| Heart failure(HF)is a syndrome characterised by symptoms(such as breathlessness,ankle swelling,and fatigue)and signs(eg,raised jugular venous pressure,pulmonary crackles,and peripheral oedema)caused by structural or functional cardiac abnormalities.According to the progress of heart failure,heart failure can be divided into acute heart failure and chronic heart failure.The high mortality and re-hospitalization rate of chronic heart failure(CHF)bring heavy social and economic burden.Currently,the incidence of HF in Europe is about 3/1000 person-years or about 5/1000person-years in adults.The prevalence of HF appears to be 1%-2% of all age-groups adults.According to the 2016 China Cardiovascular Disease Report,there are about 5 million patients with HF in China,which has become an important public health problem in the field of cardiovascular disease in China.In recent years,with the understanding of the pathogenesis of HF.The use of ACEI,ARB,β-beta blockers and aldosterone receptor antagonists significantly improved the symptoms and prognosis of HF.Recently,angiotensin receptor neprilysin inhibitor(ARNI)has become a new star in the field since SHIFT’s breakthrough(made by Ivabradine in the treatment of heart failure)in 2010.ARNI further reduced the risk of cardiovascular death,rehospitalization and all-cause death and the primary endpoint of heart failure.However,overall heart failure survival remained poor.Recently,cardiovascular protective effect of a novel glucose-lowering drug,sodium-glucose cotransporter 2 inhibitor has been identified in a cardiovascular outcome study.Several large multicenter,randomized,double-blind,placebo-controlled trials(EMPA-REG OUTCOME,CANVAS,DECLARE-TIMI 58,DAPA-HF)had shown a similar effect of SGLT2 inhibitors in reducing cardiovascular MACE events and heart failure rehospitalization rates in patients with and without diabetes.This protective effect may be independent of the hypoglycemic effect and regardless of age,sex,race,or geographic region.Dapagliflozin was approved by the US Food and Drug Administration(FDA)in 2020 for use in HFr EF adult patients with or without diabetes to reduce the risk of cardiovascular death and hospitalization.The 2021 European Guidelines for the treatment of Acute and chronic heart Failure recommend dapagliflozin and empagliflozin as class I for the treatment of HFr EF to reduce the risk of rehospitalization and death.However,the protective mechanism of SGLT2 inhibitors on cardiovascular events remains unclear.In this study,a rabbit model of chronic heart failure was established by ascending aorta constriction to observe the effects of dapagliflozin on the function of chronic heart failure and its potential molecular mechanism.Part one Established of a Normoglycemic Rabbit Model of Chronic Heart Failure by Ascending Aorta ConstrictionObjective: To explore the feasibility of establishing a normoglycemic rabbit model of CHF with ascending aorta constriction,and to observe the changes of cardiac structure,function and blood indexes after heart failureMethods:24 adult male New Zealand white rabbits,weighing3.0kg-3.5kg,were divided into 4 groups randomly: sham operation group(sham group,n=6),heart failure group(HF group,n=6),perindopril group(Peri group,n=6)and dapagliflozin group(DAPA group,n=6).In sham operation group,only thoracotomy was performed.After thoracotomy,the ascending aorta was constricted to 60% of its original diameter or circumference in other 18 animals of 3 groups for 12 weeks.The following indexes were observed before and 12 weeks after operation:(1)general conditions of each group including changes in body weight,respiratory rate,heart rate,appetite and mental state.(2)White blood cell,red blood cell,hemoglobin,albumin,potassium,sodium,blood glucose,plasma osmotic pressure and creatinine levels.(3)IVS,LVEDD,LVESD,LVPW and LVEF of heart.(4)plasma levels of NT-pro BNP.Results:1.Established of CHF rabbit model: All 6 rabbits in sham group survived after surgery.All the other 18 rabbits were successfully operated without death.All the animals showed anorexia,lethargy,decreased activity and tachypnea after operation.The above symptoms of sham group gradually recovered about 3 days.The symptoms of the other 18 rabbits were also significantly improved 3 days later,but worse than that of sham group.Echocardiography was performed to evaluate the success rate of the model,and EF≤40% was considered as successful model establishment.A total of 21 animals meted the designed standards,including 6 in Sham group,5 in HF group,5 in Peri group,and 5 in DAPA group.2.Plasma indexes of animals in each group:Before operation,there were no significant differences in body weight,WBC,RBC,hemoglobin,albumin,potassium,sodium,glucose,plasma osmotic pressure,creatinine levels among all groups(P > 0.05).12 weeks after surgery,there were no significant differences in body weight,WBC,RBC,albumin,potassium,sodium,glucose,plasma osmotic pressure and creatinine among all groups(P > 0.05).Hemoglobin in HF group,Peri group and DAPA group decreased compared with sham group(P < 0.05),but there was no statistical difference among the three groups(P > 0.05).3.Echocardiographic of animals in each group: There were no statistic difference in heart rate,IVS,LVEDD,LVPW,LVESD and LVEF among the four groups before surgery(P > 0.05).12 weeks after surgery,there were no statistic difference in IVS,LVEDD and LVPW among the four groups(P<0.05).Compared with sham group,HF group,Peri group and DAPA group showed increased heart rate,LVESD and LVEF(P<0.05).There were no significant differences in heart rate,LVESD and LVEF among the three groups(P > 0.05).4.NT-pro BNP levels in each group: There was no statistical difference in the levels of NT-pro BNP in the 4 groups before surgery(P > 0.05).12 weeks after surgery,plasma NT-pro BNP levels in HF group,Peri group and DAPA group increased compared with sham group(P<0.05).There was no significant difference among HF group,Peri group and DAPA group(P >0.05).Conclusion:1.The normoglycemic rabbit model of CHF can be successfully established by ascending aortic constriction with increased LVESD,decreased LVEF,elevated NT-pro BNP levels,increased heart rate and the corresponding clinical symptoms.2.Compared with sham group,there were no significant differences in WBC,RBC,serum albumin,potassium,sodium,glucose,osmotic pressure and creatinine in other 3 groups.Part Two Effects of Dapagliflozin on Cardiac Function in Normoglycemic Rabbit Model of Chronic Heart FailureObjective:To explore the effects of dapagliflozin on cardiac function in normoglycemic rabbit model of chronic heart failureMethods:A total of 21 animals meted the designed standards,including 6in Sham group,5 in HF group,5 in Peri group,and 5 in DAPA group.Both the sham and HF groups were given normal saline via gavage.Rabbits in the Peri or DAPA group were given perindopril(0.5 mg/kg/day)or dapagliflozin(1 mg/kg/day)by oral gavage for 10 weeks.The following indexes were observed 10 weeks after treatment:(1)general conditions of each group including body weight,respiratory rate,heart rate,appetite and mental state.(2)WBC,RBC,hemoglobin,albumin,potassium,sodium,blood glucose,plasma osmotic pressure and creatinine levels.(3)IVS,LVEDD,LVESD,LVPW and LVEF of heart.(4)plasma levels of NT-pro BNP.Results:1.General conditions of each group:All of 21 rabbits survived to the end of the experiment.Compared with sham group,animals in HF group had worse mental state,appetite,activity and respiration.Peri group and DAPA group were between Sham group and HF group,while there is no significant difference between Peri group and DAPA group.2.Body weight before and after drug treatment:There was no statistical difference in body weight among 4 groups both before and 10 weeks after treatment(P > 0.05).Body weight of all groups showed an increasing trend which reached statistical difference in sham group(P=0.016).This phenomenon may be related to the better appetite of sham group than that of other groups.3.Echocardiographic of animals before and after drug treatment:The echocardiographic in each group before drug intervention have been mentioned in part one.After 10 weeks of intervention,perindopril had no significant effect on LVESD,while dapagliflozin reduced LVESD but failed to achieve statistical significance.Both perindopril and dapagliflozin could increase LVEF(P<0.05),while dapagliflozin could further increase LVEF compared with perindopril,the difference was statistically significant(P<0.05).4.Heart rate before and after drug treatment:The heart rate of animals increased after heart failure,and the difference was statistically significant compared with that of sham group(P<0.05).Both perindopril and dapagliflozin could reduce the heart rate of CHF animal models(P<0.05),while there was no significant difference between Peri group and DAPA group(P > 0.05).5.NT-pro BNP levels before and after drug treatment:The levels of NT-pro BNP in the three groups was significantly higher than that in sham group(P<0.05).Both perindopril and dapagliflozin significantly reduced the level of NT-pro BNP in CHF animals(P<0.05),and dapagliflozin was superior to perindopril(P<0.05).6.Serum indexes before and after drug treatment:After 10 weeks of drug intervention,there were no significant differences in concentration of WBC,RBC,albumin,potassium,sodium,glucose,plasma osmotic pressure and creatinine among the four groups(P > 0.05).Compared with sham group,hemoglobin concentration of HF group,Peri group and DAPA group decreased(P < 0.05),and there was no statistical significance among HF group,Peri group and DAPA group(P > 0.05).Conclusion: Dapagliflozin can improve the state of lethargy,weak appetite and shortness of breath in CHF animals,and reduce the heart rate,plasma NT-pro BNP level and LVESD,the above effects are equal to or better than perindopril.There were no significant differences in body weight and concentration of WBC,RBC,hemoglobin,albumin,potassium,sodium,glucose,plasma osmotic pressure and creatinine among all groups.Part Three Effects of Dapagliflozin on Cardiac remodeling in Normoglycemic Rabbit Model of Chronic Heart FailureObjective: To explore the effects of dapagliflozin on cardiac remodeling in normoglycemic rabbit model of chronic heart failureMethods: The grouping of animals and drug interventions was the same as in part two.The following indexes were observed:(1)Body mass,whole heart mass,LV mass,the LV mass/body mass ratio.(2)HE staining,Masson staining,Collagen Ⅰ and Ⅲ immunohistochemistry staining of LV tissue.(3)protein levels of Collagen Ⅰ and Ⅲ,α-SMA and CTGF in myocardial tissues.Results:1.Body mass,heart mass and LV mass/body mass:There was no statistical difference in body mass among all groups(P > 0.05).HF group had higher whole heart mass,LV mass and LV/body mass than Sham group(P <0.05).The whole heart mass,LV mass and LV mass/body mass of Peri group were higher than those of Sham group(P < 0.05),but lower than those of HF group.The whole heart mass and LV mass/body mass of Peri group were statistically different from those of HF group(P < 0.05).DAPA group had significantly lower whole heart mass,LV mass and LV mass/body mass than HF group(P < 0.05),but had no statistical difference compared with Sham group(P > 0.05).DAPA group could further reduce whole heart mass,LV mass and LV mass/body mass compared with perindopril,and there was statistical significance in the decrease of whole heart mass and LV mass(P <0.05).2.HE staining: Perindopril and dapagliflozin can inhibit hypertrophy,degeneration and necrosis of cardiomyocytes in CHF animals,while dapagliflozin n is superior to perindopril.3.Masson staining: Compared with sham group,collagen fractions of HF group,Peri group and DAPA group were increased(P < 0.05),while collagen fractions of Peri group and DAPA group were significantly improved compared with HF group(P < 0.05).Compared with perindopril,dapagliflozin could further inhibit the progression of myocardial fibrosis(P <0.05).4.Immunohistochemistry staining of Collagen Ⅰ and Ⅲ:Collagen Ⅰand Ⅲ proteins in sham group were rarely expressed.Compared with Sham group,collagen Ⅰ and Ⅲ expression in HF group was increased,and the differences were statistically significant(P < 0.05).Collagen Ⅰ and Ⅲprotein expressions in both Peri group and DAPA group were lower than those in HF group(P < 0.05),and dapagliflozin was superior to perindopril(P<0.05).5.Western blot analysis of Collagen Ⅰ and Ⅲ,α-SMA and CTGF proteins: The expression levels of the above-mentioned proteins in HF group were higher than those in Sham group(P < 0.05).The expression levels of the4 proteins in Peri group and DAPA group were significantly lower than those in HF group(P < 0.05).Compared with perindopril,dapagliflozin could further decrease Collagen Ⅰ and Ⅲ expression levels(P < 0.05),whileα-SMA and CTGF protein expression levels were not significantly different between the two groups(P > 0.05).Conclusion:1.Dapagliflozin could improve the shape of the heart,reduce the total heart mass,LV mass and LV mass index of CHF animals,which was better than perindopril in reducing the total heart mass and LV mass.Dapagliflozin could inhibit hypertrophy,degeneration and necrosis of cardiomyocytes in CHF animals and was superior to perindopril.2.Masson staining showed that dapagliflozin could significantly inhibit myocardial fibrosis and decrease collagen fraction of myocardial tissue,which was better than perindopril.3.Dapagliflozin could inhibit Collagen Ⅰ and Ⅲ protein expression in myocardium of CHF rabbits,and it is better than perindopril.4.Dapagliflozin could inhibit the transformation of fibroblasts into myofibroblasts and the expression of CTGF in myocardial tissue of CHF rabbits,thereby inhibiting myocardial fibrosis.Part Four Effects of Dapagliflozin on TGF-β1/Smad pathway of my-ocardium in Normoglycemic Rabbit Model of Chronic HeartFailureObjective:To explore the effects of dapagliflozin on TGF-β1/Smad pathway of myocardium in Normoglycemic Rabbit Model of Chronic Heart FailureMethods:The grouping of animals and drug interventions was the same as in part two.The following indexes were observed:(1)Protein expression levels of TGF-β1,Smad2,Smad3 and Smad7 in myocardial tissue of experimental animals in each group.(2)Protein expression levels of MMP2,MMP9 and TIMI-1 in myocardial tissue of experimental animals in each group.Results:1.Western blot analysis of TGF-β1,Smad2,Smad3 and Smad7 proteins in myocardial tissue: Compared with Sham group,the expression of TGF-β1,Smad2 and Smad3 in HF group were significantly increased,while the expression levels of Smad7 were significantly decreased(P < 0.05).The expression levels of TGF-β1,Smad2 and Smad3 in Peri group were higher than those in Sham group,but significantly lower than those in HF group(P <0.05),while Smad7 was higher than those in Sham group and HF group(P <0.05).Dapagliflozin could reduce the expression TGF-β1,Smad2 and Smad3 and increase the expression levels of Smad7 in heart failure animals(P < 0.05),and the levels of Smad2 and Smad3 were lower than those of perindopril(P <0.05).2.Western blot analysis of MMP2、MMP9 and TIMI-1 proteins in myocardial tissue: The expression levels of MMP2,MMP9 and TIMI-1 in HF group were significantly higher than those in Sham group(P < 0.05).The expression levels of MMP2,MMP9 and TIMI-1 in Peri group and DAPA group were significantly lower than those in HF group(P < 0.05).The expression levels of MMP2 and TIMP1 in DAPA group were further lower than those in Peri group(P < 0.05).Conclusion:1.Dapagliflozin can inhibit cardiac remodeling in normoglycemic rabbit model of CHF by inhibiting the TGF-β1/Smad signaling pathway,and was superior to that of perindopril.2.The inhibitory effect of dapagliflozin on myocardial remodeling in normoglycemic rabbit model of CHF was related to the inhibitory activity of MMPs/TIMP1,and was superior to that of perindopril. |
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