Physiological and Histological Impact of Alpha-Lipoic Acid on Myocardial Ischemia Induced by Salbutamol in Rats

Cardiovascular disease (CVD) is a major cause of death and morbidity rates and remains a major global health concern. The conditions that fall under the category of CVD are hypertension, arrhythmia, ischemic heart disease (IHD), congestive heart disease, valvular heart disease, heart failure, and stroke. An imbalance between coronary perfusion and oxygen demand causes IHD, a significant form of CVD, which frequently results in myocardial infarction (MI) from prolonged oxygen deprivation. IHD, as a consequence of which myocardial tissue and contractile force are lost, is among the most frequent reasons for cardiac failure (Schwinger, 2021).

Animal models play a vital role in developing novel medications for controlling and treating a wide range of illnesses. An established standard model to investigate the beneficial effects of medicinal herbs on heart dysfunction is chemically induced myocardial ischemia in rats (Janssen and Elnakish, 2019). Catecholamines are a powerful inducer of cardiac contractility in animal models and can cause ischemia, hypoxia, myocardial hyperactivity, and coronary hypertension when administered at high doses (Ahsan et al., 2020). To induce myocardial ischemia in the animal model, Salbutamol has been used. Salbutamol is a synthetic catecholamine; its structural resemblance to isoproterenol and its manner of action cause significant oxidative stress in the heart, leading to severe myocardial stress and necrosis (Aodah et al., 2023). Salbutamol, a β2 agonist, commonly prescribed to treat asthma, chronic obstructive pulmonary disease, and bronchospasm. To delay premature labor, intravenous salbutamol might be given as a tocolytic to relax the uterine smooth muscle (Motazedian et al., 2010; Marques and Vale, 2022). When animals are given excessive salbutamol dosage causes tachycardia, which could result in myocardial ischemia. Salbutamol is less affordable and readily available when compared to other catecholamines such as isoproterenol (Afsheen et al., 2017; Abd Halim et al., 2018).

Thioctic acid, known as alpha-lipoic acid (ALA), is a naturally occurring short-chain fatty acid (1, 2-dithiolane-3-pentanoic acid), an antioxidant. In the body, it dissolves in fat and water. The gastric tract absorbs it rapidly. This antioxidant molecule has gained a lot of attention recently and is widely utilized as a dietary supplement across the globe (Superti and Russo, 2024). According to earlier research, ALA can influence both peripheral and central regulation of 5′-adenosine monophosphate-activated protein kinase. As a result, the antioxidant activity of ALA may help lower blood pressure, encourage weight loss, and improve insulin resistance and atherogenic dyslipidemia (Salehi et al., 2019).

To date, no study has systematically evaluated the effects of ALA on myocardial ischemia induced by salbutamol. This study aimed to evaluate the protective effect of alpha-lipoic acid against cardiac damage induced by salbutamol in rats.

In this experiment, adult male albino rats weighing between 160 and 200 grams were employed. The animals housed at the animal house of Basrah University Veterinary Medicine College are kept in well-ventilated cages that provide unlimited consumption of commercial diet and water. The animals were given ten days to adapt to the lab’s environment.

Alpha-lipoic acid (neutec/Turky/batch numbers: 53642), Salbutamol (S.D.I /Iraq/ batch numbers: 101025), distilled water.

Three groups of eighteen male rats each were randomly assigned (6 individuals each): Group 1: Normal rats received a daily 0.9% dose of normal saline as a control. Group 2: Positive control received salbutamol (80mg/kg) orally for 2 consecutive days (Afsheen etal.,2017), then treated with normal saline daily. Group 3: received salbutamol (80mg/kg orally) for 2 consecutive days and then received ALA (20mg/kg) orally (Cremer et al., 2006) for 28 days.

Following the experiment, blood samples were taken through the cardiac puncture and subsequently transferred into a gel tube to conduct serum biochemical analysis using commercial kits such as serum creatine kinase-myocardial band (CK-MB) (Human/Germany), Ichroma troponin I (Boditech/Korea), Aspartate transaminase (AST), Alanine aminotransaminase (ALT) and Lipid profile (Spinreact/Spain).

The eighteen heart samples, six sample from each group, were immediately removed and fixed for 12 hours, and the form fixative was eliminated using 70% alcohol. The tissues underwent gradual dehydration at increasing ethanol concentrations, followed by xylene treatment and paraffin embedding. On a glass slide, five micron-thick slices of paraffin-embedded tissues were placed, and hematoxylin and eosin stain were applied. The sections were inspected under an Olympus RPA light microscope using high-resolution camera (Sony/Japan).

Utilizing the computerized version 22 of the SPSS (Statistical Program for Social Sciences) program, statistical analysis of the data was carried out based on a one-way analysis of variance (ANOVA) and least significant differences (LSD) post hoc test with a P value (P<0.05).

The results of troponin I and CK-MB showed significant elevation (P<0.05) in the salbutamol group compared to the control group rats. While group treated with salbutamol + AlA showed a significant reduction (P<0.05) in troponin I and CK-MB compared to the salbutamol group and no significant difference was observed (P<0.05) compared to the control group (Table 1).

Table 1: Effect of ALA and salbutamol on cardiac markers.

Values are shown as mean ± SD, with n = 6/group. Different letters indicate significant differences at p < 0.05 based on LSD post hoc test.

Rats in the salbutamol group had significantly higher AST and ALT data (P<0.05) than those in the control group. The results indicate that the group treated with salbutamol + AlA reported a substantial decrease (P<0.05) in ALT and AST compared to the salbutamol group. Still, there was no significant difference (P<0.05) in ALT compared to the control group (Table 2).

Table 2: Effect of ALA and salbutamol on AST and ALT enzymes.

Values are shown as mean ± SD, with n = 6/group. Different letters indicate significant differences at (p < 0.05) based on LSD post hoc test.

The effect of ALA and salbutamol on lipid profile parameters in rats is displayed in Table 3. The findings indicate that total cholesterol, triglycerides, and LDL-C have significantly increased (P<0.05) in the salbutamol group compared to the salbutamol + AlA group and control. Whereas there was a significant decrease in HDL-C in salbutamol compared to the salbutamol + AlA group and control group. Furthermore, there were no significant differences in VLDL-C between groups.

Histological section of the heart in control rats showed that composed of elongated myofibers surrounded by epicardium. Each muscle fibers contain oval nuclei with few dense connective tissues between cardiac fibers (Figure 1A, B). Heart tissue of rats treated with SAL showed disarrangement of some myocadiac fibers with degenerated of nuclei, infiltration of inflammatory cells in the connective tissue between cardiac fibers, congestion of blood vessels was observed in the SAL group. As other section explained, degenerated with wavy some cardiac muscle, irregular sarcolemma with loss of muscle striation and karyohexis nuclei, also observed degeneration of the endothelial lining of blood vessels with pericardium spaces (Figure 1C, D).

Table 3: Effect of ALA and salbutamol on lipid profile.

Values are shown as mean ± SD, with n = 6/group. Different letters indicate significant differences at (p < 0.05) based on LSD post hoc test.

While histological section of the treated heart with (SAL₊ALA) showed clear structure of the myocardium which appear as cross striated myofibers. Few hemorrhages between cardiac fibers, clear epicardium layer with empty intercellular spaces and mild congested blood vessels, it also noted besides inflammatory cellular infiltration between the cardiac fibers (Figure 1E, F).

Troponin I and CK-MB are released into the plasma, and their level rises when there is cardiac muscles damage. The results of the present study revealed a significant decrease in cT-I and CK-MB concentration in the groups treated with ALA when compared with the salbutamol group these results are in agreement with (Tharwat and Marzok, 2024). According to the findings, catecholamine-induced oxidative stress causes beta-adrenoreceptor hyperstimulation, which leads to a complex functional and structural myocardial injury that causes cellular damage. Additionally, researchers discovered that oxidative stress could inhibit sarcolemma calcium transport, resulting in calcium overload and cardiac dysfunction. This could lead to in-situ troponin degradation and the release of low molecular weight fragments into the circulation during a period of Ca2+ overload (Ahmed and Masoud, 2024).

Because alpha lipoic acid has strong antioxidant properties, oxidative stress reduction could be one of the ways that post-reperfusion arrhythmias are decreased (Zheng etal., 2024). Alpha lipoic acid has cardioprotective effects on ischemia-reperfusion injury through a mechanism that involves activation of aldehyde dehydrogenase 2. Alpha-lipoic acid raises the sulfane sulfur content. Through scavenging ROS and activating the K-ATP channel, sulfane sulfur should be converted to H2S, shielding the heart from after-reperfusion arrhythmias (Star, 2023; Iciek et al., 2023).

According to Serbinova et al. (1992) rats hearts fed lipoic acid provided protection from ischemia-reperfusion damage in an isolated perfused Langendorff heart system. Lipoic acid also preserved vitamin E in heart tissue, enhanced post-ischemic left ventricular functional recovery, and reduced lactate dehydrogenase leakage and lipid peroxidation (Serbinova et al., 1992).

Our results supported earlier studies that showed ALA supplementation was beneficial in lowering triglycerides, total cholesterol, LDL and HDL levels in individuals with metabolic disorders, but had no effect on VLDL levels. ALA may act on rising blood and liver triglycerides by suppressing liver lipogenic gene expressions, reduce hepatic triglyceride production, and promote the removal of triglyceride-rich lipoproteins animal models currently provide evidence of a lipid-lowering response to ALA therapy (Seo et al., 2012; Akbari et al., 2018). ALA may act on rising blood and liver triglycerides by suppressing liver lipogenic gene expressions, reduce hepatic triglyceride production, and promote the removal of triglyceride-rich lipoproteins (Zhang et al., 2011). Total cholesterol and LDL was decreased by ALA most likely through the raising lipoprotein lipase activity, starting the liver’s synthesis of LDL receptors, which increases the uptake of cholesterol back into the hepatic system or increases the synthesis of apolipoprotein A component and raises serum adiponectin, which increases the β-oxidation of FFAs (Yamauchi et al., 2002).

There may be a connection between ALA’s effects on glucose consumption and lipid metabolism. When fructose is administered to rats, ALA improves insulin activity, insulin sensitivity, and glucose elimination. Reducing the levels of lipids in the blood to normal may lead to ineffective control of the major enzymes involved in lipid metabolism. One possible explanation for the decrease in plasma TG concentrations is the decreased availability of precursor FFAs and the improved peripheral tissue clearance from elevated LPL activity (Salehi et al., 2019). HDL-C plasma concentrations may have increased in ALA-treated rats because of enhanced synthesis of HDL components and delayed clearance. The ATP-binding cassette transporter A1, which is essential for the efflux of cholesterol from macrophages to HDL particles, is activated by lipoic acid. Also, elevated lipoic acid levels can promote the synthesis of ApoA-I, a significant HDL protein component that is necessary for the development of mature HDL particles. Lipoic acid may promote cholesterol mobilization and efflux by activating signaling cascades through receptors such as scavenger receptor class B type I. The preservation of normal phospholipid concentrations in tissues and plasma may be due to ALA antioxidant properties (Thirunavukkarasu et al., 2004: Lewis and Rader, 2005). The absence of significant differences in VLDL could be that the VLDL metabolic pathway may not be affected by ALA at this dose.

The results of microscopic examination of cardiac section of rats treated with SAL showed more effects on the heart tissue, including degenerated cardio fiber, degenerated nuclei and irregular with infiltration of inflammatory cells congestion of blood vessels, abnormal sarcolemma with karyohexise nuclei. Also observe wavy cardiac fibers with few of fiber striation. SAl is used blockers of β1 receptors and it acts on the heart by competing with the site of the β1receptors on the heart tissue. SAL could slow down the strength of cardiac contraction with reduce oxygen consumption, also reduce the volume of blood flow (Akhtar et al., 2023). Histological section of the heart treated (SAL and ALA) showed normal branching muscle fibers, with normal elongated nuclei and mild congested blood vessels, associated with the antioxidant activity of AlA, its cardiovascular protective properties also block pro-inflammatory genes and proteins, including cytokines, TNF-α, IL-1β-, and IL-6. To control adipogenesis, activate transcription factors and peroxisome proliferative-activated receptors (Alves et al., 2012; Suryavanshi and Kulkarni, 2017; Lee et al., 2022).

The study concluded that alpha lipoic acid has ameliorative effects in cardiac biomarker, lipid profile and cardiac tissue against myocardial ischemia induced salbutamol in experimental animals. These findings suggest a potential cardioprotective effect of ALA, but further studies are needed.

The authors would like to thank the faculty and staff of Basrah University College of Veterinary Medicine’s animal house for their support and advice.

The novelty of this study lies in demonstrating the potential therapeutic effects of alpha lipoic acid on salbutamol-induced myocardial ischemia. The study offers valuable insights by systematically exploring its impact on cardiac markers and lipid profiles in experimental animals. The key finding is the significant improvement in cardiac markers and lipid profile and decreasing harmful elements while boosting protective HDL levels. This suggests that ALA could play an ameliorative role in managing myocardial ischemia, advancing research in cardiovascular protection.

The work provided here was a collaborative effort by all authors. In addition to writing the final publication, each author had an impact on the study’s idea and design, data collecting, analysis, and interpretation.

Under IACUC ethical permission, this investigation was conducted in 2024 on animal 25/37 at the College of Veterinary Medicine, University of Basrah, Iraq.

The authors declare that no Genrative AI was used in the creation of this manuscript.

The authors have declared no conflict of interest.