham | MI | MI+acrylic acid (15) | MI+acrylic acid (30) | MI+acrylic acid (60) |
---|---|---|---|---|---|
Body weight (g) | |||||
Heart weight (mg) | |||||
Heart/body weight (mg/g) | |||||
Left ventricular systolic pressure (LVSP) (mmHg) | |||||
LV end-diastolic pressure (LVEDP) (mmHg) | |||||
The antioxidant effects of (
The antioxidant effects of (
The protective effect of (
Expression of TNF-
We next determined the inhibitory effects of (
(a) Expression of Bcl-2, Bax, caspase-3, and caspase-9 mRNA. (
Western blot analysis revealed that Akt was activated in the hearts of MI animals after 6 weeks. In the (
(a) Expression of Bcl-2, Bax, Akt, and p-Akt protein. (
Six weeks after MI, collagen deposition was detected by measuring the expression levels of TGF-
(a) Expression of TGF-
Our data indicated that treatment with (
The pathological mechanism of myocardial infarction is multifactorial. Previous studies have shown that a large number of reactive oxygen species are generated; studies also have confirmed that the production of a large number of oxygen free radicals after ischemia is one of the main mechanisms of cardiomyocyte injury. Our results show that (
In addition to the potent anti-inflammatory effects, the protective effects of (
It is well known that an imbalance of proapoptotic versus prosurvival members in the Bcl-2 family plays an important role in apoptosis of various cell types [
Ventricular remodeling is another characteristic of heart failure. Many studies have shown that failure to prevent cardiomyocyte apoptosis and suppression of proinflammatory cytokines are involved in mediating cardiomyocyte hypertrophy and ventricular remodeling. Our study indicated that treatment with (
The present study demonstrates that (
Potential mechanisms involved in the cardioprotective effect of (
The Cardiac parameter changes data, enhanced activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and the levels of glutathione (GSH) in the serum, the protein and mRNA levels of signals in the left ventricle used to support the findings of this study are available from the corresponding author upon request.
The authors have no conflicts of interest.
This work has been supported by the National Natural Science Foundation of China (No. 81872864).