Study On Protective Effect Of CDK4/6 Inhibitor Palbociclib In Diabetic Cardiomyopathy And Their Mechanisms

Background: With the development of society and economy,the incidence of cardiovascular disease has been increasing year by year,and it has become a huge health problem that the world needs to face together.Diabetic cardiomyopathy is defined as a disease of ventricular dysfunction observed in diabetic patients and has nothing to do with coronary artery disease,valvular disease or hypertension.Today,the criteria for diagnosing diabetic cardiomyopathy include left ventricular diastolic dysfunction and/or reduced left ventricular ejection fraction(LVEF),pathological left ventricular hypertrophy and interstitial fibrosis.This left ventricular remodeling may be manifested as eccentric dilation with left ventricular systolic and diastolic dysfunction.At present,the pathogenesis of diabetic cardiomyopathy is relatively complicated and not fully understood,and there is a lack of specific diagnosis and treatment methods.Changes in myocardial energy metabolism substrates caused by hyperglycemia,myocardial cell apoptosis,microvascular dysfunction,renin-angiotensinaldosterone system(RAAS)activation,oxidative stress activation,mitochondrial dysfunction,and abnormal Ca2+ signal regulation,are considered to be the mainstream pathogenesis.Cardiomyocyte apoptosis is an important pathological event during diabetic cardiomyopathy,which may lead to myocardial contractile dysfunction,heart remodeling and eventually heart failure.This event can be triggered by many factors,such as oxidative stress,inflammation,endoplasmic reticulum stress,and advanced glycation end products(AGEs).Sustained hyperglycemia will stimulate oxidative stress to activate and promote the generation of ROS,which may lead to the release of lactate dehydrogenase(LDH)and the increase of malondialdehyde(MDA)content,while inhibiting antioxidant superoxide dismutase(SOD).The expression of catalase(CAT)and glutathione peroxidase(GSH-Px)are all related to cardiomyocyte apoptosis.In addition,there are few methods to alleviate cardiomyocyte apoptosis triggered by oxidative stress.Therefore,it is of great significance to find effective anti-diabetic cardiomyocyte apoptosis drugs.Purpose: To clarify the expression of two important cell cycle regulators,CDK4 and CDK6,in STZ diabetic mice,and to evaluate the effects of CDK4/6 inhibitor Palbociclib on cardiac function,oxidative damage and cardiomyocyte apoptosis in STZ diabetic mice.To clarify whether the CDK4/6 inhibitor Palbociclib can treat DCM by inhibiting the up-regulated cell cycle kinases under pathological conditions,and explore the relevant mechanism of Palbociclib inhibiting cardiomyocyte apoptosis.Methods: Construct STZ diabetic mice,use echocardiography and other methods to analyze the effect of CDK4/6 kinase selective inhibitor Palbociclib on mouse heart damage;use enzyme kits to detect oxidative damage;use immunohistochemistry and western blotting to detect cells Apoptosis and inflammation occur.The HG-induced rat cardiac fibroblast H9c2 cell model was constructed,and the effects of CDK4/6 inhibitor Palbociclib on apoptosis and mitochondrial oxidative stress were investigated by means of enzymatic kits,flow cytometry,and western blotting.To explore the mechanism of Palbociclib’s inhibition of cardiomyocyte apoptosis.Results: This study found for the first time that the expression of cell cycle kinases CDK4 and CDK6 were up-regulated in the hearts of STZ diabetic mice,suggesting that they may be a new target for the treatment of diabetic cardiomyopathy.By using Palbociclib,a selective inhibitor of CDK4/6 kinase,to treat STZ diabetic mice,we found that this new targeted therapy can improve the heart damage of STZ diabetic mice,and reduce the oxidative stress damage and inflammation in the mouse heart.Unlike traditional treatments such as metformin,Palbociclib has no regulatory effect on blood sugar regulation;after treatment,the weight of the mice did not change abnormally.However,Palbociclib has an effect on left ventricular ejection fraction(LVEF),fractional shortening fraction(FS),mitral valve inflow velocity ratio(E/A),left ventricular intradiastolic diameter(LVIDd),and left ventricular intrasystolic diameter(LVIDs),Ventricular septal thickness(IVS),left ventricular posterior wall thickness(LVPWd),dp/dt max and dp/dt min,these key cardiac indicators have improved.Through animal and cell experiments,we found that Palbociclib,a CDK4/6inhibitor,effectively inhibited cardiomyocyte apoptosis,and explored the anti-apoptotic mechanism of Palbociclib in cardiomyocytes under diabetes.The expression of cleaved caspase-3 and caspase-9 was significantly increased in cardiomyocytes exposed to high glucose(HG),indicating that the mitochondrial pathway is involved.Interestingly,Palbociclib significantly inhibited the expression of cleaved caspase-3 and caspase-9.Because Bcl-2 protein is an important mediator of cell apoptosis,it plays an important role in stabilizing the mitochondrial membrane potential.We found that in cardiomyocytes,the overproduction of ROS increases the ratio of Bax/Bcl-2,reduces the mitochondrial membrane potential,and further mediates the expression of caspase-3.Palbociclib restores the ratio of Bax/Bcl-2 by up-regulating the anti-apoptotic protein Bcl-2 and down-regulating the pro-apoptotic protein Bax,and stabilizes the mitochondrial membrane potential of cardiomyocytes,ultimately inhibiting cardiomyocyte apoptosis under high glucose conditions.Conclusion: Therefore,this study proposes that the cell cycle kinases CDK4 and CDK6 may be potential therapeutic targets for diabetic cardiomyopathy,and proves that Palbociclib attenuates cardiomyocyte apoptosis mediated by high glucose by inactivating the Rb signaling cascade.Combined with the results of our animal experiments,Palbociclib may have more potential to treat diabetic cardiomyopathy than through conventional blood sugar regulation.Provide theoretical and experimental basis for new strategies for clinical prevention and treatment of diabetic cardiomyopathy.