Studies On The Effects And Mechanism Of Melatonin On Myocardial Apoptosis In Diabetic Rats

Objective Diabetic cardiomyopathy is a kind of disease coming after diabetes mellitus,especially type 2 diabetes mellitus.There are several characteristics,such as severe disease,poor prognosis,high mortality and so on.In order to find an effective treatment method,the present study was conducted to investigate the effects of melatonin(MLT)on myocardial apoptosis in rats with type 2 diabetes mellitus(T2DM)and its possible mechanism.Methods(1)The models of T2 DM rats: After one week of adaptation,thirty-six healthy male Sprague-Dawley(SD)rats,raised in specific pathogen free(SPF)environment,were divided into control group(n=12),model group(n=12)and treatment group(n=12)in random.Diabete models were induced by high fat diet and intraperitoneal injection of streptozotocin(STZ,25 mg/kg).Then rats in treatment group were given 10 mg/kg/d of MLT by gavage.After 24 weeks,cardiac function was detected by arterial cannulation.Abdominal aorta blood was collected to detect the fasting blood glucose(FBG),blood lipid and insulin levels.Myocardial tissue were collected,HE staining and Masson staining were used to observe the morphological changes and the accumulation of collagen fibers,respectively.Terminaldeoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)was used to observe the apoptosis of myocardial cells.The expression level of Caspase-3 was detected by immunohistochemistry(IHC).In addition,in order to explore its possible molecular mechanisms,western blot was used to detected the expression of endoplasmic reticulum stress related protein(GRP78,CHOP,PERK,ATF-6?,IRE1?,Caspase-12),mitogen activated protein kinase(MAPK)pathway related proteins(JNK,p38,ERK)and apoptosis related proteins(Caspase-9,Caspase-3,Bcl-2,Bax).H9C2 cells exposed to high glucose as the cell model.After H9C2 cells treated with different concentrations of glucose for 48 h,hoechst 33258 staining was used to detect the apoptosis of cells,and the phosphorylation levels of MAPK pathway related proteins JNK,p38 and ERK were detected by Western blot.Results T2 DM rat models were copied successfully.FBG was(19.27±1.03)mmol/L and insulin was 34.79±7.97 ?IU/ml in in the model group rats.They were all significantly higher than those of the control group.However,the insulin sensitivity index(ln ISI)was-6.48±0.23 and was significantly lower than that of the control group.These results indicated that insulin resistance(IR)was existed.MLT can not only reduced the levels of FBG and blood lipid(contain TC,LDL-c and TG),but also enhanced the sensitivity of insulin.HE staining showed that MLT could improve the disorder of myocardial morphology Masson staining displayed that MLT could decrease the accumulation of collagen fibers.In rats of the model group,the apoptosis of myocardial cells was significantly increased,and the heart function was severely damaged,which was significantly relieved after MLT treatment.IHC showed that MLT could down-regulate the expression of Caspase-3 in rats with T2 DM.Western blot showed that the expression of endoplasmic reticulum stress related protein GRP78,CHOP,PERK,ATF-6? and IRE1?,Caspase-12 were significantly increased and MAPK pathway related protein JNK phosphorylation level was significantly up-regulated in myocardial tissue of rats with T2 DM.After MLT treatment,the expression of endoplasmic reticulum stress related protein and the phosphorylation of JNK in T2 DM rats were markedly reduced,even though the expression of IRE1? was not significantly decreased.H9C2 exposed to high glucose as the cell model: Hoechst 33258 staining showed that high glucose could induce the apoptosis of H9C2 cells.Western blot showed that the phosphorylation level of JNK in the H9C2 cells was up-regulated with the concentration dependence.Conclusions The apoptosis of myocardial cells existed in T2 DM rats,which may be due to the increase of blood glucose and lipid,and the regulation of related apoptosis protein in the downstream activated by JNK pathway though endoplasmic reticulum stress.However,MLT could protect the heart of T2 DM rats by lowering blood glucose and blood lipid,enhancing insulin sensitivity and reducing myocardial apoptosis.