| Objective:Heart failure is closely related to myocardial energy metabolism disorders.Short-chain acyl-Co A dehydrogenase(SCAD)is a key enzyme in myocardial fatty acid β-oxidation,which negatively regulates heart failure and cardiomyocyte apoptosis.The physiological role of SCAD requires the participation of flavin adenine dinucleotide(FAD),and riboflavin is the precursor of FAD.However,whether riboflavin can activate SCAD by increasing the content of FAD,thereby inhibiting cardiomyocyte apoptosis and pressure overload-induced heart failure and its specific molecular mechanism remain unclear.This paper aims to observe the role of riboflavin in heart failure,to explore whether riboflavin can inhibit heart failure by increasing the content of FAD,activating SCAD,and promoting myocardial energy metabolism,and to explore its specific molecular mechanism for the prevention and treatment of heart failure.Method:1.A pressure overload heart failure model was established in C57BL/6J mice by transverse aortic constriction(TAC),and riboflavin was administered one week before and 8 weeks after surgery everyday.By observing the cardiac ultrasound indexes,various cardiac function indexes of the mice were detected.The ratio of heart weight to body weight was detected and the size of the heart were observed.The myocardial tissue sections of mice were subjected to hematoxylin-eosin staining(HE),Masson staining and picrosirius red(PSR)staining to observe and analyze morphological changes of the heart.Cardiomyocyte apoptosis was analyzed by TUNEL fluorescence staining.The expression of SCAD in myocardium was analyzed by immunofluorescence staining.The mitochondrial structure and function of mice were analyzed by detecting myocardial mitochondrial membrane potential and membrane swelling.The expression changes of related apoptotic proteins and SCAD were detected by Western blot,and the changes of SCAD m RNA were detected by real-time quantitative polymerase chain reaction(q PCR).The SCAD enzyme activity,free fatty acid(FFA),ATP and FAD contents in myocardial tissue of mice were detected,and the changes of energy metabolism in mice were analyzed.The Parkinson’s disease protein DJ-1(Park7),Kelch-like ECH-associated protein 1(Keap1),nuclear factor erythroid 2-related(Nrf2)and heme oxygenase-1(HO-1)were detected by Western blot experiment in each group of mice.The nuclear translocation of Nrf2 were also detected.2.The rat cardiomyocyte-like H9C2 cardiomyocytes were induced by tert-butyl hydroperoxide(t BHP)to construct an in vitro model of myocardial apoptosis.At the same time,riboflavin was used for pretreatment.Cardiomyocyte viability,reactive oxygen species level and degree of apoptosis were detected.The effect of riboflavin intervention on myocardial cell apoptosis was analyzed.The SCAD enzyme activity,FFA,ATP and FAD contents in cardiomyocytes were detected,and the changes of energy metabolism in cardiomyocytes were analyzed.The expression levels of DJ-1,Keap1,Nrf2 and HO-1 and the nuclear translocation of Nrf2 were detected by Western blot.Si RNA was used to interfere the expression of SCAD in H9C2 cardiomyocytes,and then t BHP was administered to stimulate cardiomyocytes.Western blot was used to detect the expression levels of DJ-1,Keap1,Nrf2 and HO-1,and to detect the nuclear translocation of Nrf2.In addition,si RNA was used to interfere with the expression of DJ-1in cardiomyocytes;H9C2 cardiomyocytes were infected with SCAD recombinant adenovirus to overexpress SCAD in cardiomyocytes,and then t BHP was administered to stimulate cardiomyocytes to construct a apoptosis model.Cardiomyocyte apoptosis rate was detected.Western blot was used to detect the expression levels of DJ-1,Keap1,Nrf2 and HO-1,and the nuclear translocation of Nrf2 was also detected.Result:1.Eight weeks after TAC in mice,various cardiac function indicators decreased;typical heart failure occurred;the ratio of heart weight and body weight and the ventricular cavity increased obviously.With extensive fibrosis and a large number of myocardial cell apoptosis,the level of reactive oxygen species in myocardial tissue increased significantly.Myocardial mitochondrial dysfunction occurred,the myocardial mitochondrial membrane potential was significantly declined,and the degree of membrane swelling was significantly increased.The ATP content,SCAD expression,enzyme activity and FAD content in myocardial tissue were significantly decreased,while the free fatty acid content in myocardium and serum was significantly increased.The above changes were significantly reversed when mice were treated with riboflavin 1 week before and 8 weeks after TAC.Compared with the Sham group,in the TACinduced heart failure model,the expressions of antioxidant stress proteins DJ-1,Nrf2 and HO-1 were markedly down-regulated,the expression of Keap1 was markedly up-regulated,and the nuclear translocation of Nrf2 was significantly decreased distinctly.After riboflavin treatment,the above protein expression changes were obviously reversed.2.Compared with the Con group,the cardiomyocytes in the t BHP group had a higher rate of apoptosis and higher level of ROS and the cell viability was significantly reduced.The content of ATP,SCAD expression,enzyme activity and FAD content were markedly decreased,and the content of FFA was obviously increased.Compared with t BHP group,Riboflavin+t BHP group had less cardiomyocyte apoptosis,increased cell viability,and significantly decreased reactive oxygen species level.SCAD protein,m RNA expression,enzyme activity,and ATP and FAD contents were significantly increased.At the same time,the FFA content was significantly reduced.It was shown that riboflavin can inhibit the apoptosis of cardiomyocytes.Compared with the Con group,in the t BHP-induced cardiomyocyte apoptosis model,the expressions of antioxidant stress proteins DJ-1,Nrf2 and HO-1 were down-regulated evidently,the expression of Keap1 was up-regulated distinctly,and the nuclear translocation of Nrf2 was obviously decreased.After riboflavin treatment,the above protein expression changes were obviously reversed.Compared with the NC+t BHP group,the expressions of DJ-1,Nrf2 and HO-1 in the si RNA-SCAD+t BHP group reduced more significantly,the expression of Keap1 increased more significantly,and the nuclear translocation of Nrf2 reduced significantly,indicating that the DJ-1-Keap1-Nrf2 signaling pathway is regulated by SCAD.Under the same conditions of t BHP-induced cardiomyocyte apoptosis,after interfering the expression of DJ-1 with si RNA-DJ-1 sequence,compared with the NC control group,the apoptosis rate of cardiomyocytes was significantly increased.The expression of DJ-1 was significantly down-regulated;the downstream of DJ-1,such as Nrf2 and HO-1 were significantly down-regulated;the expression of Keap1 was significantly up-regulated,and the nuclear translocation of Nrf2 was significantly reduced.In addition,after administration of SCAD recombinant adenovirus Ad-SCAD for 48 h,compared with the NC+Ad-GFP group,cardiomyocyte apoptosis rate was significantly reduced;the expressions of SCAD,DJ-1,Nrf2 and HO-1 in the NC+Ad-SCAD group were significantly increased;the expression of Keap1 was significantly decreased,and the nuclear translocation of Nrf2 was significantly increased.However,compared with si RNA-DJ-1+Ad-GFP group,there was no significant difference in the apoptosis rate of cardiomyocytes.The expression of SCAD in si RNA-DJ-1+Ad-SCAD group was significantly increased,but the protein expressions of DJ-1,Nrf2 and HO-1 were not significantly increased.There was no markable decrease in Keap1 expression and no significant increase in Nrf2 nuclear translocation.It was shown that SCAD can resist oxidative stress through DJ-1-mediated Keap1-Nrf2 signaling pathway.Conclusion:Riboflavin can activate SCAD by increasing the level of FAD in vivo and vitro,and then activate the downstream signaling pathway DJ-1-Keap1-Nrf2 related to SCAD,thereby inhibiting stress overload-induced heart failure and cardiomyocyte apoptosis. |
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