Comparison of the Effect of Two Moderate and High-Intensity Endurance and Resistance Training Methods on Predictors of Cardiovascular Diseases and Arteriosclerosis in Elderly Rats

INTRODUCTION One of the most important achievements of medical science in the last century is increasing life expectancy. From the beginning of the ۱۹۰۰-۲۰۲۱ century, the average life expectancy has increased from ۶۵ to ۸۵ years [۱]. On the other hand, age is the most important factor determining the health of the cardiovascular system, and according to predictions, by ۲۰۳۰, the age of about ۲۰% of the world’s population will reach more than ۶۵ years. In this age group, cardiovascular diseases are the cause of most deaths. In addition, the costs of treating these diseases will increase significantly. Therefore, it is very important to understand the importance of age and its effect on the occurrence of cardiovascular diseases. The causes of cardiovascular diseases in humans are age, gender, smoking, lipids, diabetes, and blood pressure [۲]. According to the American Heart Association, the prevalence of CVD, including high blood pressure, coronary artery disease, heart failure, and heart attacks, in men and women aged ۴۰ to ۵۹ years is ۴۰%; in people aged ۶۰ to ۷۹ years, is ۷۰ to ۷۵% and in people, ۸۰ years and over has increased to ۷۹ to ۸۶ percent [۳]. Cardiovascular diseases, especially atherosclerosis, are one of the major causes of death in most human societies, and its rate has increased with the prevalence of mechanization of today’s lifestyle and the decrease in physical activity. Atherosclerosis is a chronic inflammatory condition caused by the narrowing of the arteries due to the buildup of cholesterol-rich plaques in the walls of the arteries. This disease is associated with an increase in total cholesterol concentration, an increase in low-density lipoprotein cholesterol, and a decrease in high-density lipoprotein cholesterol [۳]. Standard lipid profile biomarkers are used to evaluate lipid disorders in people. Lipid disorder is an abnormal increase or decrease in the level of lipids, which is a very important factor in the occurrence of cardiovascular diseases in people [۴]. Coronary artery disease refers to a condition in which the deposition of fats leads to the narrowing of the coronary arteries and the reduction of blood flow to the heart. Many risk factors have been identified that may increase the risk of atherosclerosis, which mainly include total cholesterol (TC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), high blood pressure, smoking, diabetes, obesity, and sedentary lifestyle [۵]. The most important effective factor in atherosclerosis is the cholesterol in the blood circulation. LDL modulates it to reactive oxygen species when its amount increases in the blood. As a result, low-density lipoprotein is oxidized and quickly removed by macrophages of the walls of the vessels. These lipid-laden macrophages, known as foam cells, are the main cause of atherosclerosis [۶]. Since mammalian cells cannot reduce excess cholesterol, it is necessary to remove excess cholesterol inside the cell. HDL particles can remove excess cholesterol through reverse cholesterol transport. Reverse cholesterol transport is the collection of excess cholesterol from the walls of blood vessels, especially arteries, and its transfer to the liver and excretion through bile and feces [۶]. The reverse cholesterol transport cycle has several key factors, including the adenosine triphosphate-coupled transporters (ABCA۱, ABCH۱, ABCG۴, ABCG۵, ABCG۸), ApoA۱, and LCAT, which are involved in HDL biogenesis. This process finally transports cholesterol out of the veins and to the liver for excretion through bile [۷, ۸]. Today, most studies on older adults focus on the factors that cause cardiovascular diseases. Training is known as a treatment method for these diseases, which also plays a very important role in the health of the heart and its prevention [۹]. The formation of high-density cholesterol (HDL) is considered a predictor of the occurrence of atherosclerosis and an indicator of the health of the cardiovascular system [۱۰]. On the other hand, it has been reported that ApoA۱ is more sensitive and accurate than HDL in predicting atherosclerosis [۱۱]. HDL removes cholesterol and fats from the walls of cells and arteries and allows the liver to dispose of or reuse cholesterol and fats. For this reason, cholesterols that contain HDL (HDL-C) are also called good cholesterol. Finally, when the blood HDL concentration reaches more than ۶۰ ml/dL, it has protective effects against atherosclerosis and is associated with a reduction in the incidence of CHD [۱۰]. In previous studies, a strong inverse relationship between plasma HDL level and arteriosclerosis, and also the occurrence of cardiovascular diseases, has been reported. This led to the idea that interventions that increase HDL serum levels reduce CVD risk [۱۲]. After the age of ۲۰, LDL concentration increases increasingly in men and women, which is greater in men than in women [۱۳]. The level of LDL reaches in men between ۵۰ and ۶۰ years and women between ۶۰ and ۷۰ years and does not change after this age [۱]. The amount of serum HDL in men decreases during puberty, and the beginning of adulthood, but the concentration of HDL in women remains constant throughout their life [۱۴]. On the other hand, studies have shown that ApoA۱ may be a better predictor of cardiovascular disease in people than HDL and have more accuracy [۱۴]. Coronary heart disease has an inverse relationship with serum HDL level and a direct relationship with serum LDL level. Therefore, the ratio of LDL/HDL is considered a good variable to predict the rate of coronary artery disease in people, especially older adults. The appropriate level of LDL is equal to LDL≤ ۱۴۰ mg/dl, and the level of HDL is equal to HDL≥ ۴۰ mg/dl [۱۵]. Blood lipoproteins are formed by different amounts of cholesterol (C), triglycerides (TGs), phospholipids, and apolipoproteins. ApoA۱ is the most important structural protein of high-density lipoprotein (HDL), accounting for ۷۰% of the structure of HDL and exerting many of HDL’s anti-atherosclerotic actions. Conversely, ApoB is the predominant Apo used in the structure of low-density lipoprotein LDL, indicating the amount of circulating LDL associated with CHD risk. Therefore, the ratio of ApoB to ApoA۱ (ApoB/ApoA۱) is used as a surrogate index to assess the risk of CHD associated with lipoproteins. Increased or abnormal levels of lipids or lipoproteins in the blood are considered a significant risk factor for CHD [۱۶]. Several studies have supported the idea that continued activity is partially associated with reductions in serum TG and LDL-C levels and increases in serum HDL-C levels, reducing CHD risk. Also, training positively affects the maturation, formation, and recall of cholesterol from peripheral cells to the liver for metabolism and disposal. This process prevents atherosclerosis [۱۶]. According to previous research, there is a general agreement about the effect of sports activity on changes in cholesterol and lipoprotein metabolism. However, there are many contradictions regarding the appropriate intensity, type, and volume, and there still needs to be a complete consensus. Also, most of the studies have examined young samples or non-elderly diabetic samples, and in a few studies, the effect of intensity and type of training has been compared with each other. Considering the side benefits that Apo has (for example, no need for fasting sampling, the ratio of ApoB to ApoA۱ compared to LDL-C and HDL-C is a better indicator for statin therapy, and the method of measuring ApoB and ApoA۱ is standard; in While the method of measuring HDL-C and LDL-C is not the same); Therefore, it appears to be significantly beneficial to incorporate APOs into clinical practice [۲]. Most studies have investigated the effect of low and moderate-intensity training on RTC [۷]. Therefore, there is a need to conduct more studies on the effects of high-intensity training. However, they have used constant intensity to perform continuous training. However, other models and types of training have yet to be significantly investigated, and their effects need to be better understood. Therefore, in this study, the effect of two types of moderate and high-intensity resistance and interval training on TC, TG, HDL, LDL, VLDL, ApoA۱, ApoB, LDL/HDL, ApoB/ApoA۱ in elderly male rats was investigated. MATERIALS & METHODS The current research is experimental, with a pre-test and post-test design with a control group. In terms of purpose, it is part of basic research conducted in ۲۰۲۱ in the basic science laboratory of Shahrekord University in Iran. Fifty male Wistar rats with an average weight of ۴۳۷.۲±۴.۴ grams at ۲۳±۲ months were purchased from the Pasteur Institute of Iran. They were kept at a temperature of ۲۲±۳°C, with ۱۲:۱۲ hours of darkness and light, and humidity of ۴۰-۴۵%, and fed with special rat food [۱۰]. In order to acquaint the rats with the environment, they were kept and fed in the laboratory for about one week. Then, they were divided into five groups of ten based on their initial weight.  Control Group: Rats that had no physical activity and were kept in special cages. Moderate-Intensity Continuous Training Group: This group performed continuous training with an intensity of ۶۰ to ۷۰% of the maximum speed.  High-Intensity Endurance Training Group: This group performed high-intensity interval training which included a combination of high-intensity (۸۰-۱۱۰% of maximum speed) and low-intensity (۳۰-۴۰% of maximum speed) activity. Moderate Intensity Resistance Training consisted of performing resistance training with ۶۰% of the (Maximal Voluntary Carrying Capacity (MVCC) and repetitions ۱۴ to ۲۰ times per session. Furthermore, the High-Intensity Resistance Training Group included doing resistance training with ۸۰% MVCC and ۹ to ۱۰ repetitions. The training protocol of the resistance training group: Rats in both groups of moderate and high-intensity resistance training, in order to familiarize themselves with the implementation of the training protocol, in the first week, five days without weights, practiced climbing the ladder. Forty-eight hours after the last session, the maximal voluntary carrying capacity test was taken from the rats. MVCC was then defined as rats’ maximal voluntary carrying capacity [۱۷]. Both resistance training groups performed climbing a special ladder (۱۱۰ cm long, ۸۰°slope, ۲۶ steps, and two cm distance between steps) for eight weeks and five days a week, and they rested for a minute after climbing the ladder every time. According to the adaptation of the animals to training at the end of the fourth week, MVCC was retaken from the rat, and the training intensity was determined based on it from the beginning of the fifth week [۱۸]. The moderate-intensity resistance training group repeated the training protocol with ۶۰% of MVCC and ۱۴ to ۲۰ times in each session, and the high-intensity resistance group also performed the same training protocol with ۸۰% of MVCC and ۹ to ۱۰ times [۱۸]. Maximal Voluntary Carrying Capacity (MVCC): First, a weight equal to ۷۵% of the body weight of the rat was attached to their tails, then they climbed the ladder. For each successful repetition, ۳۰ grams were added to the weight. After each climb, the rats rested at the top of the ladder for two minutes. This process was repeated until each rat could not climb the ladder completely three times in a row. Finally, the maximum weight each rat managed to carry was recorded as the maximum voluntary carrying capacity for that rat [۱۷]. The training protocol of the high-intensity endurance training group (Interval group): The interval training protocol included three parts: warm-up, main training (high-intensity and low-intensity training), and cooling down. The warm-up consisted of five minutes of running on the treadmill with an intensity of ۴۰ to ۵۰% of the maximum speed. Interval training consists of a combination of high-intensity training and low-intensity training. High-intensity training, including two minutes of running at ۸۰% maximum intensity in the first week, ۹۰% of the maximum speed in the second week, ۱۰۰% of the maximum speed in the third week, and ۱۱۰% of the maximum speed from the beginning of the fourth week to the end of the training period. Low-intensity endurance training consisted of two minutes of running with an intensity of ۴۰% of the maximum speed in the first three weeks and ۳۰% from the beginning of the fourth week to the end of the training period. Moderate training was such that after warming up, the rats first performed high-intensity training and then low-intensity training. After performing the last high-intensity training, the rats cooled down for five minutes with an intensity of ۵۰% of the maximum speed. The number of high-intensity training was determined according to the training week of the rats; in the first week, two repetitions were done. In the second week, four repetitions. In the third week, six repetitions were performed, and from the beginning of the fourth week onwards, eight repetitions were performed. Therefore, the total time of high-intensity and low-intensity training with warm-up and cool-down was ۱۶ minutes in the first week, ۲۴ minutes in the second week, ۳۲ minutes in the third week, and ۴۰ minutes from the beginning of the fourth week. They also cooled down with an intensity of ۴۰ to ۵۰% of the maximum speed [۱۸]. The training protocol of the moderate intensity endurance training group (continuous training): In this group, the rats first warmed up for five minutes with an intensity of ۴۰ to ۵۰% of the maximum speed on the treadmill. Then, in the first week, with ۶۰% of the maximum speed and ۶۵% of the maximum speed in the second week, they did continuous training with ۷۰% of the maximum speed from the third week onwards. The running distance of the rats in the continuous training group was equal to the training distance of the moderate group. Therefore, the training time at the intensity of ۷۰% of the maximum speed was calculated according to the displacement value of the interval training protocol (without calculating the displacement during the warm-up and cool-down level). Ultimately, the rats cooled down for five minutes with an intensity of ۴۰ to ۵۰% of the maximum speed [۱۹]. Seventy-two hours after the last training session, rats were anesthetized by intraperitoneal injection of ketamine (۳۰–۵۰ mg/kg) and xylazine (۱۰ mg/kg). Approximately six cc of blood was collected from each rat directly from the heart and poured into normal tubes for serum separation. It was done by centrifugation at ۳۰۰ rpm for ۱۵ minutes, and the separated serums were kept at minus ۸۰ degrees. APO-A serum concentration by ELISA method and using Eastbiopharm ELISA kit (Cat No: CK-E۳۰۴۰۶) (Rat apoproteinA۱ (APO-A۱) ELISA Kit) and HDL-C serum concentration by ELISA method and using the kit (Rat High-Density Lipoprotein Cholesterol (HDL-C) ELISA Kit) (Cat No: CK-E۹۱۵۰۰) both manufactured by EASTBIOPHARM USA was measured according to the manufacturer’s protocol. Ethical Permissions: This research was registered with the ethics code IR.IAU.B.REC.۱۳۹۶.۵ in the ethics system of Shahrekord University in Iran. All the rules and how to treat the animals (introduction, training, anesthesia, and killing of the animal) were done according to the International Evaluation and Accreditation Association for the care of laboratory animals and with the approval of the Ethics Committee of the Research and Graduate Education Vice-Chancellor of Shahrekord University in Iran. Statistical analysis: The current research was conducted with SPSS ۲۲ software, and the inferential statistical method of one-way ANOVA was used at the significance level (p≤۰.۰۵). FINDINGS The results of the present study showed that the level of HDL increased significantly in both training methods in all four groups compared to the control group (p≤۰.۰۵). LDL level and LDL/HDL ratio decreased significantly in all four training groups compared to the control group (p≤۰.۰۵). TC level was significantly reduced only in the high-intensity endurance training group (۷۱.۲۵±۶.۳۲) compared to the control group (۸۸.۳۷±۷.۵۵) (p≤۰.۰۵). Even though both training methods led to changes in the levels of TG, VLDL, ApoA۱, ApoB, and ApoB/ApoA۱ ratio, the amount of these changes in both training methods compared to the control group was not statistically significant (p>۰.۰۵). Also, the difference between the two intensities in both training methods was not statistically significant in any of the measured factors (p>۰.۰۵). In addition, even though the interval training method compared to the resistance training method had a more positive effect on all the measured factors, the difference between the two training methods was not statistically significant in any of the mentioned factors (Table ۱). DISCUSSION This study aimed to compare the effect of two different training methods (continuous training and resistance training) with two different intensities (moderate and high intensity) on the predictors of cardiovascular diseases in older adults. Cardiovascular disease (CVD) rates increase with age and older adults. In this age group, the cause of more than ۴۰% of deaths is cardiovascular diseases, and they rank first among the factors that lead to the death of the elderly [۱۶]. Fortunately, it is possible to prevent people from getting CVD by controlling and managing the factors affecting CVD, such as blood pressure, lipids, glucose, and lifestyle changes such as continuous sports activities. Therefore, understanding the factors affecting health in old age is very important [۲۰]. The treatment of lipid disorders in older adults requires two approaches: lifestyle changes and drug use. To adjust the LDL level, most patients should use both approaches simultaneously [۱]. Studies have shown that compared to LDL and TG, HDL is more sensitive to training and changes more after it. Also, these changes in human samples are more than in rats [۲۱]. The effect of sports activities on HDL in human samples has been different in different studies. These differences may be due to the samples’ food habits and living conditions [۲۰]. The present study showed that training methods positively affected the measured factors and led to positive changes. It was also observed that interval training had a greater effect on all measured variables than resistance training; However, the difference between the two training methods was not statistically significant (p>۰.۰۵). The present study’s findings align with the results of Hannan et al. [۲۱], who reported that HIIT training is more effective than MIT training. Rahmati Ahmadabad et al. [۱۰] also investigated the effect of HIIT and MIT training methods on obese male rats. They reported that the difference between the two training methods was significant and that high-intensity endurance training compared to moderate-intensity endurance training resulted in a greater effect. It affects the cardiovascular health of the samples. The difference between their results and the present study is probably due to the type of samples studied (male obese rats). Fisher et al. [۲۲] reported that six weeks of HIIT training resulted in greater reductions in blood lipids compared to MIT training. Also, they have observed that both training methods improve TC, LDL, HDL, and TG levels. Therefore, they have suggested that both training methods can improve factors influencing the occurrence of cardiovascular diseases in sedentary, overweight, or obese young people. It should be noted that the samples of the present study were elderly and healthy, but those of the study, as mentioned earlier, were young and overweight. O’Donovan et al. [۲۳] have investigated the effect of training intensity on lipid changes, showing that greater intensity leads to greater changes in blood lipid levels. Overall, studies have suggested that high-intensity training has greater effects on samples’ lipid profiles [۲۳]. The level of LDL-C in the fasting state is related to the increase in coronary artery disease [۲۴]. Unlike HDL-C, the findings of studies on the effect of sports activities on LDL-C are contradictory and different. These differences may be due to the difference in the samples’ weight. Some studies have shown that performing sports activities does not change fasting LDL-C levels, although the samples’ weight changed during the training protocol. In contrast, some studies have shown that in patients with mild to moderate levels of lipid disorders, no significant change in LDL-C level is observed after several months of continuous training [۲۴]. ApoB apoprotein is the most important part of LDL particles, and it is necessary to remove LDL particles from the bloodstream. About ۹۵% of ApoB particles bind to LDL, and each LDL particle binds to only one ApoB molecule. ApoB is a more reliable predictor of circulating LDL particles. It is also a more reliable predictor of cardiovascular disease risk than LDL-C. Therefore, the ApoB concentration indirectly reflects the LDL-C concentration to some extent. An increase in ApoB levels can indicate an increased risk of a person suffering from cardiovascular diseases [۲۵]. The effect of continuous training on ApoB still needs to be better understood. Crouse et al. [۲۶] reported that ApoB concentrations decreased in men with high cholesterol levels after several months of continuous training. Leon et al. [۲۷] observed that ۲۰ weeks of continuous training did not affect ApoB concentrations. Angelopoulos et al. [۲۸] observed that long-term (۴۸ weeks) and short-term (three weeks) training did not affect ApoB concentration. The effect of continuous training on TG has been different in different studies. Some studies have reported that training can lower TG concentrations.   Others have shown that after a training session, there was no change in the TG level of sedentary people [۲۴]. The cause of these contradictions is the difference between samples regarding factors such as body weight, body fat, cardiovascular system health, sports history, nutritional changes, and genetic factors [۲۴]. Sheikh al-Islami Vatani et al. [۲۹] investigated different intensities of resistance training on fat indices after six weeks of training in healthy men. ۳۰ healthy men were randomly divided into two groups (the first group was moderate intensity training, which consisted of ۴۵-۵۰% of a maximum repetition, and the second group was intense resistance training with ۸۰-۹۰% of a maximum repetition). Both groups have shown a significant decrease after training, but only in the group with high intensity a significant increase in HDL has been observed. Fett et al. [۳۰] have studied the effect of ۱۲ weeks and three sessions of resistance and continuous training every week; continuous training with an intensity of ۶۰ to ۸۰% of reserve heart rate and resistance training with ۱۲ to ۱۵ repetitions have been performed. The duration of training in each session for both resistance and continuous training was ۳۰ minutes. Finally, the results of both groups showed a significant decrease in total cholesterol and triglycerides. Also, according to the report of some studies, the reason for this lack of change is probably related to the intensity of training because it is reported that training with an intensity of ۷۵% of MHR leads to a significant change in HDL-C level. However, when the same training was performed with an intensity of ۶۵% MHR, no significant increase in HDL-C level was observed [۳۰]. In the results of the first longitudinal study, changes in lipid and lipoprotein levels in men and women after ۱۶ weeks of strength training showed a decrease of -۹.۵% in cholesterol, -۱۷.۹% in LDL-C, and -۳.۳% in TG concentration. It has decreased by ۲۸%. Also, the ratio of LDL-C to HDL-C has decreased by -۲۰.۳%. In men, LDL-C decreased by ۱۶.۲%, the ratio of TC to HDL-C decreased by ۲۱.۶%, and LDL-C to HDL-C decreased by ۲۸.۹% [۱۱]. Studies have shown that many factors such as age, gender, diet, type of sample (human or animal and even the type of animal studied), history of diseases such as diabetes, obesity, and overweight, and even the race of the samples, as well as the type, severity, the volume and duration of the training period, are all factors that affect the results of the studies. In the studies that have examined the effect of continuous training, the minimum duration of the training period is two weeks. Finally, studies have reported that the training period of less than four weeks cannot lead to significant changes in the lipid profile and the influencing factors in the reverse transition cycle. Therefore, for the training protocol to be effective, the training period should be considered five weeks or more to affect the effective factors in the cycle of reverse transfer of cholesterol [۷]. In a study conducted by Rahmati et al. [۷] on an animal model, the effect of high-intensity endurance training on reverse cholesterol transport was studied in ۲۰ male Wistar rats. An increase in HDL has been observed after the end of the training period. In addition, they observed that high-intensity endurance training (۱۸ minutes) and moderate-intensity continuous training (one hour) significantly increased ApoA۱ in samples fed a high-fat diet. Considering that ApoB/ApoA is a suitable index for predicting cardiovascular diseases, the recommended value for this ratio is ۰.۵ or less. Increased ApoA۱ levels can increase HDL levels, and ApoB is also required for chylomicron and VLDL production [۱۹]. Studies with similar intensity on rodents have reported different results by changing the type of sample (mice or rats). Therefore, changing the type of study samples regarding gender, age, and other variables can lead to different findings. Also, the studies of the influencing factors on the results of the studies have mentioned the sex of the sample, the type of training protocol (resistance, endurance, or combined), the average age of the samples, the number of training sessions per week and the duration of the training protocol [۷]. The current research was also associated with limitations, such as lack of accurate control of the effect of possible stress on the rats during the implementation of the training protocol, lack of control of individual differences between rats, the effect of possible problems and injuries during the training period on the samples’ performance, lack of measurement of the effect of training protocols on compound rats. Future studies should investigate the effect of longer training periods but with the same training protocol to show the effects of two training methods and two intensities. Also, it is suggested that due to the influence of the gender, age, and type of subjects on the study results, similar studies should be conducted with both male and female ras at different ages and also in humans (male and female and middle-aged and old). CONCLUSION Overall, this research shows that both medium and high-intensity endurance and resistance training methods reduce cardiovascular disease risk factors and create positive changes in the lipid profile of elderly samples. Therefore, these two training methods can be recommended as an effective and suitable treatment method for changing the lipid profile of the elderly and people at risk due to lipid disorders. If they are performed under the supervision of a doctor, they can be used as an auxiliary method to reduce drug consumption, which he advised the older adults. Clinical & Practical Tips in POLICE MEDICINE: Considering that the physical fitness of the military and police is dependent on the cardiovascular health of these people and considering that both endurance and resistance training methods caused positive changes in HDL, LDL factors, and LDL/HDL ratio, to the military forces, it is recommended that in order to maintain general health and cardiovascular fitness, at least three to five sessions per week and each session for at least ۳۰ minutes, one of the two endurance or resistance training methods should be performed as desired. However, if they have access to a healthy environment and away from polluted air, they will gain more benefits by performing interval training. Acknowledgments: This research was taken from the first author’s doctoral dissertation, which was carried out under the supervision of supervisors and advisors. Therefore, we thank all the respected professors and laboratory officials of Shahrekord University in Iran who helped us conduct this study. Conflict of interest: The article’s authors stated that the present study has no conflict of interest. Authors’ Contribution: first author, ideation, study design, data collection, and data analysis; second author, study design, data collection; third and fourth authors, study design, data analysis; Fifth author, data analysis. All authors have participated in the initial writing of the article and its revision, and all of them accept the responsibility for the accuracy and correctness of the article’s contents with the final approval of this article. Financial Sources: This study had no financial support.

Keywords:

Aging , Cardiovascular diseases , Atherosclerosis , Exercise , Resistance Training , Resistance Training , Wistar Rats , سالمندی , بیماری های قلبی-عروقی , آترواسکلروز , تمرینات ورزشی , تمرین مقاومتی , تمرین استقاومتی , موش