Effects of astaxanthin supplement and acute high-intensity exercise on liver injury in rats. The enzymes that catalyze transaminase during metabolism and regulate protein synthesis-breakdown are called transaminases, widely existing in various tissues or cells, especially in hepatocytes. For instance, ALT, AST, lactate dehydrogenase, glutamyl transferase, and alkaline phosphatase are major enzymes. When the body’s metabolism is disturbed due to inflammation, drug toxicity, or other factors, transaminase enzymes will be released from cells into the blood, and serum transaminases will be enhanced subsequently. Therefore, the levels of blood aminotransferase are a very sensitive reflection of the liver function in clinical practice.
ALT, also known as glutamic pyruvic transaminase (GPT), mainly exists in the cytoplasm of hepatocytes in particular with the function of catalyzing the transamination of glutamate and alanine. Once the hepatocytes suffer from necrosis, the permeability of the hepatocyte membrane is altered, resulting in an immediate spill of intracellular ALT from the cytoplasm into the circulating blood where the levels and activity of serum ALT increase rapidly. Serum ALT levels are highly sensitive for evaluating the liver injury, and the World Health Organization also regards it as the common biomarker for liver damage [15]. AST, also known as glutamic oxaloacetic transaminase (GOT), is mainly present in the mitochondria of hepatocytes, where the transamination of glutamate and oxaloacetic acid was catalyzed. The liver is the crucial organ of detoxification and metabolism, and the levels of ALT or AST usually assist in detecting the metabolic capability and physiological state of the liver thus reflecting the different degrees of liver injury. Excessive contents of circulating ALT and AST often indicate that the membrane of hepatocytes has been damaged, resulting in hepatic breakdown, even to the level of organelles.
High activity of ALT and AST are used to predict oxidative stress and liver-related diseases [16]. Significant increases in AST and ALT levels which further caused organ damage were observed in mice serum during acute exhaustive exercise as determined by Ruhee’s study [17]. Similarly, Ding Yu’s research also demonstrated that serum AST and ALT contents in weight-loaded exhaustive swimming mice were much higher than those in quiet mice [18], which would increase the expression of tumor necrosis factor and nitric oxide synthase in liver tissues, closely related to serious liver injuries [19]. Rats with increased serum ALT were prone to hepatocyte membrane edema, disturbance or even loss of mitochondrial cristae membrane arrangement, and breakdown of rough endoplasmic reticulum arrangement [20]. our experimental results showed the activity of rat’s liver function indexes such as plasma ALT and AST in group E exceeded those in group C obviously (P < 0 01), indicating that acute high-intensity exercise might be the inescapable reason that resulted in serious damage to rats’ liver through accelerating the release of large amounts of ALT and AST from hepatocytes into the blood.
Exogenous drugs need to be metabolized through the liver to maintain the balance of internal and external circulation, as the liver is the largest digestive gland in the human body. However, the liver is more vulnerable to damage induced by some drug toxicity, which is prone to result in hepatocellular apoptosis or necrosis during the metabolism process, thus releasing ALT and AST into the blood. This is the reason for regular clinical testing of liver function in patients with long-term medication. In the results of our study, there was no significant change in the plasma ALT and AST activities between group M and group C, showing rats did not have any adverse reactions after ASTA gavage, and the liver function indexes were within the normal range, which proved the safety and no hepatotoxicity of ASTA. Oppositely, it has been reported that ASTA could accumulate in rat liver to exert its hepatoprotective effect in response to hepatocellular oxidative stress or apoptosis under specific environmental stimuli [21]. ASTA not only enhances the antioxidant system activity of the liver substance but also can alleviate liver injury by other biological mechanisms alone [22]. MA’s experiments confirmed that ASTA supplementation is beneficial for the process of chemotherapeutic drug-induced liver injury, and liver function index, ALT, and AST were reduced by ASTA intervention [23]. Here, results performed in our study showed the plasma ALT activity in group EM was significantly reduced compared to that in group E, but not to the level of group C, this condition is presumed that the unique polar structure of ASTA allows it to penetrate the cell membrane and exert a certain repairing effect on the hepatocyte membrane. However, there was no obvious change in AST activity, probably because the ASTA supplementation at this dose did not reach the effective threshold.
Effects of astaxanthin supplement and acute high-intensity exercise on liver oxidative stress in rats. The overproduction of free radicals caused by internal or external stress stimulation, along with weakened antioxidant enzymes, will boost oxidative stress and break the homeostasis of hepatocytes thereby inducing liver injury. Malondialdehyde (MDA) is one of the end-products of lipid peroxidation. When MDA is produced and released from the membrane, it will loosen the intermolecular bridge bonds of fibrin, inhibit protein synthesis, hinder the mitochondrial respiratory chain-activating enzymes and exacerbate membrane damage, which finally affects the normal progress of a series of physiological and biochemical reactions. Therefore, the degree of membrane lipid peroxidation could be detected by the assessment of MDA contents which can be used as an indirect indicator of the severity of cell damage.
A complex antioxidant defense network exists in the body where Endogenic enzymatic and non-enzymatic antioxidants act together to neutralize, scavenge, and repair the detrimental effects caused by ROS. Superoxide dismutase (SOD), one of the endogenic enzymatic antioxidants, is a component of the first-line defense system, and its significance in mediating the adaptive responses to various changes has been reported [24]. SOD can react in combination with CAT and GSH-Px to trigger bodily resistance against oxidative damage via antioxidant pathways. On the other hand, it also competes with NO for superoxide anion, indirectly reducing the levels of peroxynitrite generated by the reaction of NO with superoxide anion and increasing the bioavailability of NO [25].
Glutathione (GSH) is not only an intracellular metabolic regulator but also a non-enzymatic antioxidant. It is widely distributed in the cytoplasm, nucleus, and mitochondria along with the antioxidant and detoxifying effects. The biosynthesis of GSH is a symbiotic process, of which the liver is the main site of its synthesis [26]. It acts as an electron donor for GSH-Px to protect the body from the attack of harmful substances by decomposing H2O2 [27]. Most of the enzymatic antioxidant activity is dependent on the continuous regeneration of GSH, so it plays a critical role in the overall defense mechanism of the biological system.
A dramatic increase in ROS during high-intensity and exhaustive exercise will break above protective mechanisms, elevate oxidative damage biomarkers and lower the levels of various endogenous antioxidants. Oxidative stress indexes of athletes have been examined in a variety of exercise models including continuous running exercise (25min, 75% VO2max), 50 min intermittent exercise (15 s running and 15 s rest) as well as 50 min intermittent exercise (30 s running and 30 s rest) in Wajdi’s study where the obvious signs of plasma MDA elevation during intermittent exercise with 30s break were observed whereas there was no manifest change during intermittent exercise with 15s break. Furthermore, continuous exercise was demonstrated to increase blood SOD by 13.9%, and intermittent 30 s exercise decreased SOD contents by 19.7%, while blood SOD remained unchanged during intermittent 15 s exercise [28]. Xiaodong reported significant increases in SOD activity but decreases in GSH-Px activity in rats' serum after 3 weeks of incremental load treadmill exercise [29], whereas another rat experiment was distinguishing that one-time exhaustive treadmill exercise could notably reduce liver GSH levels, serum SOD and GSH-Px activities yet lead to significant increases of MDA contents [30] [31]. In our study, the plasma MDA levels increased significantly and GSH contents decreased significantly in group E, indicating that acute high-intensity exercise likely caused membrane lipid peroxidation and glutathione would resist the excessive oxidative stress damage as the electron door of enzyme antioxidants. However, there was no significant change in plasma SOD activity, speculating that the SOD activity of the body was sufficient enough to cope with the oxidative stress induced by exercise in this experiment. Handful studies have displayed that high-intensity strenuous exercise promotes the production of cytotoxicity by incrementing MDA over-accumulation immediately after exercise yet GSH contents, as well as antioxidant capacity of the body against oxidative stress, are inversely descendent [32]. Pedro [33] pointed out that after maximal and sub-maximal long-term exercise tests for runners or cyclists, the activity of GSH-dependent antioxidant enzymes in neutrophils decreased as well, but plasma CAT and SOD levels were basically unchanged. Correspondingly, the excessive release of by-product because of oxidative stress will expose organism to inflammatory cytokines, and then trigger chronically secondary diseases. For example, exercise-induced oxidative stress will cause severe damage to the rat liver.
Continuous oxidative stress usually leads to the overgeneration of ROS and organ degeneration, which are the main reason for long-term hepatocellular damage characterized by liver fibrosis, and this situation will gradually develop into liver cirrhosis. At this moment, natural products with exogenous antioxidants should be widely concerned to keep the ordered physiological function of cells and ameliorate impaired apparatus performance. Among them, ASTA is the most potential therapeutic agent for treating diseases related to oxidative dysfunction and protecting a variety of organs including the liver from cytotoxicity [34]. ASTA therapies could enhance mitochondrial efficiency to deal with oxidative stress, which is primarily manifested in the increase of mitochondrial quantity and the improvement of energy metabolism in the respiratory chain [35]. In addition, ASTA was able to scavenge free radicals and increase antioxidant responses timely to block the conversion of peroxidation to toxic or harmful substances [36]. For the sake of exploring the relationship between ASTA and oxidative stress, we examined relevant indicators contents, and from experimental results, we had drawn the following conclusions: in group M, the serum MDA contents decreased significantly indicating the degree of lipid peroxidation was reduced, however, levels of antioxidant enzyme SOD showed a clear upward trend after astaxanthin intervention to maintain the integrity of hepatocytes. In terms of energy supply during exercise, mitochondria are the central spot of cellular energy metabolism and ROS production. Therefore, mitochondrial disorders are major contributors to accelerate the progression of diseases, and astaxanthin intervention could play a crucial role in keeping mitochondrial quality control under different pathological conditions [34]. Polotow [37] showed astaxanthin supplementation (1mg/kg BW, 45 days) is beneficial to delaying exercise fatigue during the swimming test through improved GSH contents and SOD activities in rats’ soleus muscle, while Astaxanthin could inhibit the production of MDA both in rats’ liver and blood after exercise and enhanced the activities of the antioxidant system including SOD and GSH-Px [8]. Our result showed plasma MDA contents in group EM is obviously lower than that in group E and after intragastric administration of astaxanthin in exercise rats, serum SOD activity increased significantly while there is almost no change in serum GSH contents indicating ASTA could upregulate endogenous enzymatic antioxidants but has less effect on non-enzymatic antioxidants GSH. Moreover, ASTA tends to resist the destructive effect of MDA, which could protect the structural and functional integrity of cells effectively.
Effects of astaxanthin supplement and acute high-intensity exercise on AMPK/Nrf2/HO-1 pathway in rats. Nrf2 is the key factor that regulates the translation of multitudinous proteins with an antioxidant effect. Increasing evidence demonstrated that Nrf2 will dissociate from Keap1 into the nucleus and combine with the ARE promoter to stimulate the expression of downstream antioxidants namely HO-1 when cells suffer from oxidative stress. Thus, the Nrf2/HO-1 signal axis is closely related to the process of antioxidation. As a signal molecule that senses intracellular energy changes, activation of AMPK plays a central role both in energy homeostasis and redox responses [38]. Some studies have shown in the occurrence of oxidative stress, ROS will promote AMPK phosphorylation so that it could enhance the expression of Nrf2, which exerts antioxidant effects through the activation of downstream antioxidant pathways [39] [40]. Clinical studies pointed out that the high expression of the AMPK/Nrf2 pathway is conducive to regulating mitochondrial function [41] and it will protect liver tissue from oxidative injuries [42].
Normally, the body is capable of activating a self-protection mechanism to scavenge ROS and achieve redox homeostasis autonomously. Results of Indexes examination in our experiment indicated the repair capacity of antioxidant enzymes is not sufficient enough against the side effects of acute high-intensity exercise-induced oxidative damage in rats' liver, but the mechanism explaining this finding is indistinct. For other exercise models, endurance exercise can upregulate AMPK to activate the Nrf2-mediated antioxidant pathway, whose mechanism is closely related to the high expression of AMPKα2 protein in skeletal muscle. Apart from that, ladder climbing or free spinning exercise could improve mitochondrial function by activating AMPK/Sirt1/PGC-1α signal pathways and activating Nrf2 protein to increase downstream antioxidant enzymes, which together exert protective effects on the myocardium. For the sake of exploring the potential role of the AMPK/Nrf2/HO-1 pathway in the antioxidant process, we found that compared with quiet rats, there was no significant change in the transcription of AMPKα1, AMPKα2, and downstream Nrf2 after exercise, an obvious upward trend in AMPKα phosphorylation and HO-1 transcription but a clear downward trend in GSK-3β phosphorylation and HO-1 protein expression, indicating high-intensity exercise could determine the activation of AMPK and the downstream antioxidant pathways regulated by AMPK to resist oxidative stress. While the HO-1 protein expression was downregulated, we speculate the reason is that the liver antioxidant capacity was insufficient to cope with the excessive release of ROS from the respiratory chain. AMPKα2 is thought to be most closely related to exercise. For example, the p-AMPKα level in the skeletal muscle of mice was upregulated after a one-time exhaustive exercise and tended to be selectively highly expressed in slow-twitch muscle fibers [43]. Consistent with previous research results, there is no significant difference between AMPK α1 and α2 during acute high-intensity exercise in our study, which provides a shred of evidence that acute high-intensity exercise not only altered energy metabolism but also induced oxidative stress.
ASTA could not only clear up free radicals directly but also improve cell survival by promoting AMPK phosphorylation and the expression of antioxidant-related proteins such as Nrf2 and HO-1 indirectly [44]. The inflammation and pathological changes in the cerebral infarction rat model will be alleviated with neuroprotective and anti-apoptotic functions of ASTA by activating the AMPK/eNOS/NF-κB signal pathway [13]. ASTA supplementation will activate the Nrf2-ARE pathway in rats with subarachnoid hemorrhage so that the expression of antioxidant enzymes is upregulated and the blood-brain barrier is repaired to reduce early brain injury [45]. Other exogenous antioxidants also have remarkable efficacy against oxidative stress through AMPK/Nrf2/HO-1 pathway, for instance, gentiopicrin and corosolic acid could strengthen the antioxidant defense system via the AMPKα1/Nrf2 pathway to maintain mitochondrial mass where oxidative stress is greatly stimulated [41] [46]. However, antioxidant supplements are regarded as a “double-edged sword” with two-sided effects. Despite helpful influences mentioned above, there may be few and even undesirable effects on the application of ASTA to the normal physiological body. Here, We observed higher levels of AMPKα phosphorylation and lower levels of GSK-3β phosphorylation after astaxanthin intervention than rats in group C, however, the transcription levels of AMPKa1 and AMPKα2 in group M had an opposite trend, indicating ASTA supplementation activated the AMPK-mediated pathway, which may be more related to AMPKα1 in liver tissue. AMPK phosphorylation also regulates the downstream Nrf2 protein which is extremely sensitive to free radicals and plays an important role in the defensive capacity of antioxidants and cytoprotective enzymes. In general, Nrf2 expression is low in quiet rats as Nrf2 binds 1:2 to Keap1 in the cytoplasm. In our study, Nrf2 and HO-1 transcription, as well as Nrf2 protein expression in group M, increased significantly, but HO-1 protein expression in group M decreased significantly. The reason might be that although ASTA activated the Nrf2 pathway through AMPKα1, it had an antagonistic effect on the expression of phase II detoxification enzymes in quiet rats, which was speculated to be related to the previous studies on the bidirectional regulation and biphasic expression of antioxidants [25]. Here, the liver of acute high-intensity rats with ASTA treatment could reestablish AMPKα1 and HO-1 transcription and enhance GSK-3β contents, but phosphorylated AMPKα remains low, indicating the AMPK-mediated pathway was inhibited. While the relative expression of Nrf2 and HO-1 protein tended to increase which indicated that the supplementation of exogenous astaxanthin was fighting against oxidative stress and repairing subsequent damage, this dose of astaxanthin might not be enough to resist the ROS explosion induced by acute high-intensity exercise in a short time. In general, astaxanthin supplementation may be cross-reactive with other pathways in the antioxidant defense mechanism of organisms mainly based on the AMPK/Nrf2/HO-1 signal axis.