Quaternary amines exert anti-myocardial ischemia effects via regulation of energy metabolism and oxygen free radicals in myocardial cells in acute myocardial infarction rats

Purpose: To investigate the effect of quaternary amines on myocardial cells of a rat model of cardiac arrest, with respect to energy generation potential and oxygen free radicals. Methods: Forty-five Sprague-Dawley (SD) rats were assigned to sham, model and quaternary amine groups (each with 15 rats). After their corresponding treatments, lectrocardiogram (ECG) monitoring of the rats in the three groups at various time periods was carried out. Serum levels of myocardial enzymes, thromboxane B2 (TXB2), prostacyclin I2 (PGI2), serum carbon monoxide (CO), and changes in endothelial carbon monoxide synthase (eNOS) and endothelin (ET), were determined. Results: The levels of NO and eNOS were significantly reduced in model rats, relative to sham operation rats, while ET was significantly elevated in sham rats (p < 0.05). There were higher levels of NO and eNOS in the quaternary amine group than in model rats, but ET was higher in quaternary amine group than in model rats. Thromboxane B2 (TXB2) concentration was higher in model rats than in sham rats (p < 0.05). While PGI2 was markedly lower in quaternary group than in sham operation rats. TXB2 was lower in the quaternary amine group than in model rats, while PGI2 was significantly higher in quaternary amine group, relative to model rats (p < 0.05). Conclusion: Quaternary amines exert anti-myocardial effects by regulating energy metabolism and oxygen free radicals in myocardial cells of congestive heart failure rats, and thus are potentially useful for the management of acute myocardial infarction.


INTRODUCTION
Congestive heart failure is a life-threatening complication of coronary heart disease. Statistics have shown that about one million patients die from acute myocardial infarction every year in China [1,2]. In recent years, due to improvements in living standards and changes in
There are many clinical studies on the treatment of myocardial ischemia using corydalis, but there are limited studies on the effect of the bioactive quaternary amines on myocardial ischemia [7]. Therefore, the purpose of this study was to investigate the effect of quaternary amines on myocardial ischemia in rats, and the mechanisms involved.

Drugs and instruments
The drugs and instruments used were: quaternary amine base (

Establishment of rat model of AMI and treatment methods
Eighty SPF male SD rats were provided by Beijing Charles River Experimental Animal Co. Ltd., and they were assigned to 3 groups (sham group, quaternary amine group and model group). All rats were anesthetized with 10 % chloral hydrate, and their limbs and head were fixed in a supine position. Endotracheal intubation was used to maintain the breathing rate of the rats at 80 times/min. Electrocardiogram of the rats after anesthetics was determined, and the surgical site was depilated and disinfected. In the sham group, only thoracotomy was performed, without coronary ligation. Acute myocardial infarction was simulated by coronary ligation in model group and quaternary amine group, and the range of myocardial infarction was determined with TTG staining. Rats in the quaternary amine group were given quaternary amine (40 mg/kg) solution at a dose of 1 ml/100 g via gavage one week before operation, and operation was performed 30 min after the last drug administration. This study received approval from the Ethical Authority of Shenyang Medical College (approval no. 2021032), and was conducted according to "Principles of Laboratory Animal Care" (NIH publication no. 85-23, revised 1985) [8].

Evaluation of parameters/indices
The ECG of rats was recorded periodically at 5, 15, 30 and 45 min, and 1, 1.5, 2, 3, and 24 h after surgery. Infarct area was determined using TTC staining method. Frozen heart tissue slices were stained with TTC for 15 min (after the ECG monitoring of the rats, the rat hearts were excised and placed in the refrigerator at -20 o C for about 30 min, and then removed and sectioned). Digital imaging was used to analyze the infarct area and to calculate the degree of myocardial infarction. In the determination of serum myocardial enzymes, rat abdominal aortic blood was centrifuged and the serum was assayed for AST, LDH and CK-MB with their respective kits. Serum from rat abdominal aortic blood was assayed for levels of endothelin (ET), eNOS and NO. Thromboxane B2 (TXB2) in rat heart samples was determined using biotin double antibody sandwich enzyme immunosorbent assay.

Statistical analysis
The SPSS 19.0 software package was applied for statistical processing of the results. Measurement data are presented as mean ±

ECG at different time points before and after surgery
There were no significant differences in ST segment elevation amongst the three groups before operation (p > 0.05), and no significant differences were found in ST segment elevation amongst the quaternary amine, sham operation and model groups 0 min after operation (p > 0.05). Electrocardiogram results at each time point after operation were comparable in the quaternary amine group and model rats. These results are presented in Table 1. Table 2 indicates higher activities of CK-MB, LDH and AST in model rats than in sham rats, but the activities of these enzymes were lower in the quaternary amine group than in model rats (p < 0.05).

Serum NO, eNOS and ET levels in the three groups
There were markedly lower levels of NO and eNOS in the model group than in the sham group, but ET was significantly higher in shamoperated rats than in rats with congestive heart failure (model). In comparison, NO and eNOS levels in the quaternary amine group were significantly higher than the corresponding levels in the model group, while ET was significantly higher in model rats. These data are shown in Table 3.   Table 4 depicts markedly enhanced level of TXB2 in model rats, relative to sham rats, but there was lower PGI2 level in model rats than in sham rats (p < 0.05). In contrast, TXB2 level in the quaternary amine group was lower than that in the model group, while PG12 was markedly higher.

DISCUSSION
Recent studies have shown that quaternary amines, which are bioactive compounds in Rhizoma corydatidis, possess good watersoluble properties [9]. In clinical use, they exert anti-myocardial ischemia effect by reducing myocardial oxygen consumption, and they protect myocardial cells. The effects of quaternary amine on endogenous blood vessels and myocardial sites result in regulation of metabolism and ultimate enhancement of the tolerance of the myocardium to ischemia and hypoxia [10]. Quaternary amines reduce blood pressure by lowering heart rate and dilating blood vessels, and they prolong Q-T interval and T-wave with efficacy similar to that of ethamiodarone [11]. The results of this study showed that, on the premise that there was no obvious ST segment height in the three groups before surgery, ECG was comparable amongst the quaternary amine group, sham operation group and model group at 0 min after surgery. However, ECG values at 5, 15, 30 and 45 min, and 1, 1.5, 2, 3 and 24 h after surgery differed statistically between the quaternary amine and model groups. Electrocardiogram is the most effective method for measuring the area of congestive heart failure. It was applied for studying the efficacy of treatments on rats with congestive heart failure by monitoring ST segment elevation before and after treatment.
Myocardial tissues specifically contain CK-MB isoenzyme. Thus, measurement of blood CK-MB activity serves as an index for early diagnosis of AMI, and it is of great significance in judging the extent of myocardial infarction and post-infarction reperfusion [12]. Changes in LDH content in serum are used as important and sensitive markers of myocardial damage, and the selectivity of serum LDH diagnosis is second only to those of CK-MB [13]. The serum activities of CK-MB, LDH and AST in model group were higher than the corresponding sham group levels, but their serum activities in the quaternary amine group were markedly reduced, relative to the levels in model rats. Quaternary amines regulate the metabolism of cardiomyocytes and reduce their apoptosis, thereby minimizing the degree of damage to cardiomyocytes [14]. Nitric oxide (NO) exerts a variety of biological effects such as inhibition of platelet accumulation, reduction of leukocyte adsorption, relaxation of coronary arteries, and protection of myocardial cells. In the present investigation, 7 days after treatment, there were lower NO and eNOS levels in model rats than in sham operation rats, but ET in model rats was elevated, relative to sham group. However, NO and eNOS levels in quaternary amine group were markedly higher than model group values, and ET was markedly elevated, relative to model rats. A key enzyme in synthesis of NO is eNOS. Quaternary amine base enhances the level of eNOS and makes it synthesize more NO so as to protect the cardiomyocytes and reduce blood pressure [15]. Thromboxane A2 produced by platelets is metabolized to TXB2 which exerts a contractile effect on vascular smooth muscle. Vascular endothelial cells produce PG12 through synthesis or release, and it inhibits the proliferation of smooth muscle cells via anticoagulation [16]. In pharmacology, quaternary amine inhibits myocardial lipid peroxidation due to its antioxidant properties, thereby reducing oxygen free radical damage to myocardial cells. It was found in this study that 7 days after administration, the level of TXB2 in model rats was higher than that in sham operation rats, but PGI2 level was significantly lower than that in the sham group. However, TXB2 level in quaternary amine group was reduced, relative to model rats, while PG12 was markedly elevated, relative to model rats. These results showed that the quaternary amine increased PGI2 by decreasing the level of TXB2, thereby reducing platelet accumulation and thrombosis.

CONCLUSION
Quaternary amine mitigates myocardial ischemia by regulating energy metabolism and levels of oxygen free radicals in rats with acute myocardial infarction. It would be necessary to carry out further investigations to ascertain its usefulness in clinical practice.