Study On The Mechanism Of Anti-exercise Fatigue Of Ginseng And Purslane Compound Based On Metabonomics

Background Fatigue is a physiological and pathological phenomenon of homeostasis caused by excessive mental and physical activity,or excessive mental stimulation.It can appear as an independent disease or as a symptom.Fatigue is an "invisible killer" with a high incidence rate,which 30% of adults are in a state of chronic fatigue.Under long-term fatigued state would accelerate aging,lead to depression and anxiety,cause cancer or some immune diseases,and seriously affect people’s normal work and life.In recent years,promoting the recovery of fatigued state is a problem that need to be overcome in some fields.In the military field,the incidence and degree of fatigue are serious,which causing non-combat attrition and reducing combat effectiveness.Professor Ling generated Amaranth mixture based on theoretical basis of anti-fatigue and anti-hypoxia of "combination of tongs and tonics" and rich experience of repeated clinical verification and pharmacodynamic screening.This mixture formulation is significantly different from the currently theory of "pure tonifying without diarrhea" anti-fatigue drug formulations.It has strong innovation and could be used for the prevention and treatment of exercise-induced fatigue.The mechanism of action dose not clear.Based on the basis of establishing two different fatigue animal models to verify the anti-exercise-induced fatigue effect of Amaranth mixture,I combine the observation of relevant biochemical indicators,the possible mechanism of action initially revealed,and further research carried out through metabolomics technology to find differential metabolites and analyze metabolic pathways,to explore the possible mechanism of Amaranth mixture against exercise-induced fatigue,and to provide theoretical support for further clinical application.Objectives 1.To observe the effect of Amaranth mixture on exercise-induced fatigue by establishing two different fatigue models of static pole climbing and dynamic exhaustive swimming.2.To filter a better animal model,which could observe the liver glycogen,muscle glycogen,urea nitrogen(BUN),lactic acid(BLA),glutathione(GSH-Px),malondialdehyde(MDA),the changes of superoxide dismutase(SOD)index,and the possible mechanism of the anti-fatigue effect of amaranth mixture from different perspectives such as energy metabolism and oxidative stress 3.To use LC-MS metabolomics technology to reveal the differential metabolites of different doses of Amaranth mixture in anti-fatigue,finding the relevant action pathway,and to analyzing the possible intervention mechanism of its anti-fatigue.Methods 1.100 mice were randomly divided into swimming and pole climbing model group with 50 mice in each group.Mice in swimming model group were randomly divided into 5 groups after swimming training for 3 times,and mice in pole climbing model group were randomly divided into 5 groups.The two groups were given the corresponding drugs and doses by intragastric administration for three consecutive days on the 11 th and 7th day,respectively.When the last administration ends at 30 minute,the mice in the swimming model group swam to exhaustion with weight-bearing tails.While in the rod climbing model group,the mice were placed on the plexiglass rod.The time when the mouse fell from the plexiglass rod due to muscle fatigue was recorded,and three times were accumulated as the rod climbing time of the mouse.2.60 mice were randomly divided into 5 groups after swimming training for 3 times: model group,placebo control group,low-dose Amaranth mixture group,medium-dose Amaranth mixture group and high-dose Amaranth mixture group,with 10 mice in each group.The mice in each group were given the corresponding drugs and doses by intragastric administration for three consecutive days.When the last administration ends at 30 minutes,the weights on the tails of each mouse equivalent to 5% of their body weight were swam to exhaustion.Then,take blood,kidney tissue,liver tissue,cryopreserved for future use.After that,detect liver glycogen,muscle glycogen,BUN,BLA,GSH-Px,MDA,SOD according to the kit instructions an indicator.3.30 mice were grouped into models according to the above methods,and blood was collected after exhaustive swimming for LC-MS metabolomics detection.Multivariate statistical analysis was used to find the differential metabolites compared in different groups,and then obtain accurate information of metabolites through further matching and annotation in the relevant database.After summarizing,the differential metabolites were imported into the online platform of the Met PA database and the related metabolic pathways were analyzed through the KEGG database.Results 1.Compared with the model group,the exhaustive swimming time and pole climbing time of the mice in the middle-dose Amaranth mixture group and the high-dose Amaranth mixture group were significantly prolonged(P < 0.01).Compared with the Amaranth Mixture placebo group,the weight-bearing of the Amaranth Mixture medium-dose group and the Amaranth Mixture high-dose group did not change significantly(P>0.01).The exhaustive swimming time and pole climbing time were significantly prolonged(P<0.01),and the mice in the low-dose Amaranth mixture group had no significant changes(P>0.01).2.Compared with Xs M group,the contents of muscle glycogen and SOD in the high-dose Amaranth mixture group(Xs HD group)were significantly increased(P<0.01),while the contents of MDA,BLA and BUN were significantly decreased(P<0.01).The content of liver glycogen and GSH-Px activity increased(P<0.05);the muscle glycogen and GSH-Px activity of mice in the middle-dose Amaranth mixture group(Xs MD group)increased significantly(P<0.01).BLA and BUN were significantly decreased(P<0.01)The content of liver glycogen was increased,the activity of SOD was increased,and the content of MDA was decreased(P< 0.05).BUN content significantly decreased(P < 0.01),muscle glycogen content increased,BLA content decreased(P < 0.05),liver glycogen,content and SOD,GSH-Px activity did not change significantly(P > 0.05).There was no significant difference between Xsg C and Xsg M(P>0.05).3.The results of the LC-MS metabolomics study showed that the three dose groups of Amaranth Mixture were compared with the placebo group,and the 6common and significantly different metabolites were: Gibberellin A3,Oxoadipic acid,Aesculetin,Oxoglutaric acid,Isocitric acid,Glycochenodeoxycholic acid,involving 5related major metabolic pathways: Tryptophan metabolism,Lysine degradation,Citrate cycle,Glyoxylate and dicarboxylate metabolism,Carboxylic acid metabolism,Cholesterol metabolism,and the metabolic pathways that play a key role are tryptophan metabolism,lysine degradation,and tricarboxylic acid cycle.Conclusions 1.Amaranth mixture has anti-fatigue effect and can prolong the time of exhaustive swimming and pole climbing of mice,among which the effect of medium and high doses is the most obvious.Compared with the modeling method of climbing pole,the animal model of exhausted swimming fatigue is a more accurate modeling method.2.Amaranth mixture can play an anti-fatigue effect by improving energy reserve and supply;removing metabolites,regulating energy metabolism;accelerating free radical scavenging,reducing oxidative stress,and maintaining the balance of oxidative levels during exercise.3.The related metabolic mechanism of Amaranth mixture involves multi-target and multi-pathway interaction,among which tryptophan metabolism pathway,lysine degradation pathway and tricarboxylic acid cycle pathway play an important role,which may be the effect of Amaranth mixture.