| 1 ObjectiveFly took regular exercise by the third generation of training device and fed a high-fat-diet.The experiment used the UAS-Gal4 system on Drosophila to regulate heart dSir2 and Nmnat gene expression.By detecting flies' cardiac function,myocardial NAD+-dSir2/Nmnat signaling pathways related indicators,myocardial TAG level and oxidative stress level,myocardial NAD+-dSir2/Nmnat signaling pathways related gene expression,and the expression of myocardial lipid metabolism related genes,exploring:(1)the effect of regular exercise on the cardiolipid damage and cardiac NAD+-dsir2/Nmnat pathway in high-fat diet flies;(2)the changes in the gene expression of dSir2 and Nmnat in the heart were affected by heart lipid metabolism and NAD+-dsir2/Nmnat pathway;(3)the effect of regular exercise on cardiac dSir2,Nmnat gene knockdown or mutant flies induced cardiolipid injury and NAD+-dsir2/Nmnat pathway.Providing a theoretical basis for NAD+-dSir2/Nmnat signaling pathway to mediate or improve the damage of myocardial lipid toxicity.2 Materials and methods2.1 Male wild type w1118 with female hand-Gal4 hybridization and grouping.Male wild-type w1118 hybridized with female hand-Gal4,collecting F1 generation of virgin flies,divided into control group(C),exercise group(E),high-fat-diet group(HFD),high-fat-diet-exercise group(HFD-E).1 week old experimental group began training or high-fat diet from the 2nd day of age;The experimental group of 3 weeks started training or-high-fat diet from 15 days old;The 5-week age experimental group began training or high-fat diet from 29 days of age.1 week young fruit flies and 3 weeks adult fruit flies were given 10 seconds of regular climbing exercise after each flip to the bottom of the tube.5 weeks old fruit flies were given 15 seconds of regular climbing after each flip in the bottom of the test tube.1.5 hours exercise each time,exercise and HFD for 5 consecutive days,the indicators were tested after the end of the exercise program 24 hours.2.2 Cardiac dSir2 and Nmnat gene expression changes and grouping.Male dSir2-UAS-overexpression was crossed to female hand-Gal4,collected the F1 dSir2-UAS-overexpression>hand-Gal4 group virgin flies,divided into dSir2-overexpression group(dSir2-O)and dSir2-O-HFD group;male dSir2-UAS-RNAi was crossed to female hand-Gal4,collected the F dSir2-UAS-RNAi>hand-Gal4 group virgin flies as dSir2-RNAi group and dSir2-RNAi-E group;heterozygous mutant Sirt15.26 cn1(male)was crossed toSirt14.5 cn1/SM6b,P{eve-lacZ8.0}SBl(female),collected the F1 dSir2-mutationhomozygous mutations as dSir2-mutation group and dSir2-mutation-E group.F1 dSir2-overexpression group(male)was crossed to dSir2-mutation group(female),collected the F2 dSir2-overexpression>dSir2-mutation virgin flies as dSir2-save group;male Nmnat-UAS-overexpression was crossed to female hand-Gal4,collected the F1 Nmnat-UAS-overexpression>hand-Gal4 group virgin flies,divided into Nmnat-overexpression group(Nmnat-O)and Nmnat-O-HFD group;male Nmnat-UAS-RNAi was crossed to female hand-Gal4,collected the F1 Nmnat-UAS-RNAi>hand-Gal4 group virgin flies as Nmnat-RNAi group and Nmnat-RNAi-E group;F1 Nmnat-overexpression group(male)was crossed to dSir2-mutation group(female),collected the F2 Nmnat-overexpression>dSir2-mutation virgin flies as Nmnat-save group.5-week experiment group started training or feeding a high fat diet from 29 days of age.Flies were fed HFD five consecutive days,and then we did next stepped experiments after 24 hours after HFD program ended.Indicators detection:M-mode detected the fly's heart function.dsir2 protein level was detected by western-blot.ELISA detected the level of the total body and heart TAG,NAD+ level,dSir2 deacetylation level,SOD activity level,and MDA level.Micrographs observed the accumulation of adipose tissue around the heart.Oil red O staining observed the accumulation of lipid in myocardium.The mitochondrial ultrastructure of myocardial cells and myocardial cells were observed by transmission electron microscopy.The mRNA expression of dSir2,Nmnat,dFoxo,dTOR,BMM,dFAS,PGC-1,and Col4al genes were detected by qRT-PCR.Fruit fly climbing ability measurement and life cycle statistics.3 Results3.1 The impact of regular exercise,high fat diet,and the combination of the two combined on the heart lipid damage,cardiac NAD+-dsir2/Nmnat pathway,and climbing and longevity.3.1.1 Results of M-mode in 5 weeks old fruit fliesCompared with C group,E group heart rates decreased(P<0.05),fraction shortening increased(P<0.01),arrhythmia indexes had no remarkable change(P>0.05),fibrillationsdecreased(P<0.05);HFD group heart rates increased(P<0.05),fraction shortening decreased(P<0.01),arrhythmia indexes increased(P<0.01),fibrillations increased(P<0.01);HFD-E group heart rates,fraction shortening,arrhythmia indexes,and fibrillations had no remarkable change(P>0.05).Compared with,HFD group,HFD-E group heart rates decreased(P<0.01),fraction shortening increased(P<0.01),arrhythmia indexes decreased(P>0.05),fibrillations decreased(P<0.05).3.1.2 Results of ELIS A and Western-Blot in 5 weeks old fruit fliesCompared with C group,E group heart TAG levels decreased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),NAD+levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01),and dSIR2 protein levels increased(P<0.01);HFD group heart TAG levels increased(P<0.01),dSIR2 protein acetylation levels decreased(P<0.01),NAD+ levels decreased(P<0.05),SOD levels decreased(P<0.01),MDA levels increased(P<0.05),and dSIR2 protein levels decreased(P<0.01);HFD-E group heart TAG levels decreased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),NAD+ levels increased(P<0.05),SOD levels had no remarkable change(P>0.05),MDA levels had no remarkable change(P>0.05),and dSIR2 protein levels had no remarkable change(P>0.05).Compared with HFD group,HFD-E group heart TAG levels decreased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),NAD+ levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01),and dSIR2 protein levels increased(P<0.01).3.1.3 Results of qRT-PCR in 5 weeks old fruit fliesCompared with C group,E group heart Nmnat,dSir2,PGC-1,bmm,and dFAS gene mRNA expression up-regulation(P<0.01),dTOR gene mRNA expression down-regulation(P<0.01),Col4al gene mRNA expression down-regulation(P<0.05),and the expression ratio of dFAS/bmm decreased;HFD group heart Nmnat gene mRNA expression down-regulation(P<0.05),dSir2 gene mRNA expression down-regulation(P<0.01),dFoxo gene mRNA expression down-regulation(P<0.05),PGC-1 gene mRNA expression down-regu lation(P<0.05),bmm gene mRNA expression up-regulation(P<0.01),dFAS gene mRNA expression up-regulation(P<0.01),dTOR gene mRNA expression up-regulation(P<0.01),Col4a1 gene mRNA expression up-regulation(P<0.01),and the expression ratio of dFAS/bmm increased;HFD-E group heart Nmnat gene mRNA expression up-regulation(P<0.01),dSir2,dFoxo,PGC-1 gene mRNA expression had no remarkable change(P>0.05),bmm gene mRNA expression down-regulation(P<0.05),dFAS,dTOR,and Co14a1 gene mRNA expression had no remarkable change(P>0.05),and the expression ratio of dFAS/bmm decreased.Compared with HFD group,HFD-E group heart Nmnat,dSir2,dFoxo,PGC-1,and bmm gene mRNA expression up-regulation(P<0.01),dFAS,dTOR,and Col4a1 gene mRNA expression down-regulation(P<0-01),and the expression ratio of dFAS/bmm decreased.3.1.4 Results of heart oil red O staining,scanning electron microscopy,and microscopic imageOil red O staining on fruit flies heart,we found HFD could significantly increase lipid droplets inside the heart,but regular exercise could decrease lipid droplets inside HFD flies heart.Transmission electron microscopy observation,the HFD flies heart myocardial cell heart muscle fibers is small,irregular directions;mitochondria numbers decreased,and are abnormal morphology,and part of the matrix swelling or even form small cavity;There are a large number of vacuoles in myocardium and similar lipid droplets,which might affect myocardial fiber arrangement and direction.While E group and HFD-E group myocardial cell heart muscle fiber volume,order,direction were regular;Mitochondria numbers and volume were decreased.In the heart microscopic images,there were much lipid accumulation around the heart in HFD group flies,this makes the deputy myocardial cell morphology change.C,E,HFD-E group flies hearsrarely happen this phenomenon.When sucking the fat around the heart,HFD group flies around the heart of lipid vice myocardial cell adhesion formation as a whole.If roughly getter it,cardiac function will likely damage.3.1.5 Results of climbing indexIn 1-week-old flies,E group flies climbing indexes were higher than C group flies(P<0.01),no significant difference between HFD group flies climbing indexes and C group flies(P>0.05),no significant difference between HFD-E group flies climbing indexes and C group flies(P>0.05),HFD-E group flies climbing indexes were higher than HFD group flies(P<0.05).In 3-week-old flies,E group flies climbing indexes were higher than C group flies(P<0.05),HFD group flies climbing indexes were lower than C group flies(P<0.01),HFD-E group flies climbing indexes were lower than C group flies(P<0.01),HFD-E group flies climbing indexes were higher than HFD group flies(P<0.05).In 5-week-old flies,E group flies climbing indexes were higher than C group flies(P<0.05),HFD group flies climbing indexes were lower than C group flies(P<0.01),HFD-E group flies climbing indexes were lower than C group flies(P<0.01),no significant difference between HFD-E group flies climbing indexes and HFD group flies(P>0.05),HFD-E group flies climbing indexes were lower than E group flies(P<0.01).3.1.6 Results of lifespanE group flies lifespan was longer than C group flies(P<0.01),HFD group flies lifespan was shorter than C group flies(P<0.01),no significant difference between HFD-E group flies lifespan and C group flies(P>0.05),HFD-E group flies lifespan was longer than HFD group flies(P<0.01).3.2 The effects of cardiac dSir2 and Nmnat gene expression changes on the heart function,cardiac lipid toxicity,and cardiac NAD+-dsir2/Nmnat pathway.3.2.1 Results of M-modeCompared with Normal-expression group,dSir2-RNAi group heart rate increased(P<0.05),fraction shortening decreased(P<0.05),arrhythmia indexes increased(P<0.0i),fibrillation increased(P<0.05);dSir2-mutation group heart rate increased(P<0.01),fraction shortening decreased(P<0.01),arrhythmia indexes increased(P<0.01),fibrillation increased(P<0.01);dSir2-O group heart rate decreased(P<0.01),fraction shorteningincreased(P<0.01),arrhythmia indexes had no significant changes(P>0.05),fibrillation decreased(P<0.01);Nmnat-RNAi group heart rate increased(P<0.01),fraction shorteningdecreased(P<0.01),arrhythmia indexes increased(P<0.01),fibrillation increased(P<0.01);Nmnat-O group heart rate decreased(P<0.05),fraction shortening increased(P<0.05),arrhythmia indexes had no significant changes(P>0.05),fibrillation had no significant changes(P>0.05).3.2.2 Results of ELISACompared with Normal-expression group,dSir2-RNAi group heart TAG levels increased(P<0.01),dSIR2 protein acetylation levels decreased(P<0.01),NAD+ levels decreased(P<0.01),SOD levels decreased(P<0.01),MDA levels increased(P<0.01);dSir2-mutationgroup heart TAG levels increased(P<0.01),dSIR2 protein acetylation levels decreased(P<0.01),NAD+levels decreased(P<0.05),SOD levels decreased(P<0.01),MDA levels increased(P<0.05);dSir2-O group heart TAG levels decreased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),NAD+ levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01).3.2.3 Results of qRT-PCRCompared with Normal-expression group,dSir2-RNAi group heart Nmnat expression down-regulation(P<0.05),dSir2 and dFoxo expression down-regulation(P<0.01),PGC-1 expression down-regulation(P<0.05),bmm expression down-regulation(P<0.01),dFAS,dTOR,and Col4a1 expression up-regulation(P<0.01),and the expression ratio of dFAS/bmm increased.Compared with Normal-expression group,dSir2-mutation group heart Nmnat expression had no remarkable changes(P>0.05),dSir2,dFoxo,PGC-1,bmm expression down-regulation(P<0.01),dFAS,dTOR,and Col4al expression up-regulation(P<0.01),and the expression ratio of dFAS/bmm increased.Compared with Normal-expression group,dSir2-O group heart Nmnat,dSir2,dFoxo,PGC-1,bmm expression up-regulation(P<0.01),dFAS,dTOR,and Col4a1 expression up-regulation(P<0.01),and the expression ratio of dFAS/bmm decreased.Compared with Normal-expression group,Nnmnat-RNAi group heart Nmnat,dSir2,and dFoxo expression down-regulation(P<0.05),PGC-1 and bmm expression down-regulation(P<0.01),dFAS expression had no remarkable changes(P>0.05),dTOR expression up-regulation(P<0.01),and Col4al expression up-regulation(P<0.05),and the expression ratio of dFAS/bmm increased.Compared with Normal-expression group,Nnmnat-O group heart Nmnat,dSir2,dFoxo,PGC-1,dFAS,and bmm expression up-regulation(P<0.01),dTOR expression had no remarkable changes(P>0.05),Col4al expression down-regulation(P<0.01),and the expression ratio of dFAS/bmm decreased.3.3 The effects of regular exercise on the heart dSir2 and Nmnat gene knockdown or mutation induced cardiac lipid damage and cardiac NAD+-dsir2/Nmnat pathway.3.3.1 Results of M-modeCompared with dSir2-RNAi group,dSir2-RNAi-E group heart rate decreased(P<0.01),fraction shortening increased(P<0.01),arrhythmia indexes decreased(P<0.01),fibrillation decreased(P<0.01).Compared with dSir2-mutation group,dSir2-mutation-E group heart rate decreased(P<0.01),fraction shortening increased(P<0.05),arrhythmia indexes decreased(P<0.05),fibrillation decreased(P<0.01).Compared with Nmnat-RNAi group,Nmnat-RNAi-E group heart rate decreased(P<0.01),fraction shortening increased(P<0.05),arrhythmia indexes decreased(P<0.05),fibrillation decreased(P<0.01).3.3.2 Results of ELISACompared with dSir2-RNAi group,dSir2-RNAi-E group heart TAG levels decreased(P<0.01),NAD+ levels increased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01).Compared with dSir2-mutation group,dSir2-mutation-E group heart TAG levels decreased(P<0.01),NAD+ levels increased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01).Compared with Nmnat-RNAi group,Nmnat-RNAi-E group heart TAG levels decreased(P<0.01),NAD+ levels increased(P<0.01),dSIR2 protein acetylation levels increased(P<0.01),SOD levels increased(P<0.01),MDA levels decreased(P<0.01).3.3.3 Results of qRT-PCRCompared with dSir2-RNAi grou,dSir2-RNAi-E group heartNmnat,dSir2,dFoxo,PGC-1,and bmm expression up-regulation(P<0.01),dFAS,dTOR,and Col4a1 expression down-regulation(P<0.01),and the expression ratio of dFAS/bmm decreased.Compared with dSir2-mutation group,dSir2-mutation-E group heart Nmnat,dSir2,PGC-1,dFAS,and bmm expression up-regulation(P<0.01),dTOR and Col4a1 expression down-regulation(P<0.01),dFoxo expression had no remarkable changes(P>0.05),and the expression ratio of dFAS/bmm decreased.Compared with Nmnat-RNAi group,Nmnat-RNAi-E group heart Nmnat,dSir2,PGC-1,dFoxo,dFAS,and bmm expression up-regulation(P<0.01),dTOR and Col4a1 expression down-regulation(P<0.05),and the expression ratio of dFAS/bmm decreased.4 Conclusion4.1 High fat diet can induce myocardial lipid toxic injury,the molecular mechanism is closely related to fly heart NAD+-dSir2/Nmnat signaling pathways related factors activity were suppressed,and the dTOR,Col4a1 expression,the dFAS and bmm expression ratio increased.4.2 Regular exercise can reduce the risk of myocardial lipid toxic injury occurred,the molecular mechanism is closely related to regular exercise can increase the flies heart NAD+-dSir2/Nmnat signaling pathways related factor activity,and decrease the dTOR,Col4a1 expression,the dFAS and bmm expression ratio.4.3 Heart gene Nmnat and dSir2 were two key factors of NAD+-dSir2/Nmnat signaling pathway,which had important regulatory effect on the damage of myocardial lipid toxicity.Appropriate overexpression of Nmnat and dSir2 gene can activate NAD+-dSir2/Nmnat pathway,whichcan resist the effects of high-fat diet induced myocardial lipid toxicity;Knockdown or mutation inhibited NAD+-dSir2/Nmnat pathway and induced myocardial lipid toxicity.The NAD+-dSir2/Nmnat pathway was involved in the regulation of the expression of dTOR,Col4a1,dHAS and bmm express ratio.4.4 Regular exercise can resist a high-fat diet induced myocardial lipid toxic injury,also can improve the heart Nmnat and dSir2 knockdown or mutations induced myocardial lipid toxic injury,its mechanism is that regular exercise can actively adjust the flies heart NAD+-dSir2/Nmnat signaling pathways. |
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