Intermittent Hypoxia Combined With Regular Exercise In Early Life Promotes The Effect Of DSir2 Expression On Cardiac Function

1 research purpose:The self-developed Drosophila exercise training device and hypoxia device were used to conduct regular exercise intervention,intermittent hypoxia intervention and intermittent hypoxia combined regular exercise intervention.The results of the intervention plan were tested to achieve the expression of dSir2 and cardiac function index.To study the relationship between different training methods and dSir2 expression and cardiac function,and to explore whether dSir2participates in and regulates cardiac function.Therefore,the experiment is divided into:（1）the effects of intermittent hypoxia training,regular exercise,combined regular exercise of intermittent hypoxia and dSir2mutation expression on the cardiac function of Drosophila;（2）the effects of combined regular exercise of intermittent hypoxia training,regular exercise,combined regular exercise of intermittent hypoxia and dSir2rescue expression on the cardiac function of Drosophila.2 research methods2.1 hybridization and grouping of Drosophila2.1.1 construction and grouping of normal and mutant expression lines of dSir2Female wild-type w¹¹¹⁸ flies hybridized with male arm-Gal4,female sirt1^5.26cn¹ flies hybridized with male sirt1^4.5cn¹/sm6b,P{ever Laz}SB1,and then collected the eclosion flies within 4-6 hours.They were randomly divided into NC-dSir2 group,NE-dSir2 group,HC-dSir2 group,HE-dSir2 group,MC-dSir2 group,ME-dSir2 group,MHC-dSir2 group,and MHE-dSir2 group.From the second day to the fourteenth day,Drosophila flies were trained five days a week,two days a week for rest（9:30-12:00/day）or hypoxia（14:00-20:00/day）.All Drosophila flies were collected 24 hours after the end of the training program.2.1.2dSir2 rescue expression hybridization and groupingFemale sirt1^5.26cn¹ and male sirt1^4.5cn¹/sm6b,P{ever lacz8.0}SB1were hybridized to collect the emergence of virgin flies within 4-6 hours,and were randomly divided into MC-dSir2 group,ME-dSir2 group,MHC-dSir2 group,MHE-dSir2 group,SC-dSir2 group,SE-dSir2 group,SHC-dSir2 group,SHE-dSir2 group,400 in each group.From the second day to the fourteenth day,Drosophila flies were trained for five consecutive days every week,two days off every week（9:30-12:00/day）or with hypoxia（14:00-20:00/day）.All Drosophila flies were collected 24 hours after the end of the training program.2.2 index detectionEach group randomly selected 10 flies to detect dSir2 mRNA expression,30-40 flies to detect M-mode cardiac function,and 100 flies to detect exercise ability.3 research results3.1 results of QRT PCR3.1.1 effect of combined regular exercise of intermittent hypoxia and dSir2 mutation expression on dSir2 expression in early lifeCompared with NC-d Sir2 group,MC-dSir2 group had lower dSir2mRNA expression,and the down-regulation rate was 90.20%.These results showed that dSir2 mutation system was constructed successfully.Compared with NC-dSir2 group,the dSir2 mRNA expression of HC-dSir2 group was higher,the up-regulation rate was 50.05%;compared with MC-dSir2 group,the dSir2 mRNA expression of MHC-dSir2 group was higher,the up-regulation rate was 80.00%.These results suggest that intermittent hypoxia training may promote dSir2 mRNA expression.Compared with NC-dSir2 group,the dSir2 mRNA expression of NE-dSir2 group was higher,the up-regulation rate was 95.10%;compared with MC-dSir2 group,the d Sir2 mRNA expression of ME-dSir2 group was higher,the up-regulation rate was 80.00%.These results suggest that regular exercise training may promote dSir2 mRNA expression.Compared with NC-dSir2 group,d Sir2 mRNA expression in HE-dSir2 group was higher,up-regulation rate was 103%;compared with MC-dSir2 group,dSir2 mRNA expression in MHE-dSir2 group was higher,up-regulation rate was 90.00%.These results suggest that intermittent hypoxia combined with regular exercise training may promote dSir2mRNA expression more significantly.3.1.2 effect of combined regular exercise of intermittent hypoxia and dSir2 rescue expression on dSir2 expression in early lifeCompared with MC-dSir2 group,the dSir2 expression of SC-dSir2was higher,and the rescue rate was 100%,indicating that dSir2 rescue strain was successfully constructed.Compared with MC-dSir2 group,dSir2 mRNA expression in MHC-dSir2 group was higher,up-regulation rate was 42.00%;compared with SC-dSir2 group,dSir2 mRNA expression in SHC-dSir2 group was higher,up-regulation rate was 67.65%.These results indicate that intermittent hypoxia training promotes dSir2 mRNA expression.Compared with MC-dSir2 group,dSir2 mRNA expression in ME-dSir2 group was higher,the up-regulation rate was 78.00%;compared with SC-dSir2 group,dSir2 mRNA expression in SE-dSir2group was higher,the up-regulation rate was 98.74%.These results indicated that regular exercise training promoted dSir2 mRNA expression.Compared with MC-dSir2 group,dSir2 mRNA expression in MHE-dSir2 group was higher,up-regulation rate was 91.00%;compared with SC-d Sir2 group,dSir2 mRNA expression in SHE-dSir2 group was higher,up-regulation rate was 120.17%.These results showed that intermittent hypoxia combined with regular exercise training significantly promoted dSir2 mRNA expression.3.2 cardiac function results3.2.1 effect of combined regular exercise of intermittent hypoxia and dSir2 mutation expression on cardiac function of Drosophila melanogaster in early lifeThe heart rate of Drosophila in NC-dSir2 group was lower than that in MC-dSir2 group（P<0.01）;the cardiac cycle of Drosophila in NC-dSir2 group was higher than that in MC-dSir2 group（P<0.05）;the shortening fraction of Drosophila in NC-d Sir2 group was significantly higher than that in MC-dSir2 group（P<0.01）;the shortening fraction of Drosophila in NC-dSir2 group was significantly higher than that in MC-dSir2 group（P<0.01）;The shortening fraction of Drosophila in MHC-dSir2 group was significantly higher than that in MC-dSir2 group（P<0.01）;the shortening fraction of Drosophila in HC-dSir2 group was significantly higher than that in NC-dSir2 group（P<0.05）;the shortening fraction of Drosophila in MHC-dSir2 group was significantly higher than that in MC-dSir2 group（P<0.01）;the fibrotic tremor in MC-dSir2 group were significantly lower than those in NC-dSir2 group（P<0.01）.