Neuroprotective Benefits Of Exercise And MitoQ On Memory Function, Mitochondrial Dynamics, Oxidative Stress, And Neuroinflammation in D-Galactose-Induced Aging Rats Part 1

Abstract: 

Exercise and antioxidants have health benefits that improve cognitive impairment and may act synergistically. In this study, we examined the effects of treadmill exercise (TE) and mitochondria-targeted antioxidant menaquinone (MitoQ), individually or combined, on learning and memory, mitochondrial dynamics, NADPH oxidase activity, and neuroinflammation and antioxidant activity in the hippocampus of D-galactose-induced aging rats. 

Antioxidants are substances that fight free radicals. They help the body prevent cell oxidation. Cell oxidation may lead to various diseases, including Alzheimer's disease.

As the trend of population aging intensifies, more and more people have become interested in ways to fight Alzheimer's disease. Modern medical research has found that antioxidants can help people maintain good memory.

Studies have shown that regular consumption of foods containing antioxidants, such as strawberries, blueberries, olive oil, etc., can effectively reduce the risk of Alzheimer's disease. At the same time, the body's antioxidant system can be enhanced through proper exercise, regular life, and a balanced diet, which can also effectively protect our brains.

In addition to helping maintain memory, antioxidants have many other benefits. For example, they can improve skin conditions, reduce the risk of cardiovascular disease, and so on. Therefore, proper intake of foods containing antioxidants and maintaining a healthy lifestyle benefits our body and brain.

In short, antioxidants are inseparable from memory. By paying attention to our diet and lifestyle, we can effectively prevent Alzheimer's disease while maintaining good physical and mental health. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the level of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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TE alone and TE combined with MitoQ in aging rats reduced mitochondrial fission factors (Drp1, Fis1) and increased mitochondrial fusion factors (Mfn1, Mfn2, Opa1). 

These groups also exhibited improved NADPH oxidase activity and antioxidant activity (SOD-2, catalase). TE or MitoQ alone decreased neuroinflammatory response (COX-2, TNF-α), but the suppression was greater with their combination. 

In addition, aging-increased neuroinflammation in the dentate gyrus was decreased in TE but not in MitoQ treatment. 

Learning and memory tests showed that, contrarily, MitoQ alone demonstrated some similar effects to TE but not a definitive improvement. 

In conclusion, this study demonstrated that MitoQ exerted some positive effects on aging when used as an isolated treatment, but TE had a more effective role in cognitive impairment, oxidative stress, inflammation, and mitochondria dysfunction. 

Our findings suggest that the combination of TE and MitoQ exerted no synergistic effects and indicated regular exercise should be the priority in neuroprotection of age-related cognitive decline.

Keywords: aging; treadmill exercise; MitoQ; NADPH oxidase; mitochondrial dynamics; neuroinflammation.

1. Introduction

Aging is an inevitable process of physical and functional decline, including a gradual decrease in cognitive ability, such as reduced memory and spatial ability [1]. 

Age-related changes in the brain increase vulnerability to various diseases and ultimately result in a lower quality of life. Successful aging generally refers to the absence of disease or impairment, preservation of physical and cognitive function, and continued participation in social and productive activities [2]. Maintaining one's dignity and value as a human being is crucial, even during the aging process. 

Among several identified lifestyle changes for preventing cognitive decline with aging, recent attention has turned towards non-pharmacological treatment, such as physical activity and dietary supplements, due to the lack of pharmacological effects and the rising cost of conventional medicine. Indeed, a combination of exercise and diet with antioxidant supplementation, such as epicatechin and astaxanthin, has been shown to induce synergistic effects on the hippocampus-dependent function [3,4]. 

Contrarily, several studies have revealed that exercise alone, but not in combination with dietary changes, improves learning and memory functions [5,6], raising the question of whether a combined intervention could have greater effects on cognitive function than exercise alone. 

Although the possible mechanisms underlying exercise and/or antioxidant treatment hippocampal function involve neuronal plasticity and its related molecular levels with neurotropic factors [4,7], mechanisms, such as mitochondrial dynamics and neuro-immune system, are not fully understood. 

Mitochondria are dynamic intracellular organelles responsible for biological oxidation in most eukaryotic cells, whose dynamics are regulated by repeated processes of fusion (elongation of the mitochondria) and fission (fragmentation of the mitochondria) [8,9]. 

Several fusion- and fission-related proteins are involved in mitochondrial dynamics, which are essential for maintaining normal mitochondria function [10,11]. 

However, irregular expression of the proteins can lead to abnormal changes in mitochondria. In particular, mitochondrial dysfunction is a feature of aging [12], and persistent inflammation, oxidative stress, and mitochondrial dysfunction contribute to reduced brain function by accelerating brain aging [13,14]. 

In addition, aging is accompanied by a low level of chronic inflammation in the immune system and central nervous system (CNS), which is known to contribute to many age-related diseases [15,16]. 

Neuroinflammation in the CNS progresses when glial cells, such as microglia and astrocytes, become persistently activated in response to oxidative stress [17]. 

Reactive oxygen species (ROS) are generated by NADPH oxidase (NOX), which is a membrane-bound enzyme complex. There are several subtypes of NOX, among which NOX2 is expressed in various cell types, including neurons and endothelial cells, and is expressed at high levels in microglial cells involved in the immune response and inflammation [18–20]. 

NOX2 comprises both membrane (gp91phox, p22phox) and cytosolic (p47phox, p67phox) subunits. Elevated ROS levels due to NOX activity promote inflammation, activating glial cells, which in turn further enhance the secretion of proinflammatory cytokines, oxidative stress, and free radical injury, ultimately leading to neuronal cell death [21–23]. 

Brain aging plays an important role in cognitive impairment and is closely related to neurodegenerative disorders. Although it is impossible to prevent the general decline in brain function that occurs during natural aging, physical activity and the consumption of antioxidants are useful as noninvasive methods to maintain brain function, especially cognitive function. 

In particular, exercise reportedly ameliorates brain dysfunction by promoting neuroplasticity, improving metabolic efficiency, and increasing tolerance to oxidative stress [24,25]. 

However, the molecular mechanisms responsible for enhancing cognitive function or preventing its decline remain unknown. In terms of antioxidants, the mitochondrial-targeted antioxidant mitoquinone (MitoQ) is known to cross the inner mitochondrial membrane and accumulate within mitochondria, unlike other antioxidants, to reduce ROS levels generated in mitochondria directly [26]. 

Although few studies have been conducted on MitoQ, various reports describe its antioxidative effects. Studies by Gioscia-Ryan et al. [27] and Rossman et al. [28] demonstrated that MitoQ reduced ROS levels and increased NO production in the mitochondria of aged blood vessels. 

Vergeade et al. [29] and Braakhuis et al. [30] reported that MitoQ maintained the activities of antioxidant enzymes, such as SOD-2, catalase, and GPx, during the aging process. 

Regarding brain aging, further research is warranted to elucidate the molecular mechanisms responsible for the positive effects of exercise and MitoQ on declining brain function. 

Until now, aging research using several animal models has investigated the underlying mechanisms of brain aging. In particular, D-galactose (D-gal) administration reportedly causes aging in animals that resembles human aging, including memory loss, neurodegeneration, changes in biochemical markers of oxidative stress, decreased immune activity, and abnormal regulation of gene expression [31]. 

Rodents administered D-gal show a gradual decline in learning and memory, increased production of free radicals in the brain, impaired calcium homeostasis, and mitochondrial dysfunction [32,33], thus providing an animal model of brain aging [34]. 

Therefore, using the D-galactose-induced aging rat model, we investigated the effects of treadmill exercise (TE) and MitoQ, as independent or combined treatments, on mitochondrial fission and fusion, inflammation, and antioxidant activity, as well as hippocampus-dependent cognition. 

We hypothesized that an 8-week combined treadmill exercise and MitoQ in D-galactose-treated rats would either additively or synergistically improve the decline of learning and memory function and induce beneficial changes in the levels of proteins involved in hippocampal neuroprotection.

2. Materials and Methods

2.1. Experimental Animals

Six-week-old male Sprague-Dawley (SD) rats were obtained from JA Bio (Gyeonggi-do, Korea). The rats were reared in the Korea National Sports University Animal Laboratory (22 ± 2 ◦C, 50% ± 5% humidity, and 12/12 h light/dark cycle). 

Food and drinking water were provided ad libitum. The rats were divided into 5 groups: young control group (Y-CON, n = 12), D-galactose group (D-CON, n = 12), D-galactose plus TE group (D-TE, n = 12), D-galactose plus MitoQ group (D-MI, n = 12), and D-galactose plus TE and MitoQ group (D-COMBI, n = 12). Rats were euthanized after completing 8 weeks of TE and memory behavior tests by CO2 inhalation using a euthanasia chamber. 

The study protocol received approval from the Korea National Sports University Institutional Animal Care and Use Committee (KNSU-IACUC-2018-05).

2.2. Drug Administration

The method described by Lei et al. [31] was employed to induce the aging of the experimental animals. Specifically, D-gal (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline, and a 100 mg/kg dose was administered by intraperitoneal (IP) injection once per week for 10 weeks. 

The feeding method suggested by Smith and Murphy [26] was adapted for the administration of MitoQ in this experiment. 

MitoQ was mixed with sterile saline for a final dilution of 0.1 mM/mL. D-galactose-induced aging rats were then injected intraperitoneally with 100 µM/kg MitoQ (twice per week) for 8 weeks.

2.3. Treadmill Exercise (TE)

The D-TE and D-COMBI groups were subjected to a progressive loading exercise program using a rodent treadmill (8 Lanes, Daemyung Scientific Co, Ltd., Seoul, Korea). 

The animals first performed acclimation training for 1 week (2 m/min for the first 5 min, 5 m/min for the next 5 min, and 8 m/min for the last 20 min). 

The main exercise program was performed 5 days per week over the next 8 weeks. This program was designed as follows, concerning the progressive exercise program suggested by Hong et al. [35]: 10–12 m/min for 10 min in Week 1; 10–12 m/min for 20 min in Week 2; 18–20 m/min for 20 min in Week 3; 18–20 m/min for 30 min in Week 4; and 18–20 m/min for 50 min in Weeks 5–8. The incline of the treadmill was fixed at 0%.

2.4. Passive Avoidance Task

The passive avoidance task is a fear-motivated test used to measure the working memory ability of small laboratory animals. In the present study, the passive avoidance task was performed 3 days before the end of the TE program. 

The apparatus for the passive avoidance task consisted of two chambers: The front chamber was a well-lit, bright, white box (18 × 18 × 25 cm3 ), connected to the rear chamber, which was a dark black box (18 × 18 × 25 cm3 ). 

The rear chamber had a stainless-steel floor capable of delivering electric shocks. The wall between the two chambers had a guillotine-style door that could be opened and closed. 

The time taken for the animal to enter the rear chamber via the front chamber was measured (initial latency time), and then an electric shock (0.5 mA) was delivered for 2 s. 

The animal was removed after 5 seconds and returned to its rearing cage. The task was repeated 72 h later using an identical method, and the time for the animal to enter the rear chamber via the front chamber was recorded (retention latency time) up to a maximum of 300 s.

2.5. Morris Water Maze Test

The Morris water maze test is used to assess the spatial learning and memory of rodents. The test was performed in a room with controlled temperature (21–23 ◦C), humidity (50–60%), and lighting using a circular water tank (diameter 120 cm × height 15 cm) filled with water (20–24 ◦C). 

Skim milk powder was added to the water to obscure the escape platform (10 cm diameter), which was placed on the floor of the tank approximately 1 cm below the water surface. 

The distance traveled by the animal before reaching the platform (escape distance) and time taken to reach the platform (escape latency time) were measured before and after the TE program using EthoVision XT8 video tracking software (Noldus, Wageningen, The Netherlands) and a camera installed on the ceiling above the center of the tank. The water tank was divided into quarters labeled Zones 1–4, and the platform was placed in Zone 1. 

For each treatment group, the water maze test was performed 5 days per week. The rat always started in the same position and was allowed two 60-s practice attempts to try to reach the platform, which was always in the same location. 

After 48 hours of rest, the platform was removed on the seventh day; escape distance and escape latency time were measured for 60 seconds with the rat starting at the same position.

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