Research On Anti-fatigue In Exhausted-swimming Mice By Medium-chain Triglycerides

BackgroundIn modern society, with the quicken pace of life, the stress at work is aggravated. It isusually considered that fatigue is one of chronic diseases eroding people’s health.Theoretically, fatigue is defined as a subjectively unpleasant feeling, objectively under thesame circumstances, losing the competence to perform normal activity and work. Forexample, the deterioration in exercise performance for athletes and lower workefficiency and more mistakes for regular people. Therefore, how to delay thehappenings of physical fatigue and to accelerate the recovery have become the hot pointof Space medicine, Military medicine and Sports medicine.There are many reasons accounted for fatigue, mainly including the depletion ofenergy substances, the accumulation of metabolic products, the disturbances ofneurotransmitters and the rapid production of reactive oxygen species. Accompanied bymany changes of some physiological and biochemical parameters, the recovery of physicalfatigue requires quickly damages-restoring in body or getting rid of the accumulatedmetabolic products as soon as possible. Currently, the nutritional supplements have beengenerally recognized for the benefits in the endurance exercises.Medium chain triglycerides(MCT) has6to10carbon atoms, more readily absorbedinto the intestinal lumen due to higher soluble in water, which is different from long chaintriglycerides(LCT). Then MCT can be directly transported into liver through the portalvein and won’t be stored in the adipose tissues owing to insensitivity to thehormone-sensitive lipase. Based on those characteristics, MCT has been applied to theexperimental researches for exercise training and health effect. However, there are fewreports about MCT as a source for energy supply during endurance exercise, mainly byincreasing the fatty acids β-oxidation, reducing the depletion of glycogen in liver, thenfurther prolonging the time for exercise. Recent researches have revealed that MCT has theanti-fatigue property during physical exercises. Whether MCT can be promoted as a kindof dietary oil to ameliorate physical fatigue or not remains disputable. There is still a lackof reliable and scientific evidence for the optimal dosage of MCT.Based on the latest research progresses of physical fatigue, the topic adopted theanimal experiments methods, by supplementing MCT into the diet instead of the lard indifferent proportion,6%,18%,30%and44.4%, respectively. After3weeks, we observed the effect of MCT on improving serum LDH and creatine kinase(CK), regulating the sugar-lipid metabolism in blood, liver and gastrocnemius muscle, and reducing the liveroxidative level. By using the real-time quantitative PCR technology, the regulativefunction of MCT for PPAR-α and PGC-1α expression was also further discussed. Besides,the expression of PPAR-α in liver and AMPK-α in gastrocnemius muscle were assessedtogether by using the Western blot technology. In a word, those researches provide newexperimental evidences and theoretical supports for the alleviating-fatigue effect of MCTin endurance exercises.Objectives1. To access the alleviating-fatigue effect of MCT in endurance exercises.2. To research the mechanism of MCT alleviating physical fatigue.3. To provide scientific evidence for the relationship between MCT and physicalfatigue.Methods1. Experimental animals and GroupsMale ICR mice in these studies were provided by Experimental Animal Center ofSecond Military Medical University. First, the mice were housed for an adaptive feeding of1week before the experiment. Second, they were randomized into twelve Groups based ontheir body weight, with6mice in each group. Third, the mice were marked from1to6inone cage. Altogether, there are six big Groups(12mice in each group): untreated controlGroups(g11and g12), untreated swimming group(g21and g22), MCT6%group(g31andg32), MCT18%group(g41and g42), MCT30%group(g51and g52), and MCT44.4%group(g61and g62). Each mouse was given4.5g/d experimental diet, and had free accessto water. At the same time, all Groups were subjected to unloaded swimming training onMonday, Wednesday and Friday in one week. The mice were trained for20min withsubsequent increase of10min each time until it reached40min. The experimental periodslast3weeks.Untreated control group (UCG) and untreated swimming group (USG) were fed withD12450B diet, without containing MCT. The protein, carbohydrate and fat contentscorresponded to20%,70%and10%of metabolizable energy. Both Groups had swimmingtraining during the experimental period. UCG were considered as the before-exercises statecompared with USG. All MCT Groups were given the modified D12450B dietssupplemented different dosages of MCT. Among all the components, lard was substituted in portion by MCT, which accounted for6%,18%,30%and44.4%of the total fat. AllMCT Groups were compared with USG during the experiment.2. Weight-loaded swimming exhaustive testSelect six mice in five swimming Groups to perform the weight-loaded swimmingexhaustive test. A lead block (4%of the body weight) was loaded on the tail root of themice. Then they (six mice/group) were placed in a swimmingpool(385mm×380mm×387mm) filled with fresh water maintaining at30±1℃，andapproximately35cm deep, so that the mice could not support themselves by touching thebottom with their tails. Three mice were placed in each group. The recording of theeffective swimming time was started after dropping the mice into the pool. The mice wereaccessed to be exhausted when their head were drawn into the water and failed to rise tothe surface of water to breathe within10seconds.3. Samples collectingAll the Groups were placed into the swimming pool(385mm×380mm×387mm) insequence. The temperature of water was maintained at30±1℃.The water was4cm deepfor UCG, while other Groups kept at35cm deep. After swimming for30min, the micewere got out of water and dried. Blood were collected by extirpating the left eyeballs, andserum was prepared by centrifugation at3000r/min4℃for10min. The liver andgastrocnemius muscle of each mouse was dissected out and timely washed with icyphysiologic saline. At last, they were frozen in lipid nitrogen and stored at-80℃until foranalysis.4. Biochemical detections(1) Biochemical parameters: levels of LDH, CK, GLU and LAC were determinedusing an auto analyzer (Hitachi7600-120, Japan). The content of glycogen and NEFA inliver and gastrocnemius muscle was measured by commercial kits respectively.(2) Antioxidant enzyme markers: to detect the level of GSH-Px, MDA and SOD inliver according to the commercially available kits.(3) mRNA expression detection: to access the effect of MCT on PPAR-α and PGC-1αgene expression in the gastrocnemius muscle by real-time fluorescent quantitative PCR.(4) Western blot technology was used to detect the effect of MCT on PPAR-α in liverand AMPK-α in gastrocnemius muscle expression.5. Statistical analysisAll values are expressed as x±sand statistical analysis is done by using SPSS16.0 software. Data in different Groups were analyzed by one-way ANOVA, and thendifferences among means were analyzed using the least significant differences (LSD)multi-comparison test. The level of P <0.05was used as the criterion of statisticalsignificance.Results1. MCT can prolong the exhaustive swimming time under6%and18%dosages(P<0.01), while the swimming time were extremely reduced in44.4%MCT group(P<0.01).2. Serum LDH and CK were reduced significantly in MCT18%group (P<0.05), theserum glucose and glycogen in liver and muscle were increased (P<0.01), whereas thecontent of hepatic NEFA were reduced and NEFA in muscle were augmented(P<0.05).Those results indicate that MCT can regulate sugar-lipid metabolism anddecrease the muscle damage during endurance exercises.3. The activity of GSH-Px and SOD in liver was enhanced (P<0.05), while the levelof MDA was decreased (P<0.05) in both6%and18%MCT Groups. Thus we can concludethat the hepatic antioxidant ability is strengthened.4. The fact that hepatic PPAR-α protein expression were increased significantly under6%and18%MCT Groups (P<0.05). Besides, the muscle metabolic regulators AMPKprotein, PGC-1α and PPAR-α mRNA expression were strengthened (P<0.05).Conclusions1. MCT has the anti-fatigue effect on mice under lower dosages (6%and18%).2. During the period of exercises, MCT can accelerate the fatty acids oxidative rate,regulate sugar-lipid metabolism, and decrease the oxidative stress level in liver.3. MCT is resistant to physical fatigue mainly by mobilizing the long-chain fatty acidsand enhancing those expression metabolic regulators relative to the mitochondrialbiosynthesis.