| 1 ObjectiveIn this study,high-fat diet intervention and exercise intervention were conducted on Drosophila to explore the effects of high-fat diet and exercise on the heart function of Drosophila respectively.Then,by knocking down the Bmm gene of Drosophila fat triglyceride lipase,the relationship between Bmm and Bmm was studied.The relationship between the heart function of Drosophila and the possible molecular mechanism for the positive effect of exercise on the heart function of high-fat Drosophila.2 Materials and methodsUsing the USA/GAL4 system,the fat triglyceride lipase UAS-RNAi strain of Drosophila was crossed with W1118 strain and Arm-gal4respectively to construct the knockdown and normal expression of Drosophila Bmm gene,and divided into knockdown UAS-Bmm>Arm-gal4 group and normal expression UAS-Bmm>W1118 group.After hybridization,the F1 progeny virgin flies that emerged with 8 hours were collected and cultured with ND or HFD.UAS-Bmm>W1118 were randomly divided into normal expression quiet group（W-NC）,normal expression exercise group（W-NE）,and normal High-fat expressing group（W-HC）,normal expressing high-fat exercise group（W-HE）;UAS-Bmm>Arm-gal4 fruit flies were randomly divided into Bmm knock-down quiet group（A-NC）,Bmm knock-down exercise group（A-NE）,Bmm knock-down high-fat group（A-HC）,Bmm knock-down high-fat exercise group（A-HE）,800 F1 generation flies in each of the 8 crosses/group,20/tube.After emergence,the fruit flies in the high-fat group were raised with normal medium for 5 days,and then high-fat diet intervention was carried out on the 6th-10th day,and the exercise group started exercise training on the 11th-24th day.The exercise group uses the fruit fly exercise device independently developed by the research group to exercise every afternoon from 15:00 to 16:30.The exercise group exercises for two weeks,with two days of rest every five days of exercise.Each group of 400 flies were collected on the 25th day after the emergence of fruit flies.Drosophila culture environment setting:All fruit flies are kept at 25°C room temperature,50%humidity,12 hours/12hours light and dark cycle environment,and the medium is changed every2 days.Detection indicators:10 Drosophila were randomly selected from each group of Drosophila for RT-PCR detection;15 Drosophila were weighed,and triglyceride levels were measured by drosophila;30Drosophila were tested for cardiac function.Statistical methods:This study mainly uses independent sample t-test,two-factor and multi-factor analysis of variance to analyze the experimental samples,and all data are expressed by the mean±standard deviation（（?）±s）.3 Results3.1 Construction of high-fat Drosophila modelAfter applying 30%coconut oil medium to fruit flies for 5 days,it was found that the expression of TOR m RNA in the whole body of fruit flies in the W-HC group increased（P<0.01）,and the expression of Bmm and PGC-1 m RNA decreased（P<0.01）.The level of TG in Drosophila increased（P<0.01）.M-mode analysis found that the heart rate of the fruit flies in the W-HC group was higher than the W-NC group（P<0.01）,the heart rate of the fruit flies in the W-HC group was shorter than that of the W-NC group（P<0.05）,and the W-HC group The diastolic interval of Drosophila was shorter than that in the W-NC group（P<0.01）,the score of Drosophila in the W-HC group was reduce than that in the W-NC group（P<0.01）,and the arrhythmia index in the W-HC group was higher than that in the W-NC group Group（P<0.01）,.It suggests that the high-fat fruit fly model was successfully established.3.2 Rt-PCR detection results3.2.1 Construction of Bmm Whole Body Knockdown StrainThe expression level of UAS-Bmm>Arm-gal4 was lower than UAS-Bmm>W1118.The reduction rate is 34%.3.2.2 Test results of Bmm m RNA-expression in Drosophila induced by high fat and exerciseThe expression level of Bmm m RNA in Drosophila in the W-NE group was higher than that in the W-NC group（P<0.01）,and the up-regulation rate was about 78.367%;the W-HC group was lower than the W-NC group（P<0.01）,and the down-regulation rate was about67.07%;the W-HE group was higher than the high-fat diet group（P<0.01）,and the up-regulation rate was about 180.26%;the W-HE group and the W-NC group was no significant difference（P>0.05）.The expression level of Bmm m RNA in Drosophila in the A-NE group was higher than the A-NC group（P<0.01）,and the up-regulation rate was about 46.27%;the A-HC group was lower than the A-NC group（P<0.01）,and the down-regulation rate was about 30.44%;A-HE group is higher than A-HC diet group（P<0.01）,the up-regulation rate is about34.86%;there is no significant difference between A-HE group and A-NC group（P>0.05）.3.2.3 Test results of TOR m RNA-expression in Drosophila induced by high fat and exerciseThe level of TOR m RNA in Drosophila in the W-NE group was lower than that in the W-NC group（P<0.01）,and the down-regulation rate was about 44.40%;the W-HC group was higher than the W-NC group（P<0.01）,and the up-regulation rate was about 1875.01%;W-HE group is lower than W-HC group（P<0.01）,and the up-regulation rate is about 87.84%.UAS-Bmm>Arm-gal4 fruit flies increased the level of TOR m RNA in the whole body by 753.58%compared with UAS-Bmm>W1118drosophilas3.2.4 Test results of PGC-1 m RNA-expression in Drosophila induced by high fat and exerciseThe level of PGC-1 m RNA in Drosophila in the W-NE was higher than the W-NC（P<0.05）,and the up-regulation rate was about 20.04%;the W-HC group was lower than the W-NC group（P<0.01）,and the down-regulation rate was about It was 62.70%;the W-HE was higher than the W-HC（P<0.01）,and the up-regulation rate was about 113.76%.Compared with UAS-Bmm>W1118 drosophilas,the level of PGC-1m RNA of UAS-Bmm>Arm-gal4 fruit flies was reduced by 42.80%.3.3 Results of systemic TG level in DrosophilaThe TG level in the W-NE group was 24.29%lower than that in the W-NC group（P<0.01）.TG level in The W-HC was 24.98%higher than that in the W-NC（P<0.01）.Systemic TG level in The W-HE group was lower than that in the W-HC group（P<0.01）,which decreased by12.16%.The systemic TG level in The W-HE was higher than that in the W-NC（P<0.05）,which increased by 10.26%.TG level of Drosophila in group A-was higher than that of Group W-NC（P<0.01）;TG level in group A-NE was lower than that of Group W-NC（P<0.05）;TG level in group A-NE was lower than that of group A-NC（P<0.01）,with A-decrease of about 20.65%.TG level in HC group was 24.64%higher than that in NC group（P<0.01）.TG level in A-HE group was lower than that in A-HC group（P<0.01）,which was reduced by 13.53%.There was no significant difference in TG level between A-HE group and A-NC group（P>0.05）.3.4 Lifespan test results3.4.1 Average lifespan of fruit fliesThe average life span of fruit flies in the W-HC group was 30.45days,the W-NE group was 35.30 days,and the W-HE group was 34.09days;the life cycle of the fruit flies in the W-HC group was 8.1%shorter than that in the W-NC group,and the W-NE It was 6.5%longer than the W-NC,the W-HE group was 12%longer than the W-HC group,and the W-HE group was 2.9%longer than the W-NC group.The average lifespan of fruit flies in the A-NC group was 25.33 days,the A-HC group was 23.30 days,the A-NE group was 28.55 days,and the A-HE group was 25.00 days;the life cycle of the fruit flies in the A-NC group was shorter than that in the W-NC group The A-HC group was longer than the A-NC group by 8.0%,the A-NE group was longer than the A-NC group by 12.7%,the A-HE group was longer than the A-HC group by 7.3%,and the A-HE group It was 1.3%shorter than the A-NC group.3.4.2 Half of the time to death of fruit fliesThe half days of death of fruit flies in the W-NC group were 29 days,the W-HC group was 26 days,the W-NE group was 33 days,and the W-HE group was 30 days.The half days of death of fruit flies in the A-NC group were 19 days,the A-HC group was 18 days,the A-NE group was 23 days,and the A-HE group was 21 days.3.5 Results of M-mode in detecting Drosophila heart functionThe heart rate of fruit flies in the W-HC was higher than the W-NC group（P<0.01）;the heart rate of fruit flies in the W-HC group was higher than that of the W-NC group（P<0.01）,and the heart rate of fruit flies in the A-HE group was higher than that of A-HC diet group（P<0.05）;The systolic cycle of W-NE group was lower than that of W-NC group（P<0.05）;the cardiac cycle of Drosophila in W-HE group was lower than that of W-NC group（P<0.05）;the diastolic interval of Drosophila in W-HC group was lower than W-NC group（P<0.01）,W-HE group was lower than W-NC group（P<0.01）,A-HC group Drosophila heart diastolic interval was lower than W-NC group（P<0.01）,A-The HE group was higher than the A-NC group（P<0.01）,and the A-HE group was higher than the A-HC group（P<0.01）.The heart shortening score of Drosophila in W-HC group was significantly lower than that in W-NC group（P<0.01）,W-HE was higher than W-HC group（P<0.05）;A-HC was lower than A-NC group（P<0.05）.The index of arrhythmia of fruit flies in the W-HC was higher than that of the W-NC group（P<0.01）,and that of the W-HE group was lower than that of the W-HC group（P<0.01）;the index of arrhythmia of fruit flies in the A-NC group Lower than W-NC group（P<0.01）,A-NE lower than A-NC group（P<0.01）,A-HC higher than A-NC group（P<0.05）,A-HE group lower than A-HC group（P<0.01）.4 Conclusion1.A high-fat diet of 30%coconut oil on fruit flies for 5 days will up-regulate the expression of TOR,down-regulate the expression of Bmm and PGC-1,promote lipid accumulation throughout the body of fruit flies,induce heart dysfunction in fruit flies,and shorten the life of fruit flies.2.Regular exercise on fruit flies for two weeks suggests that exercise can down-regulate the expression of TOR,up-regulate the expression of Bmm and PGC-1,promote lipolysis of fruit flies,improve heart function of fruit flies,and prolong life of fruit flies.3.Bmm knock-down will cause Drosophila to produce systemic lipid accumulation similar to high-fat diet,cardiac dysfunction and shortened life expectancy.High fat will further aggravate these phenomena.And exercise can improve these phenomena.It suggests that exercise may improve the heart function of high-fat flies by up-regulating the expression of Bmm. |
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