Study On The Effect Of Resisting Decoction On Diabetic Cardiomyopathy Based On Mitochondrial Division And Fusion

Diabetic cardiomyopathy(DCM)is a cardiomyopathy caused by diabetic patients when the blood sugar is at a high level for a long time or when lipid metabolism is disturbed.The early manifestations of diabetic cardiomyopathy mainly include left ventricular stiffness,left ventricular diastolic dysfunction,and reduced left ventricular compliance function.With the development of the disease and the aggravation of the disease,the patient’s systolic function will be impaired,and symptoms such as dyspnea will appear one after another,and finally the clinical manifestations of congestive heart failure will appear.Abnormal glucose metabolism can cause myocardial lesions in patients,but the specific inducing mechanism is still not very clear at this stage.Studies have shown that its possible pathogenesis mainly includes:hypertension,insulin resistance,abnormal metabolic function of myocardial cells,yin and yang Ion imbalance,abnormal activation of renin-angiotensin,mitochondrial dysfunction,etc.,cause cardiomyocytes to produce symptoms such as nutritional disorders and interstitial fibrosis.Mitochondria,commonly known as "the " power factory " in the cell",are the main synthesis site of energy substances(ATP),and play a very important role in many organelles of cardiomyocytes.Abnormal mitochondrial function can reduce the energy supply capacity of cardiomyocytes,which can have a significant impact on the heart function of patients with DCM.According to data,disorders of glucose metabolism not only lead to changes in the structure of mitochondria,but also have a major impact on the function of mitochondria.Therefore,in-depth study of the mechanism of diabetic cardiomyopathy plays an important clinical guiding significance for finding a new entry point for the treatment of DCM and preventing heart failure.Objective:In this project,by giving different doses of resisting decoction and the positive control drug simvastatin,the mitochondrial fusion protein 2(Mfn2),optic neurotrophin 1(Opa1)and mitochondrial dynamic related protein 1(Drp1)of mice with diabetic cardiomyopathy,Re-regulate the expression of human mitochondrial fission protein 1(Fis-1)protein,and explore the mechanism of action of resisting decoction in the treatment of diabetic cardiomyopathy,so as to open up new areas in the treatment of diabetic cardiomyopathy and realize the treatment of diabetic cardiomyopathy New breakthroughResearch methods:1.One week of adaptive feeding was carried out to 100 C57BL/6 mice aged 4 weeks.Randomly select 85 adaptively fed mice,and use high-fat diet(sucrose 20% + cholesterol 1%+ lard 10% + egg yolk powder 5% + sodium cholate 0.2% + Ordinary feed 63.8%)feeding.After 4 weeks,85 mice were injected with streptozotocin(STZ)intraperitoneally according to the standard of 50mg/kg,and the injections were divided into 5 times for 5 days.After 5 days,blood was collected from the tail vein of the fed mice,and the postprandial blood glucose of the mice was tested.If the random blood glucose of the mice exceeds the standard blood glucose level(16.7mmol/L),and there is an increase in drinking water,With the clinical manifestations of increased eating and weight loss,it is believed that the mouse model of type2 diabetes needed for this experiment was successfully modeled,and subsequent experiments can be carried out one after another.At the same time,select the mice of the same series that have been adaptively fed,adopt the conventional feeding method,and give them intraperitoneal injection of normal saline to form a control group,which increases the convincing and accuracy of the experiment.Continue to feed the successfully modeled type 2diabetic mice for 8 weeks with high-fat feeding,and then perform echocardiographic testing of the mouse heart function.If the mice have cardiac dysfunction at this time,the DCM mice used in this experiment Modeling was successful.The 60 successfully modeled DCM mice were randomly divided into 5 groups: diabetic cardiomyopathy model group,simvastatin group,resisting decoction low-dose group,resisting decoction medium-dose group and resisting decoction high-dose group.Prepare for follow-up experiments.2.The type 2 diabetic cardiomyopathy mice were treated with drugs after they were modeled.Three different doses of resisting decoction,high,medium,and low,and simvastatin were administered by gavage.The intervention was continued for 8 weeks until the end of the experiment.3.Blood glucose and body weight of mice were measured.4.Perform HE staining on mouse myocardial tissue and observe its pathological changes..5.Observe whether the structure of mitochondria in the myocardial tissue of mice changes under electron microscope6.Western-blot to detect the protein expression levels of Mfn1,Opa1,Drp1,and Fis-1 in myocardial tissue.7.RT-PCR detection of myocardial tissue Mfn-2,Opa-1,Drp-1,Fis-1 m RNA expression levels8.The expression of ROS in cardiomyocytes was detected by fluorescence.Results :After the experiment,the mice in the model group and the drug intervention group(simvastatin group,resisting decoction low,medium,and high dose groups)decreased in weight compared with the normal control group,and the difference in the experimental results was statistically significant.(P<0.05);Compared with the model group,the weight gain of the mice in the resistance decoction group and the simvastatin group was less,and the difference in the experiment was not statistically significant(P>0.05);the model group,Xin Compared with the normal control group,the mice in the Vastatin group,the resisting decoction low-dose group,the resisting decoction medium-dose group,and the resisting decoction high-dose group had significantly higher blood glucose levels.The difference was statistically significant(P<0.05).Compared with the model group,although the blood glucose levels of the mice in the resisting decoction group and the simvastatin group decreased to different degrees,and the decrease index was more obvious,the experimental results were not statistically significant(P> 0.05).1.Echocardiographic testing of mice,the results showed that compared with the normal control group,the other experimental groups of mice(model group,simvastatin group,resisting decoction groups)cardiac function index ejection fraction(ejection fraction),EF)value and left ventricular short axis shortening rate(left ventricular fractional shortening,FS)value decreased significantly,and statistically significant(P <0.05),left ventricular end diastolic dimension(left ventricular diastolic dimension,LVIDd),The value of left ventricular end-systolic dimension(LVIDs)increased significantly,and the experimental results were statistically significant(P<0.05),indicating that the experiment had an impact on the ventricular structure of DCM mice,which made DCM mice The ventricle was reconstructed during the experiment.Compared with the DCM group,the EF and FS values of the mice in the Simvastatin group and the resistance decoction group increased to varying degrees,and were statistically significant(P<0.05);compared with the normal control group,the DCM group,The heart mass index of mice in the Simvastatin group,the resistance decoction low-dose group,the resistance decoction medium dose group,and the resistance decoction high-dose group were significantly higher than that of the normal control group.The experimental results have obvious statistical significance.(P<0.05),but the mice in the Simvastatin group,the resisting decoction low-dose group,the resisting decoction medium-dose group,and the resisting decoction high-dose group compared with the DCM group mice,the mice’s heart weight index all have different degrees Decrease,but the difference is not obvious,and it is not statistically significant(P>0.05).Studies have shown that different doses of resisting decoction can protect the heart function of DCM mice and can effectively improve ventricular remodeling,but it cannot restore the heart function of DCM mice to normal levels.2.Observe the HE staining display of each group of mice in the experiment.In the control group,no degeneration and edema of cardiomyocytes were observed.The cardiomyocytes were evenly distributed and arranged neatly,and the nuclei in the cells were evenly distributed and single.The boundary is clear and there is no abnormal phenomenon;the myocardial fibers of the DCM model group mice have different degrees of rupture,ventricular degeneration and edema,uneven distribution of nuclei in the cells,disorganized,and unclear boundaries,and local necrosis occurs;Simva Compared with the mice in the DCM experimental group,the myocardial fibers of the mice in the statin group and the resisting decoction low-,medium-,and high-dose groups have reduced myocardial fiber breakage,and the nuclei in the cells are arranged neatly and the structure is clear.The DCM model group is more clear.3.Observe the myocardial fibers of each group of mice under an electron microscope.The myocardial fibers of the mice in the normal control group are not broken,and they contain a large number of muscle fibers and the Z-lines of muscle segments,and they are evenly arranged and clearly visible;mitochondrial structure Standard,elliptical,with an orderly and complete cristae structure;the DCM model group has obvious changes,myofibrils appear flaky necrosis and dissolution,cell nuclei appear pyknosis,necrosis and other pathological changes,and mitochondria appear swelling,degeneration,Structural changes such as cristae rupture,thickened capillaries of the basement membrane,and vacuoles of different sizes can be seen;in the high,medium,and low-dose groups and simvastatin groups,all the indicators of mice appear Significant improvement.The Z-line arrangement of myofibrils and myofibrils is more orderly,and the distribution is more uniform.Myofibrils no longer show obvious necrosis and dissolution.The situation is improved.The swelling of mitochondria is slowed down and degeneration.The degree is reduced,and the cavitation phenomenon has also been improved to varying degrees.4.Western blotting was performed on mice.The results showed that compared with the control group,the expression levels of Mfn2 and Opa1 in the myocardial tissue of the model group mice increased significantly,and both were statistically significant(P<0.05).),while the expression levels of Drp1 and Fis-1 all showed a downward trend during the experiment,and both were statistically significant(P<0.05).Compared with the model group,the dose groups of resisting decoction and simvastatin In the myocardial tissue of mice,the expression levels of Mfn2 and Opa1 in mouse cells were reduced to varying degrees,and were statistically significant(P<0.05),while the expression levels of Drp1 and Fis-1 in myocardial tissue Significant increase,and statistically significant(P<0.05).5.Observing the polymerase chain reaction(PCR)of mice,the results showed that compared with the normal control group,the expression levels of Mfn2 and Opa1 m RNA in the cardiomyocytes of the model group increased,and it was statistically significant(P <0.05),while the m RNA expression of Drp1 and Fis-1 decreased and was statistically significant(P<0.05);Compared with the model group,the m RNA of Mfn2 and Opa1 were in the high,medium and low-dose groups of Danangtang and Xin The expression levels in the cardiomyocytes of the mice in the Vastatin group decreased to varying degrees and were statistically significant(P<0.05),while the expression levels of Drp1 and Fis-1 m RNA in the cardiomyocytes increased to varying degrees.And it is statistically significant(P<0.05).6.Detect the expression of reactive oxygen species(ROS)in mouse cardiomyocytes using fluorescence staining detection technology.Compared with the control group,the content of ROS in the mouse cardiomyocytes of the model group was significantly increased,and It is statistically significant(P<0.05).Compared with the model group,the content of ROS in the myocardial cells of the mice in the resisting decoction high-dose group,the resisting decoction medium-dose group,the resisting decoction low-dose group and the simvastatin group was significantly reduced.And it is statistically significant(P<0.05)Conclusion:Resistant Decoction has a certain inhibitory effect on the expression of Mfn2 and Opal protein in myocardial cells of diabetic cardiomyopathy mice,and can also have a certain inhibitory effect on the fusion function of mitochondria.At the same time,by promoting the expression of Drp1 and Fis-1 in the cardiomyocytes of diabetic cardiomyopathy mice,it greatly improves the division function of mitochondria.In the end,the division function and fusion function of mitochondria always maintain a high degree of dynamic balance in a hyperglycemic environment,which is of great significance to maintaining the structure and function of mitochondria,thereby protecting the function of the myocardium.