Introduction
By increase of mechanized life and reduction in mobility, there has been an increase in some diseases such as Cardiovascular Diseases (CVDs), which is directly related to inactivity and poor nutrition (use of unhealthy foods). Major CVDs include atherosclerosis and its complications [1]. Atherosclerosis is the inflammation of coronary arteries, and occurs due to the accumulation of macrophage foam cells and the inability to remove the excess of cholesterol. Macrophage foam cells are enlarged cells in the artery wall consisting of macrophages rich in Low Density Lipoproteins (LDL), and are the cause of vascular occlusion. increase of cholestrol can jeopardize cell function, although it is useful for continuity of some vital cell processes. When the level of cell cholesterol exceeds compared to phospholipids in the membrane or the cholesterol is converted to cholesterol ester, the excess of cholesterol inside the cell causes toxicity and destroys the endothelial cells [2]. In other words, Reverse Cholesterol Transport (RCT) is an anti-atherosclerotic process when excess cholesterol is removed from surrounding tissues, including arterial wall macrophages, and is returned back to the liver by the formation of High Density Lipoproteins (HDL) [3]. Regulation of the genes from ATP-Binding Cassette (ABC) family by the Liver X Receptor (LXR) is one of the important components of RCT. There are 49 different genes encoding ABC transporters in humans. All ABC genes are divided into seven subfamilies named ABCA-ABCG according to the Cytochrome p450 enzyme gene (CYP) nomenclature system. In vascular diseases like atherosclerosis, factors such as ABC transporters play a significant role due to involvement in cholesterol homeostasis, blood pressure regulation, endothelial function, vascular inflammation, and platelet production and accumulation. ABCC9, ABCC6, ABCC1, ABCB6, ABCG4, ABCG1 are among ABC transporters that directly or indirectly affect cellular cholesterol excretion, inflammatory response in macrophages, megakaryocyte proliferation and thrombus formation, which may affect vascular function and blood pressure and play a role in CVDs and their complications. 
Studies have indicated that exercise increases the expression of some families of ABC genes including ABCA1, ABCG5, ABCG4, ABCG1, ABCG5 /8 and ABCA5 [4, 5, 6, 7, 8]and, thus, improves some key steps in the RCT process like the increase and synthesis of HDL and the increase of cholesterol transport out of the cell. Therefore, exercise is important for cardiovascular rehabilitation and reduces cardiovascular complications and mortality from CVDs. Ray et al. reported that the increased ABCA5 gene expression in patients with dyslipidemia decreased cholesterol levels [9]. Ye et al. reported that the ABCA5 knockout was associated with an increase in LDL level [10]. Although, studies have reported that the ABCA5 is involved in the removal of fat from cells, its role as a transporter is unclear for atherosclerosis [9]. Many studies are needed to determine the role of ABCA5 in high cholesterol-related diseases such as CVDs. Few studies have been conducted on the rehabilitation phase of CVD patients and the hybrid effects on ABCA5 gene expression. This study aims to answer the question that whether eight weeks of combined exercises affect the expression of ABCA5 gene of white blood cells in middle-aged men after Coronary Artery Bypass Graft (CABG) surgery. 
Materials and Methods 
This is a quasi-experimental study with a pre-test/post-test design. The statistical population consists of all male patients aged 50-65 years referred to Javad Al-Aemeh Cardiovascular Hospital in Mashhad, Iran. The sample size was determined according to previous studies and patient availability. In this regard, 26 men who underwent CABG surgery were selected using a convenience sampling method and according to the inclusion criteria (no cognitive, vision and hearing problems, no blood pressure level higher than 160 mmHg, and diastolic above 100 mmHg, no drug use, no use of assistive devices such as walkers). They were randomly divided into exercise and control groups. Exclusion criteria were: Absence from more than three consecutive sessions and four non-consecutive sessions, unwillingness to continue participation, injury or clinical symptoms, participation in other sports activities simultaneously, and not allowed for attending the study according to the physician. Anthropometric variables include age (year), height (cm) measured by a digital scale (SEKA, Germany) with an accuracy of 0.1 Kg, body fat percentage, and body mass index (kg/m2) measured by a digital scale (Inbody720, South Korea), heart rate (per minute) measured by a heart rate monitor (F1tm Polar, Finland), blood pressure, measure by a blood pressure monitor (ALPK2-500, Japan), as well as training times measured by a digital timer with an accuracy of 0.01 seconds. 
Before starting the combined exercises, 5 cc of blood sampling was collected from brachial vein at the beginning and after 10-12 hours of fasting (between 8 and 9 a.m) and put in experimental tubes containing ethylenediamine tetraacetic acid as an anticoagulant. Lymphocytes were isolated at this stage. Real-time Polymerase Chain Reaction (PCR) method was used to evaluate the expression of ABCA5 gene. Subjects then participated in a combined exercise programs for eight weeks. After eight weeks, blood sampling was done again during fasting and the changes were re-evaluated. Blood sampling time in the pre-test phase was 24 hours before the start of the first exercise session, while in the post-test stage, it was 48 hours after the end of the last exercise session (to prevent the acute effect of exercise on study variables). 
Combined exercise program
Patients in the exercise group performed combined exercises (aerobic and resistance exercises) at in the rehabilitation ward of Javad Al-Aemeh Hospital at 24 sessions, three days a week, as well cardiac rehabilitation at each session according to the assessment results (cardiopulmonary status, exercise tolerance, etc.) for 1-1.5 hour. In each exercise session, stretching exercises were performed to warm up and there was a time for cooling down at the end of session. The exercises included: walking on a treadmill (20-30 minutes), riding a stationary bike (10-12 minutes), and using cycle ergometer (10 minutes). All patients in the exercise group performed the above exercises during each rehabilitation session. The intensity and duration of exercises gradually increased based on the ability of individuals. In the last 7-10 sessions, the intensity reached 80% of their maximum Heart Rate (HR). Before and after aerobic exercise and once during cooling down, the HR was measured and then the target HR was calculated using the Karvonen formula: 
Resting HR+[(55, 75%)×(Resting HR-maximum HR)]=Target HR
Each week, the target HR increased approximately by 5% through increasing the intensity of exercise. The resistance exercise was performed at 8 repetitions in the initial sessions and increased to 15 repetitions in the next sessions in three sets. The movements included: squat with a physio ball, shoulder flexion, hip flexion, shoulder abduction, hip abduction, elbow flexion, ankle plantar and ankle dorsiflexion. The movements were initially performed using a weak thera band at 8 repetitions and by adding two more repetitions in each session, the number of repetitions increased to 15. Then, the resistance of thera band increased where the movements initially performed at 8 repetitions and gradually increased to 15 repetitions in subsequent sessions. There was no specific arrangement for aerobic and resistance exercises; patients performed resistance exercises during an interval between aerobic exercises or separately after aerobic exercises. At the end of each session, for cooling down, stretching movements were performed for 5-10 minutes and relaxation movements for 5-10 minutes. Fluctuations in the patient’s HR during exercises were controlled by a monitoring system, the researcher and a cardiologist. Blood pressure was measured and recorded by the rehabilitation nurses after using each device. The control group did not participate in postoperative combined exercises and had no regular physical activity. 
Measurement of gene expression 
For mRNA purification, peripheral blood mononuclear cells were immersed in liquid nitrogen and completely shredded by mortar & pestle. To obtain mRNA, degraded tissue was homogenized in RLT buffer and then tissue powder and liquid nitrogen were poured into a 2-mL Rnase-free microcentrifuge tube. The liquid nitrogen was let to be evaporated, but not let the lymphocytes got out of the frozen state. RLT buffer was added sufficiently. Lysate was transferred directly into a QIAshredder column placed in the tube and centrifuged at high speed for two minutes. For cDNA synthesis, 200 ng mRNA was evaluated using oligo dT primer and a special kit. Real-time PCR method was used to assess the gene expression. Finally, after obtaining the results using a UV-single device and obtaining beta-actin values for each sample, the obtained numbers were divided by the beta-actin values for each sample and then multiplied by 100 to mRNA values related to gene expression for each sample to be obtained by percentage (Table 2).


The PCR was performed using BIO RAD (C1000TM Thermal Cycler) in 96-well plates. Table 3 presents the thermal cycle of PCR.


The Pfaffl method was used to measure the copy number of target and reference genes. 
Statistical analysis 
Descriptive statistics including mean and Standard Deviation (SD) were used to describe data. The normality of data distribution was examined using Shapiro-Wilk test, and parametric paired sample t-test was used for data analysis in SPSS v.21 software at a significance level of P<0.05. 
Results 
According to the results in Table 4, the pre-test results showed no significant difference in the ABCA5 gene expression between the groups (P=0.457).


The expression of ABCA5 gene from pre-test to post-test phases increased significantly in the combined group (P=0.001) and showed an insignificant in the control group (P=0.60). A significant difference was observed between the two groups (P=0.001) (Figure 1).  Discussion
The results of this study showed that eight weeks of combined exercises increased the expression of ABCA5 gene in patients with CABG surgery. There are few studies that have examined the effects of combined exercises on the level or expression of ABCA5 gene in patients with CABG surgery. Our results are consistent with the results of Ray et al. [9] and Fu et al. [3]. No disagreement between our results and the results of other studies was found. 
The ABC proteins, which are a superfamily of membrane proteins and use ATP energy to transport various substances across membranes, play a prominent role in the mechanism of RCT [11]. ABCs are divided into separate A-G categories. All of them except G2 play an important role in the RCT process. One of the ABC subfamilies is the ABCA complex, which is responsible for removing cholesterol from cells [13]. One of the members of this complex is ABCA5 which plays an important role in RCT and is expressed in cardiomyocytes, oligodendrocytes, astrocytes, and Leydig cells [9]. Moreover, ABCA5 is found on endosomes and lysosomes, the Golgi apparatus, and plasma membranes. Studies have indicated that the lack of ABCA5 impairs lysosomal function in the heart, decreases HDL level, and increases cholesterol flow to ApoA-1. Decreased ABCA5 level is associated with increased neurodegenerative diseases such as Alzheimer and Parkinson [14]. According to a study, mice with ABCA5 knockout suffered from cardiopathy and lysozyme problems after 10 weeks [14]. Studies have indicated that ABCA5 is associated with ABCA1 in lowering cholesterol, and mice with low ABCA5 level suffered from atherosclerosis and hyperlipidemia [15]. ABCA5 stimulates the peroxisome proliferator-activated receptors PPARy and PPARa to reduce the cholesterol level and lipolysis. In fact, the increase in cholesterol level by 100 μM increases the expression of ABCA1 and ABCA5, and when the cholesterol level increases by 150 μM, the ABCA1 level decreases; however, the ABCA5 level continues to increase [9, 15]. ABCA1 has been specified as the major regulator of RCT, but when ABCA1 is blocked due to high cholesterol, ABCA5 is considered as the main regulator of RCT. This cholesterol excretion by ABCA5 is done by facilitating the diffusion. It has also been suggested that the cholesterol transport to HDL is mediated by ABCA5 [9]. On the other hand, increasing endothelial cell apoptosis is very evident in patients with atherosclerosis and CABG surgery [15]. Libby et al. reported that the increase in ABCA5 level reduced apoptosis in endothelial cells; this reduction plays a vital role in the viability of these cells [16]. According to a study, increased ABCA5 level is associated with decreased apoptotic factors (such as Bax and BCL), and increased LDL [18]. Increasing level of ABCA5 is very effective for CABG patients since it lowers cholesterol, increases HDL, reduces apoptosis, and stimulates lipolysis by activating PPARa and PPARy. Probably, the reason for the increase in ABCA5 gene expression following combined exercises in the present study is the increase in peroxisome proliferator-activating receptors that play an important role in regulating the expression of genes involved in RCT [19, 20]. Moreover, ABCA5 has been introduced as an inhibitor of atherosclerosis that can increase LXR expression. Due to the fact that transcription of some genes in the ABC family (including ABCA5 involved in RCT) is regulated by heterodimer system named LXR/Retinoid X Receptor (RXR) [21, 22], it is possible that the expression of the ABCA5 gene can be increased by regulating these receptors following combined exercise. As a result, PPARa seems to activate LXR and positively regulate transporters of ABC family, including ABCA5 by the LXR. 
The results of this study confirmed the increase of ABCA5 gene expression in patients with CABG. The increase in the levels of peroxisomes family was one of the reasons for increasing expression of the ABCA5 gene. Lack of peroxisomes’ level measurement, individual differences, and the differences in diet and motivation of patients were the limitations of the present study; therefore, it is recommended to design exercises protocols for this group of patients by considering these factors. 
Conclusionon 
Eight weeks of combined exercise program (aerobic and resistance exercises) can improve the RCT status by increasing the expression of ABCA5 gene in middle-aged men after CABG surgery. 

Ethical Considerations
Compliance with ethical guidelines
This study has an ethical approval obtained from the ethics committee of Islamic Azad University of Neishabour branch (Code: IR.IAU.NEYSHABUR.REC.1400.001). All study methods were explained to the participants, and they were assured of the confidentiality of their information.

Funding
This study was extracted from the PhD thesis of first author. It received support from Javad Al-Aemeh Cardiovascular Hospital in Mashhad and Islamic Azad University of Neishabour branch. 

Authors' contributions
Writing the initial draft: Mahboubeh Sazgar; Review and final approval; Amir Rashidlamir; Data analysis; Rambod Khajehi; Discussion and conclusion; Ameneh Barjasteh Yazdi. 

Conflicts of interest
The authors state that there is no conflict of interest in this article.


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