Study On The Difference Of The Effect Of Single Aerobic Exercise On Gene Expression In Gastrocnemius Of Different Strains Of Mice

Objective:Exercise induces complex and extensive molecular changes in multiple tissues and organs,and the increasing maturity of transcriptomics techniques provides the possibility to understand these alterations in depth.Mice are commonly used as model animals in exercise physiology research,and although the similarities and differences between the genomes of multiple mouse species have long been clear,the molecular phenotypic characteristics of these mouse species have rarely been studied,and the differences in molecular changes in skeletal muscle after exercise in different mouse species are rarely studied.Moreover,whether in humans or experimental animals,the study of molecular changes in skeletal muscle after exercise is mostly carried out at a single time point,and information about gene changes at any time is relatively lacking.The acquisition of these molecular information has certain application value and theoretical significance for clarifying the molecular mechanism of motor phenotypic differences between rat species,guiding the selection of optimal animal models for different research objectives,and in-depth discussion of the molecular mechanism of exercise producing health effects.In this study,two common laboratory rat species BALB/c mice and C57BL/6 mice with similar maximum oxygen uptake and certain differences in exercise capacity were selected as research objects,in an attempt to analyze the characteristics of gene expression in skeletal muscle after a single acute exercise of each mouse species,and to compare and analyze the similarities and differences of these molecular changes in different mice.Research methods:This study used two different strains of mice,BALB/c and C57BL/6,with 32 males in each group at 8 weeks old and weighing 22±2g.The mice were placed under standard breeding conditions and allowed to adapt to the laboratory environment for one week（temperature at 23±2℃,12-hour light/dark cycle,and free access to standard diets）.During this period,the mice underwent an adaptive training with an exercise intensity of 10 m/min for 30 minutes per day.After one week,the mice of each strain were randomly divided into four groups,with eight mice in each group.The exercise groups underwent acute exercise for 30minutes,with a running speed of 20 m/min and a slope of 5°.The gastrocnemius muscles of the mice were collected at three time points after exercise:immediately after exercise（T0 group,n=8）,30 minutes after exercise（T30 group,n=8）,and 60minutes after exercise（T60 group,n=8）.The control group（C group,n=8）did not exercise,and their gastrocnemius muscles were collected.Total RNA samples were extracted using the TRIzol method and stored at-80°C.High-throughput sequencing analysis was performed on the samples using bioinformatics methods such as differential gene expression analysis,pathway and function enrichment,and temporal analysis to describe and compare the significantly changed genes,their functions,and the temporal patterns in skeletal muscles after exercise between the two strains of mice.Research results:1.Significant gene expression changes after exercise in two strains of miceThere were significant changes in the expression of a large number of genes in the gastrocnemius muscles of both strains of mice after exercise,with significant differences in the patterns of changes.C57BL/6 mice showed a total of 610significantly changed genes after exercise,with 423 upregulated genes and 187downregulated genes,whereas BALB/c mice showed a total of 1534 significantly changed genes after exercise,with 1158 upregulated genes and 376 downregulated genes.The most significantly changed genes after exercise in C57BL/6 mice were EGR3(T0,P<9.29x10^-24),FOSB(T0,P<2.50x10^-10),EGR1(T0,P<1.33x10^-9),ATF3(T30,P<1.08x10^-17),SKIL(T30,P<2.03x10^-22),MAFF(T30,P<8.03x10^-19),IRS2(T30,P<8.13x10^-19),NR4A2(T30,P<2.91x10^-12),etc.The most significant genes in BALB/c mice mainly included:ERRFI1(T0,P<2.17x10^-96),SIK1(T0,P<5.92x10^-91),BTG2(T0,P<1.94x10^-57),NR4A1(T0,P<1.37x10^-42),NR4A2(T0,P<1.16x10^-41),MAFF(T0,P<1.70x10^-33),FOS(T0,P<1.99x10^-36),DDIT4(T0,P<7.85x10^-44),SLC25A25(T30,P<3.09x10^-36),DUSP10(T0,P<2.54x10^-57),etc.the above are the P values after multiple test correction.2.Network node analysis of significantly changed genes at each time pointThe network of differentially expressed genes was constructed through the correlation between differentially expressed genes,and the network node genes were discovered and visualized with Cytoscape software,and it was found that the network node genes in C57BL/6 mice included NR4A1,NR4A2,NR4A3,MAFF,MT1,MT2and CSRNP1.The network node genes in BALB/c mice included NR4A1,NR4A2,NR4A3,MAFF,MT1,MT2,CSRNP1 and SIK1.Interestingly,although the genes and signaling pathways of the two mice changed significantly after exercise,their key node genes had certain similarities.It was further found that these genes and some common exercise-related genes changed to different degrees after exercise in the two mice,but their trends over time were mostly very similar.3.Enrichment analysis of signaling pathways that significantly change after movement in two mouse speciesAfter gene function enrichment analysis,it was found that metabolism-related pathways in C57BL/6 mice,such as apoptosis signaling pathway,c AMP,estrogen signaling pathway,and circadian rhythm,were significantly changed after exercise,but no significant changes in these signaling pathways were found in BALB/c mice.Conversely,signaling pathways associated with intercytokine,cancer,and viruses were found to be significant in BABL/c mice.FOXO signaling pathway,TNF signaling pathway,MAPK signaling pathway,heat shock protein B,circadian rhythm and other signaling pathways were significant signaling pathways in both mice.Conclusion:Through the transcriptome sequencing analysis of C57BL/6 mice and the gastrocnemius muscle of BALB/c mice after a single acute exercise,it was found that acute exercise would exert local or systemic stress on the body to activate or inhibit the expression of a large number of genes with different functions in any kind of mouse.Although the genes and signaling pathways of the two mice changed significantly after exercise were quite different,the key node genes had certain similarities,and the expression of these node genes and common exercise-related genes in the gastrocnemius muscle of the two mice was very similar,suggesting that the relatively conserved motor core genes in terms of function and expression regulation in different mouse species,and the existence of motor variable genes with large differences.