Introduction
Combined with a sedentary lifestyle, modern life is imbued with the aggravation of health problems, including cardiovascular diseases. Outbreak studies using factors, such as nutrition, lifestyle, and exercise have shown that increased cardiovascular disease is typically associated with decreased physical activity and unhealthy dietary habits, including processed and fatty substances [1]. Cardiovascular diseases, which often begin with a background of atherosclerosis, are the leading cause of death and disability in many countries, including Iran. The high contribution of substances, such as cholesterol to the development of coronary diseases has been identified as one of the most prominent factors related to cardiovascular problems [2].
Despite the usefulness of cholesterol for sustaining several vital cell processes, its overproduction can compromise cellular function [3]. In the human body, about two-thirds of cholesterol is transported by low-density lipoproteins (LDL), approximately 20% by high-density lipoproteins (HDL), and the remaining 14% by very-low-density lipoproteins (VLDLs) [3]. When cellular cholesterol levels exceed the levels of phospholipids present in the membrane or when cholesterol is converted to cholesterol ester, the additional cholesterol in the cell creates toxicity [2]. Several mechanisms for cholesterol toxicity have been proposed so far, including cholesterol crystals, apoptotic pathway condensation, toxic Oxysterol formation, and membrane abnormalities that are critical for the function of certain specific enzymes and signaling molecules. However, as stated above, cholesterol removal from the cell is necessary to avoid the harmful effects of increased cholesterol in the cell [4].
The reverse cholesterol transfer (RCT) from the vessel wall binds to apolipoprotein A1 to produce HDL and aims to remove cholesterol from the cell environment via adenosine triphosphate-bound transporter proteins [5], mediated by adenosine triphosphate (ATP)-binding cassette (ABC) transporters [6]. The main transporters of bile cholesterol secretion are adenosine triphosphate (ATP)-binding cassette transporters G5/G8 (ABCG5 and ABCG8), and their encoding is located adjacent to each other on chromosome 2p21, where mutation of these genes or dysfunction of these two proteins results in a lipid disorder called sitosterolemia. These disorders usually cause cholesterol accumulation, resulting in coronary atherosclerosis, which may eventually lead to acute myocardial infarctions [7, 8]. Nevertheless, these two proteins secrete plant sterols and cholesterol from the enterocytes into the intestinal tract and excrete them from the hepatocytes into the bile, thereby lowering excess cholesterol from the coronary walls and lowering blood cholesterol levels, which decreases the risk of atherosclerosis and myocardial infarction [9]. Therefore, exercise is considered an inexpensive and effective treatment to reduce cholesterol and consequently, treat atherosclerosis [2]. 
Recent studies have shown that physical activity can improve RCT phases, such as improving HDL levels via enhanced lecithin: cholesterol acyltransferase (LCAT) enzyme activation and cholesterol withdrawal from cells through ABC protein activity [10, 11, 12, 13, 14]. These mechanisms suggest that exercise is a potential strategy for patients with atherosclerosis, being recommended even after coronary artery bypass graft surgery (CABG) [15], considering that regular exercise with low to moderate intensities reduces the risk of coronary artery diseases [16, 17]. Several studies addressed the exercise capacity to significantly reduce blood lipid levels, lowering low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), with subsequent increase of HDL, improving the health condition of these patients [13, 18, 19, 20, 21]. Therefore, given the effect of exercise training on cholesterol levels, associated with the reduction of cardiovascular risk factors and the inconclusive reports regarding ABCG5 and ABCG8 gene expression, we aimed to investigate the effect of eight weeks of aerobic training combined with resistance exercise in specific gene expression of CABG patients.
Materials and Methods
The study employed a quasi-experimental control. The research follow all the recommended guidelines proposed by the Declaration of Helsinki according to ethical principles regarding human experimentation, where all subjects were informed about the possible benefits and risks related to the protocols applied during the present intervention. That research was approved by the Ethical Committee board (protocol number, IR.IAU.NEYSHABUR.REC.1398.013). Thirty men were selected by a convenient sampling method in the rehabilitation ward of Jawad Al-Aemeh Heart Hospital. All selected participants underwent bypass surgery in the last year. In the current study, the inclusion criteria included individual cognitive, visual, and hearing health, and the exclusion criteria included no blood pressure greater than 160 mmHg and diastolic blood pressure greater than 100 mm Hg, not using the same medication, not using mobility assistance devices e.g. walker. Therefore, 30 CABG men (body mass index [BMI]: 25.11±1.57 kg/m²; age: 55.37 ±6.90 years; body mass: 75.45±5.87 kg/m²) were recruited to participate in the present study and 15 men were randomly allocated into an experimental group (BMI: 24.94±1.8 kg/m²; age: 54.58±6.47 years; body mass: 74.75±6.6) that underwent through an exercise intervention, and the others 15 subjects were randomly allocated to control group (BMI: 25.28±1.34 kg/m²; age: 56.16±7.5 years; body mass: 76.16±5.23). 
Experimental design
Aerobic training: The training protocol was performed three days a week, with a 24 hours interval between each exercise session, and a total of 24 meetings during the study protocol. Each exercise session lasted up to 42 minutes by the cardiopulmonary status and physical tolerance of each subject. The exercise session included treadmill walking (20 to 30 minutes), pedaling on a stationary bike (10 to 12 minutes), and ergometer use (10 minutes). Before the beginning of each training session, stretching exercises were used to warm up. The same stretching technique was used to cool down at the end of the session. The exercise intensity of 55% of patients’ maximum heart rate was considered the target heart rate during the initial phases of the training, with gradually increased based on the patient’s ability to reach 75% of the maximum heart rate in the last 7 to 10 meetings. 
Resistance training: The resistance exercise program was performed three times a week for eight weeks, alternately with aerobic training. The initial sessions included three sets of up to eight repetitions, which increased up to 15 repetitions in the subsequent sessions. The resistance training session included Scott with a physioball ball, shoulder flection, hip flection, shoulder abduction, hip abduction, elbow flection, ankle plantar, and dorsi ankle flection. The movements were initially performed with eight repetitions using a light traband (yellow). Then, in each session, two repetitions were added to each movement to increase the number of repetitions to 15. Then the traband strength increased (pink) and the movements increased again with eight repetitions and gradually increased to 15 repetitions in the subsequent sessions. Each session was followed by stretching exercises for 5-10 minutes and cool-down movements for 5-10 minutes. The fluctuations of the patient’s heart rate during the exercise were monitored by a monitoring system.
Instruments and procedures 
Anthropometric Components: At the first visit to the laboratory, the participant’s body weight and body fat (%BF) were measured with an Inbody720® digital device manufactured by South Korea. The height was assessed by a SEKA® digital scale device (cm/cm) manufactured in Germany. The BMI was calculated as the body weight, in kilograms, divided by the square of the height, in meters (kg/m²).
Cardiac Measurements: The heart rate was measured using Polar® F1tm Polarimeter and analyzed using the Karunen formula on three different occasions, at the beginning of the session, at the cool down, and at the end of the session. As the intensity of exercise increased, approximately 5% of the heart rate was added to the target heart rate every week [22, 23, 24]. The blood pressure, as well as the resting blood pressure, was analyzed using an ALPK-2 -500 device, recording the data that are later processed by the rehabilitation nurses after each training session 
Gene Analysis: The subjects were instructed to arrive in the laboratory in a fasting condition 48 hours before and 48 hours after the last training session. During the visits, ten cc venous blood samples were obtained, in which a monocyte separation was performed using Ficoll Monocyte messenger ribonucleic acid (mRNA) purification. The samples were homogenized to obtain mRNA in RLT buffer and then combined with liquid nitrogen (2 mL) in an RNA-free microcentrifuge tube. Real-time PCR was used to evaluate the relative expression of ABCG5/8mRNA. Table 1 presents the sequence of primers used to measure the ABCG5 and ABCG8 genes.


Statistical analyses
All measurements and analyses were reported in mean±SD and were obtained using SPSS software, version 21 (SPSS™, Inc., Chicago, IL) at the significant level of P<0.05. After examining the normality of data distribution through the Shapiro test, the research findings were analyzed using paired t test and dependent t test to compare pretest and posttest within and outside the group.
Results
The real-time PCR observed in the present study showed a significant difference between the aerobic training mixed with resistance exercises compared to the control group in the expression of ABCG5 (P=0.001) and ABCG8 (P=0.001) genes. Correlation t test results showed significant differences between pretest and posttest in the experimental group (t=-4.517, P=0.001) (Table 2).


Figure 1 shows that changes in the expression of ABCG5 and ABCG8 genes in the aerobic-resistance training group compared to the control group increased significantly. Discussion 
The present study investigated the impact of eight weeks of aerobic training combined with resistance exercises on the expression of ABCG5 and ABCG8 genes in CABG patients. The present study showed that eight weeks of aerobic training combined with resistance exercises increase the expression of ABCG5 and ABCG8 genes in CABG patients, which may reduce cardiovascular diseases. 
One of the most dangerous cardiopathy is related to coronary artery disease (CAD), commonly reported as atherosclerosis cases, decreasing vessel wall elasticity due to the formation of atherosclerotic plaques, and thickening arteries due to an increased amount of cholesterol accumulation [22]. Increased endothelial abnormalities of some cholesterol transporters at the endothelial membrane surface are suppressed in expression and function, leading to increased cardiovascular diseases [23], including decreased expression and impaired function of RCT factors called ATP-dependent (ABC) box proteins in a way that these patients are resistant to interventions designed to regulate transcription to increase cholesterol transporters. Therefore, mechanisms that impair ABC transporters or affect actual cellular pathways should be directly treated [24]. 
The ABC family is subdivided into sequences, and ABCG is a subset of the large ABC family, which is a significant lipid transporter and has several subclasses, including ABCG1, ABCG2, ABCG3, ABCG4, ABCG5, ABCG8, ABCG11, ABCG12, and ABCG26 [25, 26, 27, 28, 29, 30]. All of these ABCGs except ABCG2 play a crucial role in the RCT process. Two members of the ABC transporter family, ABCG5 and ABCG8, which are heterodimeric and are often expressed in the liver and small intestine, restrict sterol uptake by the intestine and increase biliary secretion by the cholesterol-induced liver. The ABCG5, and ABCG8 transporters are both regulated by the hepatic X receptor [25, 31, 32, 33, 34 ,35]. In other words, the liver X receptor (LXR), by upregulating ABCG5 and ABCG8, produces bile acid and secretes cholesterol into the bile, promoting fecal excretion of cholesterol in the intestine [17, 36, 37, 38]. 
Overexpression of ABCG5 and ABCG8 transporter genes in mice reduces dietary cholesterol up to 50% by increasing biliary secretion of sterols. Conversely, abnormalities of both genes in mice led to a 3-fold increase in dietary plant sterol uptake, increasing plasma cytosterol levels by 30%, combined with decreased biliary cholesterol levels [26]. The beneficial effects of these transporters on cholesterol homeostasis have made them vital as therapeutic targets to prevent atherosclerotic cardiovascular disease. Nevertheless, although most studies have shown that exercise activities increase the expression of cholesterol transporters, and affect blood cholesterol levels [29, 30, 31], the most significant part of these studies was conducted to investigate the effects of physical activity on the mechanisms of expression of genes involved in the process of RCT, including the family of ABC transporters, especially type G often focused on animals, demonstrating a lack of knowledge regarding these effects in humans [27, 28]. 
In this context, a team of researchers reported that the expression levels of ABCG5 decreased in Sprague–Dawley female rats following moderate-intensity aerobic training on a treadmill (six weeks of incremental running from 15 m/min and 0% slope for 15 minutes a day [two weeks] to 60 minutes a day at 26 m/min 10% [four weeks] and five times [weekly]) [28]. Besides, another study examined the impact of training on the expression of the ABCG5 gene in intestinal tissue of female mices, and the findings showed the increased expression of this gene following exercise training (six weeks of incremental running at 15 m/min and 0% slope for 15 minutes a day to 60 minutes a day at 26 m/min at 10% slope, five times a week) [39]. In another study, Ghanbari Niaki et al reported an increase in the relative expression of ABCG5 visceral fat following aerobic exercise on the treadmill [40]. Nevertheless, Hosseini et al. examined the effects of aerobic training and high-intensity exercise on the expression of ABCG4, ABCG5, and ABCG8 genes in athlete muscles. Their results showed that one session of aerobic and high-intensity exercise induced the expression of genes involved in cholesterol transfer compared to the control group, but no significant differences were observed between the two experimental groups [41]. 
Rehabilitation programs are a crucial part of the treatment process aiming to return the patient to their optimal physical, psychological, and social conditions [42]. Life expectancy after cardiac rehabilitation has been shown to significantly increase, demonstrating the efficacy of these strategies. Training protocols reduce cardiovascular mortality by 20% to 25% and cardiovascular disease death by approximately 22% to 25% after a three-year follow-up study. Rehabilitation is also considered a facilitator in controlling risk factors in these individuals because it can slow or delay the disease process [42, 43, 44, 45].
Based on the results of the present study and combined with several studies in animals, it can be suggested that the combination of aerobic training and resistance exercise is effective to enhance the RCT via ABCG5 and ABCG8 gene expression, and shows that it can be considered an effective and preventive treatment for CABG patients.

Ethical Considerations
Compliance with ethical guidelines
This study has been approved by the ethical code IR.IAU.NEYSHABUR.REC.1398.013 in Islamic Azad University, Neishabour branch.

Funding
This article is taken from the PhD thesis of Farida Sadeghi Fazel, Department of Physical Education, Islamic Azad University, Neishabour branch.

Authors' contributions
Conceptualization and Supervision: Rambod Khajei and Amir Rashid Lamir; Methodology: Amir Rashid Lamir; Data collection and Funding acquisition and Resources: Farida Sadeghi Fazel; Data analysis: Frida Sadeghi Fazel and Akbar Safipour Afshar; Investigation, Writing–original draft, and Writing-review & editing: All authors. 

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors of this article appreciate the participants in this study.

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