| ObjectiveExercise fatigue is a temporarily reduced physical performance of the human body after overload exercise.When exercise starts,a large amount of lactate is produced in skeletal muscle,and the material for gluconeogenesis in the liver is lactate.MCT1,a major lactate transport channel,facilitates the entry of lactate into the liver for gluconeogenesis in the liver.MCT1 also activates the CRTC2/CREB pathway in the liver and the following factors,FoxO1,HNF4 α and PGC-1α,which upregulate the gene expression of PEPCK and G-6-Pase,the core rate-limiting enzymes of gluconeogenesis,to promote hepatic gluconeogenesis and produce hepatic glycogen,which is transported to muscle and blood via GLUT1 and GLUT4 in the liver,to provide continuous energy for last exercise.In order to clarify the efficacy and mechanism of Zusanli(ST36)for exercise fatigue,the bibliometric analysis,acupoint selection rules and target network research were conducted.More important,,acupuncture at ST 36 intervened in the exercise fatigue of MCT1+/-mouse model.By observing the exhaustion swimming time,basic features,biochemical indexes,histopathological changes of skeletal muscle and liver,ultrastructure of skeletal muscle and mitochondria,and genes and proteins expression of gluconeogenesis in model mice,clarify the effects and mechanisms of action of acupuncture at ST36 for exercise fatigue which provided a reference for acupuncture therapy on exercise fatigue.Methods1.The bibliometric analysis,acupoint selection rules and target network research(1)Articles related to exercise fatigue from 2012 to 2021 on China National Knowledge Infrastructure were researched,and the current status,hotspots and frontiers through CiteSpace software were analyzed and summarized.(2)The clinical publications of acupuncture for exercise fatigue from 2022 to 2021 were searched on China National Knowledge Infrastructure,etc.,and were analyzed by SPSS model 14,SPSS Statistics 26 for cluster analysis and association rule analysis,and summarize the clinical acupoints for exercise fatigue.(3)The publications of acupuncture at ST36 were collected by searching databases such as China National Knowledge Infrastructure,etc.from 2012 to 2021,and acupuncture targets were obtained by Uniformprot database and intersected with disease targets in Gene database.Then,protein interaction network analysis and KEGG were performed on targets of ST36-cxercise fatigue to clarify the mechanisms,that related to acupuncture for exercise fatigue and provided a basis for subsequent experiments.2.Exploring and establish the model of exercise fatigue in C57BL/6J mice12 C57BL/6J mice in each group were given control group,1-week model group,2-week model group,3-week model group and 4-week model group.Except for control group,the rest groups were given swimming exhaustively.The time of swimming exhaustion,body weight,diet and water consumption were recorded.After executing the mice,HE staining was performed to observe the histopathological manifestations of gastrocnemius muscle and liver in each group of mice.The histopathological manifestations of gastrocnemius and liver of each group of mice were observed by HE staining.The mitochondrial structure of skeletal muscle of each group were observed by ultramicroelectron microscopy.The lactate,glycogen,AST and ALT in blood,lactate,glycogen,SOD,MDA and ATP in gastrocnemius,and glycogen and lactate in liver were measured by ELISA.3.Confirming that MCT1+/-C57BL/6J mice are an ideal model for regulating gluconeogenesis to study exercise fatigueC57BL/6J control group,C57BL/6J model group,MCT1+/-C57BL/6J control group,and MCT1+/-C57BL/6J model group were given 12 mice in each group.C57BL/6J model group and MCT1+/-C57BL/6J model group received swimming exhaustion and times were recorded.The basic data of swimming exhaustion time,body weight,food,and water consumption were recorded.Tissue and chromogen staining was used to identify the pathological changes in skeletal muscle and the liver.ELISA was used to determine lactate and glycogen in blood,lactate and glycogen in gastrocnemius muscle,glycogen and lactate in liver.PCR method was used to detect the gene expression of PEPCK and G-6-Pase in gastrocnemius MCT1 and liver.WB was used to measure MCT1,GLUT1and GLUT4 protein expression.4.Acupuncture up-regulation of CRTC2/CREB pathway induces glycoisogenic counteracting exercise fatigue mechanism in MCT1+/-miceMCT1+/-C57BL/6J mice were divided into each in control group,model group,acupuncture group and sham acupuncture group with 12 mice.After 3 weeks of load swimming,weight,diet and water consumption basic indications of each group were recorded,HE staining was used to observe the pathological changes of skeletal muscle and liver,transmission electron microscopy was used to observe the skeletal muscle of mice.Blood,gastrocnemius and liver tissue lactate expressions,blood glucose,myoglycogen and hepatic glycogen level were tested by ELISA,skeletal muscle MCT1 content and liver PEPCK and G-6-Pase level were measured by PCR.CRTC2,CREB,PGC-1α,HNF-4α,FoxO1,GLUT1 and GLUT4 expressions were measured by WB.Results1.The bibliometric analysis,acupoint selection rules and target network research(1)1100 publications related to exercise fatigue has received attention from scholars in recent years.Its research hotspots are oxidative stress,lactic acid accumulation,and acupuncture prevention,and its frontiers are sports injury,glucose metabolism,and Chinese Medicine therapy.Chinese medicine therapy,especially acupuncture therapy,has been applied to the prevention and treatment of exercise fatigue.And ST36 has the highest frequency and centrality.(2)35 publications related to ST 36,as an acupuncture prescription was the highest in clinical trail,the frequency of application of foot Yangming Stomach meridian and the Ren meridian.The support for zusanli-sanyinjiao was the highest frequency.The analysis concluded that the frequency of ST 36 with a separate prescription for exercise fatigue was with the highest frequency.(3)129 publications include 159 gene targets,which intersected with 997 gene targets of exercise fatigue to obtain 68 common targets.Through protein interaction network analysis,41 interaction nodes were obtained,and CREB and PGC-1α with high BC value.186 signaling pathways were collected for KEGG pathway,which were related to TNF signaling pathway,cAMP signaling pathway,AMPK signaling pathway,insulin resistance,mTOR signaling pathway,etc.in combination with the characteristics of exercise fatigue.2.Exploring and establish the model of exercise fatigue in C57BL/6J miceWhen compared with the 1-week model group and the 2-week model group,the mice in 3 and 4-week model groups had significantly lower swimming exhaustion times than the rest groups(P<0.05).3 and 4-week model groups had significantly more weight loss values than the 1 and 2-week model groups(P<0.05).there was a reduction in food and water consumption(P>0.05).Mice in 3-week model group had shapeless hair and less willingness to swim,and mice in the 4-week model group had shapeless hair,depression,resistance to swimming,and death happened.The blood,muscle and liver glycogen contents in the 3 and 4-week model groups were significantly lower than those in control group,1-week model group and 2-week model group(P<0.05).The blood,gastrocnemius and liver lactate contents in 3 and 4-week model groups were significantly higher than those in the remaining groups(P<0.05);the SOD and ATP contents in 3 and 4-week model groups were significantly lower than those in the remaining groups(P<0.05).The MDA content of 3 and 4-week model group was significantly higher than that of rest groups(P<0.05).The ALT and AST in 3 and 4-week model group increased more than that of the rest groups.The muscle texture of the mice in 3 and 4-week model group was unclear,the muscle fibers were disordered and swollen with different shapes and sizes,and the cells were scattered,disorganized and irregularly arranged.The hepatocytes in 3 and 4 weeks model group were intact and regularly arranged,some hepatocytes had irregular morphology,the liver had ischemic areas,and the hepatic cords were neatly arranged.The number of mitochondria in 3 and 4 weeks model group were less than the rest of the groups.The mitochondrial morphology was of different sizes and had enlargement,more vacuole-like changes,and some cristae disappeared.3.Confirming that MCT1+/-C57BL/6J mice are an ideal model for regulating gluconeogenesis to study exercise fatigueMCT1+/-C57BL/6J model group mice had significantly less swimming exhaustion time than C57BL/6 model group mice(P<0.05),their body weight loss values were significantly more than C57BL/6 model group(P<0.05),and their food and water consumption were lower than C57BL/6 model group(P>0.05).MCT1+/-C57BL/6J model group mice had more pathology changes than C57BL/6 model group mice(P<0.05).C57BL/6J model group mice had unclear skeletal muscle pathology muscle texture,scattered,disorganized and swollen muscle fiber arrangement,scattered cell arrangement,irregular morphology,and more obvious pathology.MCT1+/-C57BL/6J model group had intact and regular hepatocyte morphology,and some hepatocytes had irregular morphology.There were ischemic areas in the liver and the hepatic cords were neatly arranged,which were consistent with the pathology of the mice in C57BL/6J model group.The blood and gastrocnemius lactate in the mice in the MCT1+/-C57BL/6J model group were significantly higher(P<0.05)and liver lactate content was significantly lower(P<0.05)than the rest of the groups.MCT1 gene and protein expression,and GLUT1 and GLUT4 protein in MCT1+/-C57BL/6J model group mice was significantly lower than that in 57BL/6J model group mice(P<0.05)and significantly higher than rest two groups(P<0.05).The PEPCK and G-6-Pase genes expressions in MCT1+/-C57BL/6J model group mice was significantly lower than that in the 57BL/6J model group mice(P<0.05)and significantly higher than that in rest two groups(P<0.05).4.Acupuncture up-regulation of CRTC2/CREB pathway induces glycoisogenic counteracting exercise fatigue mechanism in MCT1+/-miceIt was found that the mice in the acupuncture group had significantly more swimming exhaustion time than model group and sham acupuncture group(P<0.05),their body weight loss values were significantly lower than rest groups(P>0.05),and food and water consumption were more than the model group and sham acupuncture group.At the end of the acupuncture intervention,the blood and muscle contents of MCT1+/-C57BL/6J mice were significantly lower than those of the other groups and the liver lactate contents were significantly higher than those of the other groups(P<0.05).The blood,muscle and liver glycogen of the acupuncture group were significantly higher than those of the other groups(P<0.05).The skeletal muscle pathology of mice in acupuncture group had clear myofilaments,neatly arranged muscle fibers and tightly arranged cells,which were greatly improved compared with the model group.The skeletal muscle fibers of mice in acupuncture group were neatly arranged,uniform in morphology,with clear M-line and H-band in the muscle nodes and inconspicuous Z-line,which were close to the blank group.The mnumber of mitochondria in the skeletal muscle of mice in the acupuncture group was normal and the morphological structure was intact,which was consistent witih the blank group.In acupuncture group,the hepatocytes were normal and intact in morphology.The hepatic cords were clear,non-infiltrated and regularly arranged,which were greatly improved compared with model group and sham acupuncture group.The expressions of MCT1 gene and protein,PEPCK and G-6-Pase gene and protein in acupuncture group were significantly higher than rest groups(P<0.05).GLUT1 and GLUT4 proteins were significantly higher in acupuncture group than in rest groups(P<0.05).The expressions of CRTC2,CREB,PGC-1α,HNF-4α,and FoxO1 proteins in the liver of acupuncture group was significantly higher than that of the rest groups(P<0.05).Conclusion1.3 weeks of loading exhaustion swimming can construct the exercise fatigue model of C57BL/6J mice.2.It has confirmed that MCT1+/-C57BL/6J mice are ideal animal models for the construction of glycoisogenic anti-exercise fatigue.3.Acupuncture at ST 36 can promote more lactate into the liver to promote gluconeogenesis by elevating MCT1 expression.4.Acupuncture at ST 36 can up-regulates MCT1 expression to promote CRTC2/CREB pathway-induced gluconeogenesis in MCT1+/-mice against exercise fatigue. |
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