| Objective:Fatigue affects the quality of life,physiological health,and mental status of modern people.The development of efficient anti-fatigue functional nutritional preparations is a current research hotspot.The literature research and the results of the group’s previous studies suggest that blueberry extract has anti-fatigue effect,but its anti-fatigue mechanism of action is still unclear.In this study,we applied transcriptomics technology and bioinformatics analysis to explore the key nodes of the biological process of the anti-fatigue effect of blueberry extract and the core target genes,and conducted experimental validation to provide new ideas to study the anti-fatigue effect mechanism of blueberry and provide some theoretical reference for the development of anti-fatigue functional nutritional preparations of blueberry.Methods:1.Comparative study of the anti-fatigue effect of blueberry extract and cyanidin-3-O-glucoside(Cy3G).Sixty 5～6-week-old male BALB/c mice were randomly divided into exercise control group(EC),low-dose blueberry extract intervention group(BL),high-dose blueberry extract intervention group(BH),low-dose Cy3G intervention group(SL)and high-dose Cy3G intervention group(SH),and 12 mice in each group were gavaged with pure water,blueberry extract(10Omg/kg·bw·d),blueberry extract(400mg/kg·bw·d),Cy3G(25mg/kg·bw·d)and Cy3G(10Omg/kg·bw·d).After 30 days of gavage,each group of mice was subjected to weight-exhaustion swimming experiment,and the weight-exhaustion swimming time was recorded.2.Comprehensive evaluation and validation of the anti-fatigue effect of blueberry extract.Sixty 5～6-week-old male BALB/c mice were randomly divided into weight-exhaustion swimming exercise control group(EC0,purified water),weight-exhaustion swimming exercise+low-dose blueberry extract intervention group(BL0,blueberry extract 100mg/kg·bw·d),isochronous swimming blank control group(NC,purified water),isochronous swimming exercise control group(EC,purified water),and isochronous swimming exercise+low-dose blueberry extract intervention group(BL,blueberry extract 100 mg/kg·bw·d),10～14 animals in each group.After 30 days of gavage,the swimming time of mice in the weight-exhaustion swimming group was recorded.Blood,liver and muscle tissues were collected from mice in the isochronous swimming group.And serum lactate(LA),urea nitrogen(BUN)and glucose(GLU)levels,lactate dehydrogenase(LDH)and creatine kinase(CK)activities;liver glycogen(LG)and myoglycogen(MG)levels,Na+-K+-ATPase,Ca2+-Mg2+-ATPase activities and other fatigue-related markers in muscle tissue were measured.3.Transcriptomics-based study on the mechanism of anti-fatigue effect of blueberry extract.The muscle tissues of isochronous swimming mice in the experiment of the second part were collected,total RNA was extracted and sequenced on machine for differentially expressed gene analysis,and the functions of differentially expressed genes were annotated using GO database,and related signaling pathways were enriched by KEGG database.The screened differentially expressed genes were then validated by real-time fluorescence quantitative PCR experiments and Western Blot experiments.Results:1.Blueberry extract and Cy3G interventions had no adverse effects on body weight and growth and development of experimental animals;blueberry extract and Cy3G interventions could prolong the time of weight-exhaustion swimming in mice(P<0.01),and there was no statistical difference between the intervention groups.2.In the weight-exhaustion swimming experiment,blueberry extract significantly prolonged the time of weight-exhaustion swimming in mice(P<0.01).In isometric swimming experiment,compared with NC group,serum LA,BUN,LDH and CK levels were significantly increased in EC group(P<0.05),muscle glycogen,liver glycogen and Na+-K+-ATPase and Ca2+-Mg2+-ATPase levels in muscle tissue were significantly decreased(P<0.05),and blood glucose levels tended to decrease;compared with EC group,serum LA,BUN,LDH and CK levels were significantly decreased(P<0.05)in BL group.Muscle glycogen,liver glycogen,blood glucose and muscle tissue Na+-K+-ATPase and Ca2+-Mg2+-ATPase levels were significantly increased(P<0.05)compared with the EC group.3.Transcriptomic results and bioinformatics analysis:① 381 differentially expressed genes between NC and EC groups,of which 114 were up-regulated and 267 were down-regulated;404 differentially expressed genes between EC and BL groups,of which 323 were up-regulated and 81 were down-regulated.②Annotated by GO analysis,the difference genes between NC and EC groups were related to various motor functions such as "muscle regulation","skeletal muscle adaptation" and "muscle fiber contraction",as well as various regulatory functions related to energy metabolism such as "glucose metabolism","lipid metabolism" and "regulation of calcium homeostasis".The difference genes between EC and BL groups were mainly related to energy metabolism regulation functions such as "sugar metabolism","lipid metabolism","calcium ion transport" and physiological functions such as "muscle fiber".③The KEGG enrichment analysis revealed that there were 2 enriched pathways with up-regulated expression levels between NC group and EC group and down-regulated expression levels between EC group and BL group,which were metabolic pathway and cGMP-PKG signaling pathway,respectively;16 enriched pathways with down-regulated expression levels between NC group and EC group and up-regulated expression levels between EC group and BL group,including glycolysis/gluconeogenesis,MAPK signaling pathway,HIF-1 signaling pathway,TNF signaling pathway,sphingolipid metabolism,type Ⅱ diabetes and other pathways related to energy metabolism.④By comparing the differentially expressed genes with up-regulated expression levels between NC group and EC group and down-regulated expression levels between EC group and BL group,and the differentially expressed genes with down-regulated expression levels between NC group and EC group and up-regulated expression levels between EC group and BL group,35 differentially expressed genes were found in total.After checking the relevant literatures and researches,seven differentially expressed genes related to exercise or energy metabolism were screened out,namely Gpam,Slc8a1,Mt2,Mtl,Kif2 7,Cdh10 and Zfp92.4.Verification of partial transcriptome results:the seven identical differentially expressed genes were verified by real-time fluorescence quantitative PCR and Western Blot.①The results of significant difference in mRNA expression of Gpam,Slc8a1,Mt2 and Mt1 among the groups were consistent with the transcriptome results;the trends of difference in mRNA expression of Kif27,Cdh10 and Zfp92 among the groups was also largely consistent with the transcriptome results.②The results of significant differences in protein expression levels of Gpam,Slc8a1 and Mt2 between groups were consistent with the results of real-time fluorescence quantitative PCR experiments and transcriptome;the differences in protein expression levels of Cdh10,Mtl,Kif27,and Zfp92 between groups were not statistically significant.Conclusion:1.Both blueberry extract and Cy3G interventions significantly improved the exercise endurance of mice,and Cy3G may be the main active component of the anti-fatigue effect of blueberry extract.2.The intervention of blueberry extract could improve glycogen reserve,reduce metabolite accumulation,enhance energy metabolizing enzyme activity and reduce muscle damage in swimming mice,which had significant anti-fatigue effects.3.The anti-fatigue effect of blueberry extract may be achieved by regulating the expression of genes in muscle tissue,such as Gpam,Slc8a1 and Mt2,which modulate metabolic pathways and cGMP-PKG signaling pathway. |
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