One of the key factors in skeletal muscle fatigue is the accumulation of reactive oxygen species (ROS) and other metabolites that result from their active contraction. Currently, exogenous antioxidants are commonly used to reduce muscle fatigue. The purpose of the study was to investigate the development of fatigue of rat skeletal muscle under the action of water-soluble С60 fullerene nanoparticles. Electrophysiological, biochemical methods, behavioral tests on the model of experimental hemiparkinsonism and methods of mathematical statistics were used to achieve this goal. The experimental work consisted of three main series: I – study of animals that were administered С60 fullerene aqueous colloidal solution (C60FAS) at a dose of 0.15 mg/kg during the development of skeletal muscle fatigue; II – study of animals that were administered C60FAS or N-acetylcysteine (NAC) or β-alanine (0.15 mg/kg dose) an hour before the electrical stimulation that caused fatigue; III – study on non-anesthetic animals (experimental hemiparkinsonism model) that were administered C60FAS or NAC (0.15 mg/kg dose), prior to an hour before chemical stimulation, caused intense circulatory movements and, as a consequence, led to muscle fatigue.
To cause muscle fatigue of the three-headed calf muscle (i. e. gastrocnemius - soleus), rats were administered 3 and 5 series of high-frequency electrical stimulation for 30 min, separated by 15-min rest intervals. Fatigue was considered a decrease in the level of effort that was developed by the muscle by 50% or more of the initial values.
Using the electrophysiological method of research, it was revealed that with the development of skeletal muscle fatigue, caused by electrical stimulation, there was a decrease in the level of muscular effort in experimental rats. However, after the use of C60FAS injection, a partial recovery of the muscle force contraction was observed with its subsequent maintenance at a certain level for a long time.
After electrophysiological experiments, biochemical studies of the most informative markers of fatigue were conducted, in particular, the number of products of muscle metabolism (lactic acid, LA), markers of oxidative stress (thiobarbituric acid reactive substances, TBARS; hydrogen peroxide, H2O2), as well as the activity levels of such endogenous antioxidants such as reduced glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were evaluated in fatigued muscle tissue. Electric muscle stimulation has been found to lead to an increase in metabolism (LA) in the functioning muscle and intensification of oxidation processes, namely a significant increase in ROS and lipid peroxidation, which was associated with an increase in endogenous CAT and GSH antioxidants activity. Analysis of changes in the metabolic processes that accompany muscular fatigue showed an increase in the markers of oxidative stress H2O2 and TBARS in relation to intact muscles. After the administration of C60FAS, the concentration of TBARS and the level of H2O2 significantly decreased.
In behavioral tests the use of an animal model with experimental hemiparkinsonism allowed us to detect the effect of C60 fullerenes on their physical activity dynamics during muscle fatigue development. The positive effect of C60 fullerenes on the development of fatigue in hemiparkinsonian rats was established, as evidenced by the relatively constant rate of circulatory movements of animals throughout the experiment, with a significant decrease in the number of revolutions in animals of the control group. This confirms the significant antioxidant effect of C60FAS at the level of the whole organism.
A comparative analysis of the C60FAS effect depending on the administration method into the organism (systemic or oral) has been studied. In the electrophysiological and biochemical experiments, the effectiveness of C60 fullerene antioxidant properties was investigated under conditions of the muscle fatigue development compared to the action of known antioxidants such as NAC and β-alanine. It was shown that in animals that drank C60FAS and in rats that were injected with NAC or β-alanine, there was only a slight decrease of the muscle contraction force followed by long-term maintenance of such force level. In animals, with C60FAS oral administration, as well as in groups of animals with injections of N-acetylcysteine or β-alanine, the amount of ROS and concentrations of TBARS, H2O2 and LA under fatigue conditions were also significantly lower (p<0,05) compared to the rats of the control group. Levels of endogenous antioxidant concentrations (GSH and GPx) and enzyme activity (CAT and SOD) in animals of these groups were also significantly lower compared to the control group.
