Study On The Protective Effect Of Ketone Bodies On Acute Cardiac Is Chemia/Reperfusion Injury And Chronic Aging-Related Myocardial Remodeling And Heart Dysfunction In Mice

BackgroundKetone body is the general name of acetoacetic acid,?-hydroxybutyric acid and acetone,which mainly comes from fatty acids and is the intermediate product of lipid metabolism.It can be used as an alternative energy source for glucose.Ketosis acidosis is a pathological ketosis,harmful to the human body.However,mild ketosis induced by ketogenic diet(KD)or intermittent fasting has been proved beneficial in animal model experiments,which can improve metabolism,prolong life span and improve nervous system function.Ketone bodies are involved in regulating the expression activity of many known key proteins affecting aging.They can not only serve as auxiliary fuel for heart failure,but also improve endothelial function,relieve oxidative stress and improve mitochondrial function.However,there is still debate about the role of ketone bodies in heart-related diseases,very little has been studied in heart senescence,and the mechanisms involved have not been clarified.In recent years,the role of ketone body has become a medical research topic in heart-related diseases.The current animal models of heart-related studies include models of ischemia/reperfusion injury,chronic heart failure,and age-related cardiac aging.Our group has completed the study of the role of ketone body supplementation in cardiac ischemia/reperfusion injury and age-related cardiac aging models.The study in the chronic heart failure model is still under way.We also designed different ketone body supplementation methods for the characteristics of cardiac ischemia/reperfusion injury and age-related cardiac aging models:in myocardial ischemia/reperfusion injury mice,we used D-?-hydroxybutyrate(BHB)for treatment;in age-related cardiac aging models,we used a long-term intake of KD to supplement ketone bodies.We initially observed and explored the mechanism of ketone bodies in two experimental models.Part ? The protective effect of exogenous ketone body therapy on cardiac ischemia / reperfusion injury in mice and the preliminary exploration of relevant mechanismsPurpose:The current work was designed to test the protection of D-?-hydroxybutyrate(BHB)against myocardial I/R injury and initially explored the possible mechanisms of actionExperimental approach:1.Male adult mice were randomly divided into three groups: sham operation treatment group(Sham),blank test group(I/R)and BHB treatment group(I/R BHB).Twenty-seven animals were housed in each group,and 9 animals were taken for each group at the detection index.The mice in the surgical treatment group underwent ligation of the left anterior descending(LAD)coronary artery to cause partial ischemia.Thirty minutes later,the operator disconnected the LAD ligation surgical line to perform regrouting.The mice in the sham operation treatment group used a similar procedure.But there is a difference that the silk thread is only bypassed LAD but not ligated.5 min before reperfusion,the mice were subcutaneously implanted with osmotic pumps.The BHB treatment group received exogenous D-?-hydroxybutyrate(1?l/h,1.6 mmol/kg/24 hours for 24 hours).The sham operation treatment group and the blank test group are equipped with saline pumps.2.After 24 hours,we removed the mouse heart and treated it with slices.Then Evans blue and TTC were given double staining and area statistical analysis were carried out to calculate the area of myocardial infarction.3.We used echocardiography and left ventricular catheterization to detect cardiac function in mice.The main indexes were ejection fraction(EF),short axis shortening rate(FS),ventricular pressure rising rate(DP/dt)and ventricular pressure decreasing rate(-DP/dt).4.The biochemical parameters of mice were measured 24 h after reperfusion: lactate dehydrogenase(LDH)and creatine kinase(CK)levels in serum and troponin I(c Tn I)levels in serum.The apoptotic cells were counted after myocardial TUNEL staining and the activity of Caspase-3 in myocardium was detected to evaluate the degree of apoptosis in myocardium.Detection of autophagy in myocardial tissue,including LC3-II/LC3-I ratios,p62 lysosomal-associated membrane protein-2(Lamp2)protein expression,to assess the flow of autophagy in cardiomyocytes.Myocardial mitochondrial reactive oxygen species(ROS)formation,ATP production,mitochondrial swelling,and mitochondrial membrane potential(MMP)levels were measured to assess myocardial mitochondrial function changes.Dihydrogen ethidium(DHE)staining and malondialdehyde(MDA)levels to assess oxidative stress in mouse myocardium.The expression levels of glucose regulatory protein 78(GRP78),C/EBP homologous protein(CHOP)and X box protein 1(XBP-1)were measured to assess endoplasmic reticulum stress in myocardium.Key results:.1.BHB effect of treatment on myocardial I/R injury in mice: the area of infarction in BHB treatment group was significantly reduced.BHB treatment significantly reduced serum c Tn I?CK and LDH levels in I/R mice.BHB treatment significantly reduced the number TUNEL positive cells and Caspase-3 activity in the myocardium of I/R mice.2.BHB treatment significantly increased the EF?FS? DP /dt?-DP/dt level of I/R mice and alleviated the decline of cardiac function in I/R mice.3.The effect of BHB therapy on myocardial mitochondrial function in I/R mice:BHB therapy significantly reduced myocardial mitochondrial ROS in I/R mice,increased mitochondrial ATP,reduce mitochondrial swelling,MMP.4.The effect of BHB therapy on myocardial oxidative stress in I/R mice: BHB therapy significantly reduced the intensity of myocardial DHE staining fluorescence,reduced myocardial MDA generation.5.The effect of BHB therapy on endoplasmic reticulum stress in the myocardium of I/R mice: immunohistochemistry and RT-PCR detection showed that BHB therapy significantly reduced the expression of GRP78?CHOP and XBP-1 in myocardial tissue of mice.6.The effect of BHB therapy on autophagic flow in the myocardium of I/R mice:BHB therapy reduced the ratio and p62 expression in the myocardium of I/R mice and increases the protein expression in the myocardium of I/R mice,suggesting that BHB therapy promotes autophagy flow levels in the myocardium of I/R mice.Conclusions and Implications:Exogenous BHB reduced myocardial I/R damage and improved cardiac function in I/R mice.The mechanisms involved include improving mitochondrial function in cardiomyocytes,mitigation of oxidative stress and ER stress,and promoting levels of autophagic flow in the myocardium of I/R mice.Part ? Study on the protective effect of ketogenic diet on chronic aging-related myocardial remodeling and cardiac dysfunction and preliminary exploration of relevant mechanismsPurpose:In this part,we propose a diet therapy.By long-term intake of KD,we maintained a high level of ketone body in mice,tried to improve the aging associated myocardial remodeling and cardiac function decline in mice,and preliminarily observed and explored the molecular mechanism.Experimental approach:1.The 20-month-old aged male mice and 8-week-old young were fed with KD or standard chow for four months.Twenty-eight animals were housed in each group,and 9animals were taken for each group at the detection index.Divide the male mice into four groups: adult group with normal diet(Adults),adult KD group(Adults KD),elderly normal diet group(Aged),and elderly KD group(Aged KD).2.?-hydroxybutyrate levels in plasma were measured regularly to determine KD ketogenic effects.3.After 4 months of feeding,cardiac function was evaluated by echocardiography.Major detection indicators included left ventricular end-systolic diameter(LVESD),FS.4.After four months of feeding,the myocardial indexes were detected.The m RNA expression of myocardial cross-sectional area(CSA),cardiac weight,and atrial natriuretic peptide(ANP)was detected to assess myocardial hypertrophy.Myocardial fibrosis and m RNA expression of ?1-I collagen genes(Col1A1)and ?-smooth muscle actin(?-SMA)were detected.The MDA,3-nitrotyrosine(3-NT),catalase,glutathione peroxidase(GPx)and glutathione/oxidized glutathione(GSH/GSSG)ratios were examined to assess oxidative stress.CHOP,GRP78,cleaved activated transcription factor 6(ATF6)and XBP1 s expression were detected to assess endoplasmic reticulum stress.Mitochondrial function was assessed by detecting mitochondrial ROS formation,ATP production,MMP,nicotinamide-adenine dinucleotide cytochrome reductase(NCCR)and succinate cytochrome c reductase(SCCR)activity.Myocardial P62 protein,Lamp2 protein expression and LC3-II/LC3-I ratio were measured to assess autophagy flux.Key results:1.The level of ?-hydroxybutyrate in plasma of mice increased significantly after KD intake.2.The effect of KD feeding on cardiac function in aged mice: echocardiographic results showed that long-term intake of KD could significantly reduce LVESD,increase FS levels in aged mice.3.The effect of KD feeding on myocardial hypertrophy and fibrosis in aged mice: in aged mice,KD can significantly reduce cardiomyocyte cross-sectional area(CSA),cardiac weight,and myothelin volume fraction;at molecular level,KD can significantly reduce the m RNA expression levels of ANP,Col1A1 and ?-SMA in aged mice myocardium.4.The effect of KD feeding on myocardial oxidative stress in elderly mice: in elderly mice,KD can significantly reduce myocardial MDA and 3-NT levels,increase myocardial SOD and CAT enzyme activities,and increase myocardial GPx activity and GSH/GSSG ratio.5.The effect of KD feeding on myocardial endoplasmic reticulum stress in aged mice:long-term ingestion of KD can significantly reduce myocardial cleaved ATF6,CHOP and XBP1 s expression levels in aged mice.6.The effect of KD feeding on the function of myocardial mitochondria in elderly mice: in elderly mice,long-term intake of KD can significantly reduce mitochondrial ROS production,increase ATP synthesis,and increase the activity III mitochondrial respiratory chain complex enzymes.Maintain the activity of electron coupling between complex I and III and between complex II and complex.7.The effect of KD feeding on myocardial autophagy in elderly mice: in myocardial tissue of elderly mice,long-term intake of KD can significantly increase the LC3-II/LC3-I ratio,increase Lamp2 expression,reduce the expression of P62 protein,suggesting that KD can promote myocardial autophagy.Conclusions and Implications:Long-term intake of KD reduces cardiomyocyte hypertrophy and myocardial tissue fibrosis in aged mice and alleviates decreased cardiac function in aged mice.Long-term intake of KD can reduce oxidative stress and ER stress in the elderly mouse myocardium,improve mitochondrial function,and can promote autophagic flow.