Effect Of Melanin Nanoparticles On Myocardial Injury Induced By Hyperglycemia And The Underlying Mechanisms

BackgroundDiabetic cardiomyopathy（DCM）is an important cause of death in diabetic patients,which is independent of hypertension,heart valve disease,coronary artery disease and congenital heart disease,and is characterized by left ventricular diastolic and/or systolic dysfunction and eventually progresses to heart failure.There is great evidence that DCM involves multiple pathogenic mechanisms,among which oxidative stress is the common mechanism of many pathological cases,which can promote myocardial fibrosis and myocardial remodeling and cause left ventricular diastolic and systolic dysfunction.Therefore,it is important to explore the pathogenesis of DCM from the perspective of oxidative stress and to develop related therapeutic drugs.Melanin contains various reducing functional groups such as catechol,amine and imine in its molecular structure,which can effectively scavenge reactive oxygen and nitrogen species as antioxidants.However,the application of natural melanin is limited by its low solubility,low content and tedious extraction.Melanin nanoparticles（MeNPs）,a biomimetic nanoparticle material developed by combining natural melanin and nanotechnology,possess the antioxidant ability of natural melanin and the low cytotoxicity and biocompatibility of ordinary nanoparticles.It has been shown that MeNPs have good antioxidant effects in cardiovascular diseases such as ischemic stroke and myocardial ischemia/reperfusion injury.However,the role and mechanism of MeNPs in DCM are still unclear.Therefore,this study intends to explore the therapeutic effect of MeNPs on DCM in combination with animal experiments and cell experiments.Objectives1.To explore the ability of melanin nanoparticles to scavenge reactive oxygen species.2.To explore the protective effect of melanin nanoparticles on myocardial injury caused by hyperglycemia.MethodsConstruction of MeNPs:Dopamine hydrochloride was oxidized and polymerized to polydopamine（PDA）in alkaline solution,and then modified with polyethylene glycol surface to formMeNPs.Physical Characterization Detection of MeNPs:The morphology was observed by transmission electron microscopy,the zeta potential was analyzed by nanoparticle size potentiometry,the characteristic functional groups were resolved by Fourier transform infrared spectroscopy,the elemental composition was analyzed by X-ray photoelectron spectroscopy,and the ROS scavenging ability in vitro was detected by nitrotetrazolium blue chloride and H₂O_2.Animal experiment:Sprague Dawley（SD）rats were fed high-fat diet for8 weeks,and a single intraperitoneal injection of 25 mg/kg was used to establish a diabetic rat model.After the establishment of diabetic rat model,rats were fed with a normal diet for four weeks,and then MeNPs at the dose of 5 mg/kg and 10 mg/kg was administrated by tail intravenous injection every other day for two weeks.The general condition of the rat,body weight,fasting blood glucose and serum insulin were observed.Liver and kidney function（ALT,AST,Bun and Cr）,blood lipids（TC,TG,HDL-C and LDL-C）and myocardial injury indicators（CK and LDH）were detected by fully automated biochemistry,and animal ultrasound was used to detect the function of left ventricle.H&E staining was used to observe the morphological structure of heart,liver and kidney.Western blot was used to detect the protein expression of Bax and Bcl-2.Colorimetric assay was used to detect the levels of O₂^·-,H₂O₂and MDA,and the activity of GSH-Px and Caspase-3.Cell experiments:H9c2 cells were treated with D-glucose（33 mM,24 h）,and then treated by MeNPs at the doses of 0.625μg/ml,1.25μg/ml and2.5μg/ml for 24 h.Cell viability was detected by CCK-8,and the injury of H9c2 cells were observed by LDH release assay.The levels of O₂^·-,H₂O₂,MDA,GSH-Px and Caspase-3 were detected by colorimetric assay,and the protein expression of Bax and Bcl-2 was detected by Western blot.Mitochondrial membrane potential was detected by JC-1 staining.Results1.The physical characterization test results showed that MeNPs was about 200 nm spherical particles;The results of Zeta potential,FTIR, and XPS confirmed that MeNPs were successfully constructed from the aspects of system stability,characteristic functional groups, element valence and element content.2.In vitro experiments showed that MeNPs had strong scavenging effects on O₂^·-and H₂O₂.3.In DM group,the body weight and serum insulin levels were decreased,and blood sugar and blood lipids levels were increased, accompanied by obvious symptoms of polydipsia,polyuria and polyphagia,indicating that diabetes rat model was successfully established.Compared with DM group,the body weight and insulin level in MeNPs group were significantly improved,but there were no significant changes in the levels of blood glucose and blood lipids.4.The ultrasound data showed that compared with DM group,Tei index in MeNPs group was decreased,and LVFE and LVFS were increased, indicating that both diastolic and systolic functions of left ventricle were improved.5.The results of CK,LDH and H&E staining showed that compared with DM group,the levels of CK and LDH in MeNPs group were significantly decreased,and myocardial injury was alleviated.6.Compared with DM group,the content of O₂^·-,H₂O₂and MDA in myocardial tissue of MeNPs group were decreased,the activity of GSH-Px was increased,and the level of MDA was decreased.7.Apoptosis index results showed that the level of Bax protein in the myocardial tissue of MeNPs group was decreased,the level of Bcl-2 protein was increased,and the activity of Caspase-3 was decreased.8.The results of ALT,AST,Bun,Cr and H&E staining of liver and kidney tissue indicated that MeNPs at the dose of 10 mg/kg had good biological safety.9.The levels of O₂^·-,H₂O₂and MDA in H9c2 cells by high glucose were increased,and the activity of GSH-Px was decreased,while the contents of O₂^·-,H₂O₂and MDA in H9c2 cells treated by MeNPs group were decreased,the activity of GSH-Px was increased.10.JC-1 result showed that the mitochondrial function in H9c2 cells by high glucose was damaged,while MeNPs group improved mitochondrial function.The level of Bax protein was decreased,the level of Bcl-2 protein was increased,and the activity of Caspase-3 was decreased.ConclusionMelanin nanoparticles exerts anti-hyperglycemia-induced myocardial injury by scavenging reactive oxygen species and inhibiting mitochondrial damage and apoptosis.