Effect And Potential Mechanism Of Hyperbaric Oxygen Combined With Astaxanthin On Eliminating Exercise-induced Fatigue Of Rugby Players

Objectives:The generation of exercise-induced fatigue is a necessary prerequisite for the growth of athletic ability,but excessive accumulation of fatigue will weaken the quality of movement completion,disturb the benefit of training preparation,increase the risk of sports injury,and even sudden death in sports.Therefore,how to effectively eliminate exercise-induced fatigue is the key problem to be solved on the road of competitive sports development.Existing methods for relieving exercise-induced fatigue include massage,acupuncture,tight clothing,intermittent negative pressure of lower limbs,water bath,and whole-body cryotherapy.After years of research and practice,the efficacy and scope of application of these methods have become clearer and more saturated,and the development of exercise fatigue elimination field has slowed down,so it is the development trend and inevitable choice to explore new exercise fatigue recovery methods under the current situation.Hyperbaric oxygen has multiple effects such as anti-inflammatory,improvement of oxygenation,neuroprotection and improvement of metabolism.It shows great potential as an auxiliary means to eliminate exercise-induced fatigue,but it may produce excessive free radicals and other side effects when applied.Antioxidants are often used in sports science to combat excess free radicals.Objective 1:To compare the differences between natural recovery,hyperbaric oxygen intervention and hyperbaric oxygen combined with astaxanthin intervention in eliminating acute exercise-induced fatigue of rugby players,and to analyze the effects of hyperbaric oxygen intervention combined with astaxanthin on eliminating exercise-induced fatigue.Objective 2:Long-term hyperbaric oxygen intervention combined with astaxanthin intervention was used to compare natural recovery and hyperbaric oxygen intervention to explore the effect of four-week intervention on exercise-induced fatigue elimination caused by high-intensity sports training during winter training of rugby players.At the same time,in order to explore the potential mechanism of the influence of hyperbaric oxygen combined with astaxanthin intervention on human long-term exercise fatigue,the mediating effect of hyperbaric oxygen combined with astaxanthin intervention on human body was discussed from six aspects,namely body composition and muscle strength,blood cell changes,antioxidant capacity,inflammatory response,hormone and metabolic effects,and behavioral psychology.It provides a way to further explore the internal mechanism of hyperbaric oxygen combined with astaxanthin to eliminate exercise-induced fatigue.Objective 3:According to the main mediating factors of the effect of hyperbaric oxygen combined with astaxanthin on exercise-induced fatigue in the results of mediating effect test in Study 2,the role of related signaling pathways and proteins in the intervention of hyperbaric oxygen combined with astaxanthin was explored through animal experiments,in order to provide a basis for the application of hyperbaric oxygen combined with astaxanthin on exercise-induced fatigue.Methods:This study consisted of acute intervention in humans（Study 1）,four-week intervention in humans（Study 2）and four-week intervention in animals（Study 3）.As part of the study,30 active rugby sevens players（15 males,15 females;Sixteen first-level national athletes,14 elite athletes）were recruited as subjects after screening,and matched according to gender,age and sport grade.Then,the matched athletes were randomly assigned to blank control group（n=10）,hyperbaric oxygen group（n=10）and hyperbaric oxygen+astaxanthin group（n=10）with computer-generated numbers.Acute exercise fatigue was induced by exhaustive exercise with increasing load,and then the blank control group recovered naturally for 60min.The hyperbaric oxygen group received 60min hyperbaric oxygen intervention（ATA=1.3）,and the hyperbaric oxygen+astaxanthin group received60min hyperbaric oxygen intervention after taking astaxanthin（16mg）（ATA=1.3）.Indicators were collected in quiet state,immediately after exercise and immediately after intervention to explore the differences of natural recovery,hyperbaric oxygen intervention and hyperbaric oxygen combined with astaxanthin intervention in eliminating acute sports fatigue of rugby players.The effects of hyperbaric oxygen intervention combined with astaxanthin on reducing the side effects of hyperbaric oxygen intervention and eliminating exercise fatigue were analyzed through indicators including HR,Sa O₂,Sm O2,oxidative stress damage markers such as CK,LDH,oxidation and antioxidant indexes such as MDA,SOD,GSH-Px,inflammatory markers such as CRP,blood routine markers such as WBC,RBC,Hb,HCT,metabolic indicators such as Bla,BU,UA,Glu,hormone indicators such as T,C,T/C and RPE.Using descriptive statistics,one-way analysis of variance（ANOVA）compared basic information（such as age,height,weight,body mass index,exercise class,training years and weekly training time,etc.）and exhaustion indicators such as the time to exhaustion（TLim）,maximum power（Pmax）whether there is a difference.The other results were analyzed by repeated-measure ANOVA.In the second part of the study,the information of the subjects is consistent with that of the first part of the study.By monitoring the fatigue condition of high-intensity training during the winter training,the groups were divided into groups after class for natural recovery,hyperbaric oxygen intervention and hyperbaric oxygen combined astaxanthin intervention.The effects of intervention on aerobic capacity,muscle strength and behavioral psychological performance were investigated from the aspects of blood routine indexes,metabolic indexes,markers of oxidative stress damage,antioxidant indexes,inflammatory indexes,hormone indexes and psychological exertion state,so as to evaluate the effect of peripheral hyperbaric oxygen combined with astaxanthin on eliminating exercise fatigue.In addition,the mediating effect models were constructed from six aspects:body composition and muscle strength,blood cell changes,antioxidant capacity,inflammatory response,hormone and metabolic effects,and behavioral psychology.Results were analyzed by repeated-measure ANOVA.Based on the results of study 1 and 2,exercise-induced fatigue model of mice was established through four-week high-intensity training to simulate human exercise fatigue state,and exhaustive exercise time,oxidative stress indexes of serum and skeletal muscle tissue were measured to explore the antioxidant and anti-fatigue effects of blood and tissue oxidative stress and hyperbaric oxygen combined with astaxanthin.To investigate the effect of hyperbaric oxygen combined with astaxanthin on tissue damage caused by exercise-induced fatigue,the morphological changes of skeletal muscle were compared by H&E staining and immunofluorescence staining.Immunohistochemistry and western blotting showed the expression and distribution of Keap1/Nrf2/HO-1 pathway related proteins in tissues,to explored the effect of hyperbaric oxygen combined with astaxanthin on Nrf2pathway in mice after exercise fatigue.Descriptive statistics and repeated-measure ANOVA were used to compare groups.Results:As part of the study,results showed that acute intervention improved exercise-induced fatigue to varying degrees.Immediately after intervention,erythrocyte indices Hb and HCT in hyperbaric oxygen+astaxanthin group were significantly higher than those in control group（P=0.033;P=0.033）,skeletal muscle damage index CK,Oxidative stress index MDA were significantly lower than control group（P<0.001;P=0.008;P=0.004）.In addition,metabolic product Bla in hyperbaric oxygen group and hyperbaric oxygen+astaxanthin group was significantly lower than that in control group（P=0.010;P<0.001）.In terms of hormone indexes,male testosterone and testosterone/cortisol ratio and female testosterone/cortisol ratio in hyperbaric oxygen group and hyperbaric oxygen+astaxanthin group were significantly lower than those in control group.In the second part of the study,14 subjects（11 males and 3 females）reached the seventh stage of exhaustion exercise test after four weeks intervention.After intervention,TLim in hyperbaric oxygen group was higher than that before intervention（P=0.019）,and VO2max in hyperbaric oxygen group and hyperbaric oxygen+astaxanthin group was significantly higher than the corresponding value before intervention（P=0.001;P=0.003）.During the four-week intervention,Hb level in the hyperbaric oxygen+astaxanthin group was significantly higher than that in the control group at four weeks and two weeks after intervention（P=0.019,P=0.005）.In terms of metabolites,the UA value of hyperbaric oxygen group and hyperbaric oxygen+astaxanthin decreased gradually,and was significantly lower than the corresponding value before intervention two weeks after intervention,and lower than that of control group at the same period.LDH level increased first and then decreased,and was significantly lower than that of control group four weeks after intervention（P<0.001）.The CK level of each group showed a trend of significant increase.The CK level of the hyperbaric oxygen group was significantly lower than that of the control group at four weeks after intervention（P=0.044）,while the CK level of the hyperbaric oxygen+astaxanthin group was significantly lower than that of the control group at two weeks after intervention（P=0.024）.MDA level in the hyperbaric oxygen+astaxanthin group was significantly lower than that in the control group four weeks after intervention（P=0.001）.SOD and GSH-Px indexes in all groups decreased first and then increased,and SOD levels in all groups were lower than before intervention at two weeks（P=0.001;P<0.001;P=0.005）;After four weeks of intervention,SOD and GSH-Px levels in the hyperbaric oxygen group and the hyperbaric oxygen+astaxanthin group recovered to the pre-intervention levels,and were significantly higher than those in the control group（P<0.01）.In addition,CRP levels in the hyperbaric oxygen group and the hyperbaric oxygen+astaxanthin group were significantly lower than those in the control group at 4 weeks after intervention（P=0.011;P=0.007）.In terms of hormones,the testosterone/cortisol ratio of male subjects in the hyperbaric oxygen group and the hyperbaric oxygen+astaxanthin group showed an increasing trend,and was significantly higher than the corresponding value before intervention two weeks and four weeks after intervention（P<0.05）,but no significant difference was found between groups.In female subjects,cortisol levels in the hyperbaric oxygen group were significantly lower four weeks after intervention than before intervention（P=0.024）,but no significant differences were seen between groups.The results of mediating effect test showed that the mediating effect of SOD and LDH on fatigue was significant in hyperbaric oxygen group and hyperbaric oxygen+astaxanthin group when control group was taken as reference.In the third parts of the study,after four weeks of intervention,Tlim in astaxanthin exercise group and hyperbaric oxygen combined astaxanthin exercise group increased by73%and 95%compared with exercise control group,respectively（P<0.001,P<0.001）.The Tlim of hyperbaric oxygen combined with astaxanthin exercise group was significantly higher than that of hyperbaric oxygen exercise group（P=0.001）.The levels of Bla and BUN in the exercise control group were significantly higher than those in the control group（P<0.001）,indicating that the exercise-induced fatigue model of mice was successfully established after four weeks of treadmill exercise training.The levels of Bla and BUN in hyperbaric oxygen group,astaxanthin exercise group and hyperbaric oxygen combined with astaxanthin exercise group were significantly lower than those in exercise control group（P<0.001）.In addition,SOD,GSH-Px and T-AOC levels in serum and skeletal muscle of mice in exercise control group were significantly decreased compared with control group.After four weeks of intervention,SOD and GSH-Px levels in serum and skeletal muscle of mice in hyperbaric oxygen group,astaxanthin exercise group and hyperbaric oxygen combined astaxanthin exercise group were increased,which was significantly higher than that in exercise control group.Serum MDA and T-AOC levels were recovered（P<0.001）.H&E staining showed that after four weeks of high-intensity exercise training,the gastrocnemius fibers of exercise control mice were thickened and enlarged,but the muscle fibers were deformed and slightly pale.After training,hyperbaric oxygen intervention can improve the damage of gastrocnemius muscle fibers,make the arrangement of muscle fibers more regular,and improve the color,but some muscle fibers still exist deformation.Astaxanthin intervention after training made the gastrocnemius fibers thickened,but still slightly pale,and some of them were accompanied by inflammatory cell infiltration.Hyperbaric oxygen combined with astaxanthin significantly improved the histological changes of the gastrocnemius muscle of mice,and the arrangement of the muscle fibers was regular,compact and orderly,and the shape was normal and uniform.The proteins Keap1,Nrf2 and HO-1 in the gastrocnemius of mice were detected by immunofluorescence single stain technique,which showed small particles scattered in the cytoplasm of skeletal muscle cells.The expressions of Keap1 and Nrf2 and HO-1 were significantly increased in gastromius cells of mice in exercise control group,while the expressions of Nrf2 and HO-1 were significantly decreased in hyperbaric oxygen group,astaxanthin exercise group and hyperbaric oxygen combined with astaxanthin exercise group after four weeks of intervention.Western blotting showed that the intervention of peripheral hyperbaric oxygen combined with astaxanthin could significantly reduce the expression of Keap1 protein by regulating Keap1/Nrf2/HO-1pathway,further promote the expression of Nrf2 and HO-1,improve the antioxidant capacity of skeletal muscle tissue and promote the elimination of exercise-induced fatigue.Conclusion:In summary,hyperbaric oxygen combined with astaxanthin could improve the related indexes of red blood cell system,promote the clearance of metabolites,regulate hormone secretion,and prevent oxidative stress,and have a good effect on eliminating acute exercise-induced fatigue of rugby players.Four-week intervention can improve red blood cell index,optimize body metabolism,inhibit oxidative stress injury and inflammatory response,play a role in the long-term exercise-induced fatigue of rugby players,and have a positive impact on improving athletic ability.Through exercise-induced fatigue mice model,it was found that four-week hyperbaric oxygen combined with astaxanthin affected the expression of Keap1/Nrf2/HO-1 signaling pathway related proteins,significantly improved the damage of skeletal muscle tissue,and played an anti-fatigue role in regulating oxidative damage.