| According to the latest data from the International Diabetes Federation Organization published in 2015 in the "World Diabetes Congress",a total of 415 million adults with diabetes mellitus in the world.It means one among 11 people suffer from diabetes.UP to 2040,the number of the patients will reach 642 million.There are also a number of patients worldwide who have been diagnosed with "undiagnosed diabetes",all of which are potential diabetic patients in the future.Diabetes Mellitus(DM)becomes a severe health problem in the world,which has brought huge economic burden to the world.So it is worth of our attention.According to the International Diabetes Federation(IDF)data,China is the top one contury with the largest number of diabetic patients in the world.We have about 110 million diabetic patients now.Therefore,the prevention and research of DM has become one of the most important and urgent problems.DM is a metabolic disorder,,which is mainly results in the damage of the cardiovascular and nervous system.Diabetes usually impacts multiple systems and organs,including cardiovascular system,cerebrovascular system,urinary system,peripheral nervous system,and so on.Among them,cardiovascular complications are with the highest risk,which is the main cause of high morbidity and mortality.Although people realized the dangers of diabetes gradually,the methods and strategies of prevention and treatment of diabetic complications are still limited,especially for the prevention of highrisk cardiovascular system and cerebrovascular system complications.Traditional treatment strategies still can not control the occurrence and development of diabetic cardiovascular complications.More and more clinical data have shown that the incidence and mortality of Diabetes Mellitus Cardiomyopathy(DMC)is increasing year by year.However,the specific mechanism of diabetic cardiomyopathy remains unclear.Further research is needed.Autophagy eliminates the senescence and damaged proteins and organelles through lysosome.It is a very conservative process in the cell.By removing these harmful substances,the cells can inhibit the cell death.It is important to clear these cumulated substance and maintain the normal structure and function.The decrease of autophagy can lead to the pathological process of many diseases,such as cardiomyopathy,coronary heart disease,heart failure,hypertension and so on.Mitochondria is the only energy resource in the heart.In addition to participate oxidation energy supply in cell,it is also involved in other cellular processes,such as cell differentiation,the transfer of genetic information,and cell apoptosis.A number of studies have shown that mitochondrial dysfunction is one of the major causes of DMC.Mitochondrial dysfunction leads to the damage of respiratory chain function and the decrease of oxidative phosphorylation.It leads to apoptosis of cardiomyocytes eventually.The abnormal accumulation of damaged mitochondria aggravates oxidative stress and further reduces oxidative phosphorylation.Therefore,the effective elimination of damaged mitochondria(mitochondrial autophagy)is likely to play an important role in diabetic myocardial injury.The mechanism of mitochondrial autophagy is complex,but it is not clear yet.However,it is very important to clarify the regulation mechanism of mitochondrial autophagy for the treatment and prevention of disease.The current study showed that Drp1 or DNM1 L molecules play an effective protection role in ischemia reperfusion injury.through upregulation of autophagy,effectively removing damaged mitochondria and maintaining mitochondrial homeostasis.Therefore,we assume that in diabetic cardiomyopathy,Drp1 protects against diabetic cardiomyopathy by increasing the level of mitochondrial autophagy,to maintain myocardial mitochondria dynamic balance,and to maintain the balance of energy metabolism in cells.To sum up,we mainly aim to clarify the role of Drp1 in DMC and to explore the possible mechanism for it..Objective 1.Diabetes Mellitus Cardiomyopathy model was constructed,observation of diabetes on cardiac structure and function of the role,as well as the influence on myocardial cell mitochondria autophagy level;2.Confirm that the effect of diabetes on the expression of myocardial cell Drp1,role in the occurrence and development of diabetic cardiomyopathy;3.Explore Drp1 abnormal cardiac function of diabetic cardiomyopathy in the mitochondrial autophagy barrier,the influence of clarify Drp1 protective mechanisms of diabetic cardiomyopathy.Methods 1.8-12 week old C57 and Drp1-KO male mice were randomly divided into control group and diabetic group.The diabetic group was given intraperitoneal injection of streptozotocin(STZ,50mg/kg)for five consective days.One week after the last injection,random blood glucose levels(via tail veins)were determined by using a Glucometer.The random blood glucose of mice was measured two times at different time >11.1mmol/L.It was considered successfully diabetic.The successful model mice and the control mice were feed for 3 months under the same conditions and to establish diabetic cardiomyopathy model.2.All mice were randomly divided into four groups and marked as normal group(Con),diabetic group(DM),gene knockout group(Drp1-KO),gene knockout + diabetic group(Drp1-KO+DM).To detect of mouse myocardial cells autophagy quantity,LC3 immunofluorescence distribution,determination of autophagy related proteins(P62,LC3),to observe the morphology of myocardial cells,fibrosis,myocardial mitochondria morphology and distribution of the level and degree of myocardial cell apoptosis.3.To detect of autophagy related proteins in mouse cardiomyocytes by Western blotting.The number of autophagy in myocardial cells was detected by transmission electron microscopy and immunofluorescence.The distribution of LC3 was observed by immunofluorescence.4.The heart function of each group was detected by Doppler ultrasound.5.Masson(Masson)staining was used to observe the level of cardiac fibrosis in mice.6.The morphology of myocardial mitochondria was observed by transmission electron microscope.7.Mitochondrial distribution was observed by mitochondrial staining(Mito-Tracker)staining.8.TUNEL method was used to observe the apoptosis of myocardial cells in each group.9.The myocardial cells were extracted from Kunming child mice.The cells were randomly divided into four groups and marked as normal glucose group(NG,5.5mmol/L),normal glucose + gene over expression group(NG+Drp1-OE),normal glucose + gene over expression +3MA group(NG+Drp1-OE+3MA),high glucose Group(HG,33mmol/L),high glucose + gene over expression group(LG+Drp1-OE),high glucose + gene over expression+3MA group(LG+Drp1-OE+3MA).10.To detect of autophagy related proteins in myocardium of mouse by Western blotting.11.TUNEL method was used to observe the apoptosis of myocardial cells in each mouse.12.The number of autophagic cells in myocardium of mice was detected by transmission electron microscopy and immunofluorescence.13.JC-1 staining was used to observe the level of mitochondrial membrane potential.14.The level of autophagy in myocardial cells of mice was observed by LC3 immunofluorescence.Results 1.The cardiac function of diabetic mice was decreased,myocardial fibrosis was elevated,cardiomyocyte apoptosis was significantly increased,mitochondrial number was increased,compared with the control group.The cardiac function of DRP1-KO group was significantly lower than that of the control group.There was no significant difference between the DRP1-KO group and the diabetic group.Compared with the control group,the level of fibrosis increased.The level of fibrosis in DRP1-KO group was lower than that in diabetic group.Compared with the control group and the diabetic group,the number of mitochondria increased significantly in DRP1-KO group.Compared with control group,the level of autophagy decreased in DRP1-KO group.The level of autophagy in DRP1-KO group was higher than that in diabetic group.Compared with the diabetic group,the DRP1-KO+DM group had lower cardiac function,increased fibrosis,increased number of mitochondria,and lower levels of autophagy.2.Compared with the control group,the level of mitochondrial membrane potential was decreased,and the level of mitochondrial membrane potential was higher in NG+Drp1-OE group than that in high glucose group.Compared with the control group,the level of autophagy increased in Drp1-OE group.Compared with the control group,the level of autophagy decreased in high glucose group.Compared with high glucose group,the level of autophagy increased in NG+Drp1-OE group.Compared with the control group,the number of apoptotic cells increased significantly.Compared with high glucose group,the number of apoptotic cells in NG +Drp1-OE group was significantly lower.Conclusion For the first time,it is proved that the dynamic related protein 1(Drp1)protects the Diabetes Mellitus Cardiomyopathy(DMC)against diabetes mellitus by inducing mitochondrial autophagy. |
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