| Diabetic cardiomyopathy(DCM)is primarily caused by diabetes,which is independent of coronary artery disease and hypertension,which leading to cardiac diastolic dysfunction at the forepart and systolic dysfunction in terminal.The main pathological characteristics of DCM is suppressed glucose metabolism,elevated fatty acid metabolism and lipid accumulation within myocardial cell.Because of no significant clinical symptoms in the early stage of DCM,it is hard to identify until presenting with heart failure.At that time,DCM is difficult to control effectively,so it is crucial to act at the early stage of DCM.Liraglutide,a glucagon like peptide-1(GLP-1)receptor agonist,has been proved effectively for glycemic control,lipid profile control,body weight management,and safety for diabetes mellites treatment.It also has cardiovascular benefit.However,there are limited studies illustrate the effect of Liraglutide on cardiac lipid disorder and whether reduced cardiac steatosis will protect diabetic heart.Besides,whether give liraglutide at the early stage of DCM will reverse the progress is also still unknow.Aerobic interval training(AIT)has been proved to improve cardiac function,reverse left ventricular remodeling,so it can be used at rehabilitation of heart failure patients.Can AIT regulate cardiac steatosis in DCM rats and protect diabetic heart is still unknown.The safety of AIT intervention on DCM in early phage is also unknown.As the “pharmacological therapy” is the core of the hygiene system,which increased the pressure of national economy.Apart from pharmacological treatment to DCM,exercise intervention also works.However,no study has been conducted to investigate the protective effects of AIT combined with liraglutide on DCM.Whether the two have superimposed effects is unknown.Objectives:This study is aimed to investigate the cardiac morphology and function changes during DCM pathological progress through establishing animal model of DCM.Then compared the effects of 8 weeks liraglutide and/or AIT intervention on reducing cardiac lipotoxicity to explore whether the effects of combination therapy are better than single pharmacological treatment or exercise intervention.Finally,further explore the mechanisms of cardiac lipotocity improvement and the internal mechanisms of the feasibility of drug and exercise intervention,to provide experimental basis for the clinical application of exercisedrug combination intervention.Methods:(1)Establishment of DCM model.110 male adult Wistar rats(250-280 g,8 weeks old)rats were randomly divided into control group(CON),diabetic cardiomyopathy group(DCM),DCM with high dose of liraglutide group(DH),DCM with low dose of liraglutide(DL),DCM with aerobic interval training group(DE)and DCM with low dose of liraglutide and AIT(DLE).All the subgroups of diabetic rats were injected with small dose of STZ 35 mg/kg by intraperitoneal style after 4 weeks of high fat diet.Diagnostic criteria of type 2 diabetic rats: two consecutive fasting glucose?11.1 mmol/L after STZ injection.Two weeks after model stabilization intervention program was began.During intervention schedule,blood glucose,body weight,food intake and water intake were measured.Pre-,during and post-intervention,cardiac function was measured by echocardiology.(2)Compare the effects of glucose control and cardiac protection of different intervention.Serum samples were analyzed for triacylglycerol(TAG),total-cholesterol(TC),high density lipoprotein cholesterol(HDL-c),low density lipoprotein cholesterol(LDL-c),fasting blood glucose(FBG),glycosylated hemoglobin(Hb A1c),fasting insulin(FINS)to compare the effects of different intervention on serum metabolic parameters.Through echocardiology and serum creatine kinase(CK),lactate dehydrogenase(LDH),B-type natriuretic peptide(BNP),cardiac troponin T(c TNT)level to evaluate cardiac function.Heart mess index HMI)cross sectional area(CSA)were used to identify cardiac hypertrophy.HE staining,oil red O staining and electron microscope were measured to observe cardiac lipid accumulation.The FFA and DAG concentration in the supernatant was determined using the enzyme-based colorimetric assay to measure lipotocity.(3)Explore the mechanisms underlying the cardiac protection function of liraglutide combined with AIT intervention on DCM.The expression levels of cardiac protection related genes ?-MHC,?-MHC,PPAR?,GSK3?were detected with real-time quantitative PCR,the expression levels of lipid metabolism related genes and protein CD36,CPT-1,PPAR?,AMPK,FOXO1 were evaluated to explore the mechanisms of cardiac protection.To measure serum GLP-1 level and cardiac tissue GLP-1 and GLP-1R protein expression level to explore the mechanisms of combination therapy.Results:1.Establishment of DCM model.(1)General situation of all the rats.After the intervention,54 rats finished the experiment.The CON group had a good mental state,quick reaction.DCM rats showed poor spirit,slow weight gain,dur dirty and dull.After 8 weeks intervention,body weight increased,fur become clean.(2)8 weeks blood glucose changes.There were no significant changes of DCM rats before intervention(P>0.05),but they were still higher than CON group(P<0.01).During intervention period,FBG levels in different groups had varying degrees of reduction.According to two-way ANOVA,Liraglutide and AIT have a significant effct on FBG levels during intervention(P<0.05),interection effect of Liraglutide and AIT have non-significant effect on FBG levels(P > 0.05),which mains Liraglutide and AIT may reduce FBG in different ways.(3)8 weeks cardiac function changes.During 8 weeks intervention,the heart function of DCM rats gradually changes from impaired diastolic function to impaired systolic function.Before intervention,diastolic parameters E/A ratio,IVRT and EDT significantly increased(P<0.05);During intervention,no significant differences was found in the diastolic and systolic function parameters in DCM group and CON group(P > 0.05).After intervention E/A ratio and EDT increased with LVEF significantly decreased(P<0.05).2.The effects of different interventions on DCM.(1)Liraglutide combined with AIT reduced DCM rats FBG and blood lipid profiles.Compared with the DCM groups,FBG,Hb A1 c,TAG,LDL-c levels were decreased(P<0.05)in 4 intervention groups,with no significant changes between 4 intervention groups(P>0.05).The serum levels HDL-c levels were significantly increased when compared with DCM group(P<0.05).According to twoway ANOVA,Liraglutide,AIT and interaction effect have significant effects on FBG ?Hb A1c?HDL and LDL levels(P<0.05),Liraglutide and AIT have significant main effect on TG levels(P<0.05),not interaction effect(P>0.05).(2)Liraglutide combined with AIT reduced insulin resistance in DCM rats.FIN levels in DH group increased significantly when compared with DCM groups(P<0.05),while FIN levels in DL,DE and DLE group were not significantly elevated(P>0.05).HOMA-IR levels in 4 groups were significantly lower than DCM group(P<0.05),but they were no different between subgroups(P>0.05).According to two-way ANOVA,Liraglutide,AIT and interaction effect have significant effects on HOMA-IR(P<0.05),which means that pharmacological treatment and AIT may reduce insulin resistance and the combination of the two has a strengthening effect.(3)Liraglutide combined with AIT reduced of serum myocardial injury biomarkers.When compared with group DCM,the c TNT and CK levels in 4 groups were significantly reduced(P<0.05).BNP levels were significantly lowered in DH,DL and DLE group than DCM group(P<0.05),no statistical differences between DE and DCM group(P>0.05).According to two-way ANOVA,Liraglutide,AIT and interaction effect have significant effects on c TNT and CK(P < 0.05),which means that combination therapy have a strengthen effect;Liraglutide have significant main effects and interaction effect on BNP levels,AIT have nonsignificant effect on BNP(P>0.05),which means that Liraglutide may reduce BNP levels,but combination therapy has a offset effect.(4)Liraglutide combined with AIT improved cardiac function.Compared to the DCM group,4 intervention groups showed decreased E/A ratio and improved LVEF(P<0.05),without statistical changes among all groups(P>0.05).According to two-way ANOVA,Liraglutide,AIT and interaction effect have significant effects on E/A ratio(P<0.05),which means that combination therapy have a strengthen effect;AIT have significant main effects and interaction effect on LVEF levels(P<0.05),Liraglutide have non-significant effect on LVEF(P > 0.05),which means that the improvement of systolic function is rely on AIT.(5)Liraglutide combined with AIT reduced cardiac lipid accumulation.From the results of HE staining,oil red O staining and electron microscope,we found that many lipid droplets were deposited in the myocardium of diabetic rats,mainly under the myofibrillar membrane and around the mitochondria.Comparison with the control group,high HMI with normal CSA demonstrated that DCM heart present eccentric hypertrophy.DH,DL,DE and DLE group reduced ectopic accumulation of lipid,HMI were reduced in DH and DLE group(P<0.05),CSA in group DE were significantly higher than DCM group(P < 0.01).According to two-way ANOVA,Liraglutide and AIT have significant effects on HMI(P<0.05),but no interaction effect(P>0.05);Liraglutide,AIT and interaction effect have significant effects on CSA(P <0.05),which means that both two may alleviate centrifugal cardiac hypertrophy,but combination therapy may have offset effect in some extent.(6)Liraglutide combined with AIT reduced FFA and DAG content within cardiac tissue.Consistent with the histological findings,the heart FFA and DAG content were significantly higher in DCM group than in the CON group and were significantly reduced by Liraglutide and/or AIT treatment(P<0.05).There were no significant changes of cardiac steatosis between 4 groups(P>0.05).According to two-way ANOVA,Liraglutide and AIT have significant effects on FFA(P<0.05),but no interaction effect(P >0.05);Liraglutide,AIT and interaction effect have significant effects on DAG levels(P<0.05),which means that combination therapy have strengthen effect.3.The mechanisms underlying the heart protection function of liraglutide combined with AIT intervention.(1)Liraglutide combined with AIT improved indicators related to heart protection.Comparison with the DCM group,?-MHC m RNA levels were significantly reduced and ?-MHC m RNA levels were significantly increased in DH,DL,DE and DLE group(P<0.01),with increased PPAR? m RNA expression and decreased GSK3? m RNA expression(P<0.01).According to two-way ANOVA,AIT have significant main effects and interaction effect on ?-MHC m RNA expression(P < 0.05),Liraglutide have non-significant effect on it(P>0.05);Liraglutide,AIT and interaction effect have significant effects on ?-MHC m RNA expression(P < 0.05),which means that the combination therapy have strengthen effect.Liraglutide have significant main effect on PPAR? and GSK3? m RNA expression(P<0.05),AIT and interaction of AIT and Liraglutide also have non-significant effect on PPAR? and GSK3? m RNA expression(P>0.05).(2)Liraglutide combined with AIT inhibited lipid metabolism related gene and protein expression.Compared with DCM group,m RNA and protein levels of CD36 and CPT-1 were significantly reduced in 4 intervention groups(P<0.01).Likewise,the m RNA and protein levels of PPAR? markedly increased in diabetic heart(P<0.01),and this increased reverted by Liraglutide and/or AIT intervention(P<0.01).According to two-way ANOVA Liraglutide,AIT and interaction effect have significant effects on CD36,CPT-1 and PPAR? m RNA and protein expression(P<0.05),which means that the combination therapy have strengthen effect.(3)Liraglutide combined with AIT activate AMPK-FOXO1 signaling pathway.Compared with group DCM,m RNA expression of AMPK was significantly increased and FOXO1 m RNA expression was significantly decreased in the hearts of rats in 4 intervention groups(P<0.01).The protein levels of AMPK did not change remarkable among these groups(P>0.05),but P-AMPK protein expression significantly increased in 4 intervention groups(P<0.05).FOXO1 protein expression was inhibited in DH,DL,DE and DLE group when compared with group DCM,so inhibited “fatty acid induced lipid metabolism”.According to two-way ANOVA Liraglutide,AIT and interaction effect have significant effects on AMPK m RNA expression and FOXO1 m RNA and protein expression(P<0.05),which means that the combination therapy have strengthen effect;Liraglutide and AIT have significant main effects on p-AMPK/AMPK protein relative expressions(P < 0.05),without interaction effect(P>0.05),which means the activation of AMPK by Liraglutide and AIT were different.(4)The effects of liraglutide combined with AIT on GLP-1/GLP-1R signaling pathway.Compared with DCM group,serum GLP-1 levels elevated in varying degrees in 4 treatment groups(P<0.05).Serum GLP-1 levels in DH group were significantly higher than DL,DE and DLE group(P<0.05),with no significant difference among these 3 groups(P>0.05).From western blot results we can know that compared with DCM group,the expression of GLP-land GLP-1R increased in 4 treatment groups(P<0.05).GLP-1 expression in DH group is significantly higher than DE group(P<0.05),with no difference between DL and DLE group(P>0.05).GLP-1R protein expression in DLE group significantly higher than DH and DL group(P<0.05).According to two-way ANOVA Liraglutide,AIT and interaction effect have significant effects on serum GLP-1 level and cardiac GLP-1 protein expression(P<0.05),which means that the combination therapy have strengthen effect;Liraglutide and AIT have significant main effects on cardiac GLP-1R protein expressions(P<0.05),without interaction effect(P > 0.05),which means the activation of GLP-1R by Liraglutide and AIT were different.Conclusion:(1)Aerobic interval training can significantly reduce FBG,improve cardiac function and myocardial contractility in DCM rats.It acts through activating AMPK-FOXO1 signaling pathway,reduce CD36 and CPT-1 expression,so inhibited fatty acid induced PPAR?,which further inhibited the uptake and metabolism of fatty acids in cardiomyocytes,reduce the heterotopic deposition of lipids and alleviate myocardial lipotoxicity.(2)AIT and Liraglutide have independent function on glycemic control and cardiac protection,combination therapy have better effect than single pharmacological treatment or exercise intervention,the reason is that on the one hand,it reduces the stress of exercise imposed on heart or offset the side effect of medicine,on the other hand,it improves sensitivity of GLP-1R in cardiomyocytes,which avoids the decrease in receptor sensitivity induced by long-term drug intervention.Therefore,liraglutide combined with AIT intervention has superposition effect. |
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