A Experimental Study On The Expression Of Connective Tissue Growth Factor And Protein Kinase C In Myocardium Of Diabetic Rats

Objective:The existence of diabetic cardiomyopathy(DCM) referring to myocardial disease in diabetic subjects that cannot be ascribed to hypertension,coronary artery disease,or any other known cardiac disease.DCM is the specific pathological changes of diabetic cardiac diseases.The diabetic state can directly induce abnormalities in cardiac tissue independent of coronary artery defects.The pathological changes are cardiac muscle reconstitution and microangiopathy.In the earlier stage of DCM,diabetic patients maybe asymptom;but in the later stage,patients with diabetes develop in the presence of ischemic heart disease,arhythmia and symptomatic heart failure. Cardiomyopathy in diabetes is associated with a cluster of features,including left ventricular hypertrophy,increased wall thickness and decreased diastolic compliance.Common finding in pathologies of DCM are cardiac tissue fragmentation,fibroplasia,focal necrosis,deposition of collagen and fibrosis in perivascular and among cadiocyte,and deposition of PAS positive substance in intercellular. Triglyceride and cholesterol deposite in left ventricle and interventricular.Cardiocytes appear mitochondria cristae fragme- ntation,vacuolation,inocomma dislocation,and deposition of glucogen.DCM is a complicated disorder,and several factors have been associated with its development.Clinical research indicates DCM presense of diastolic heart failure in early stage,that is degraded of myocardial relaxation and increased stiffness of the left ventricular wall associated with accumulation of connective tissue and insoluble collagen.The interstitial fibrosis plays an important role in the development of heart failure.The finding in examine of the patients with diabetes which are neither hypertension nor coronary artery disease is confirmed interstitial fibrosis.So,Interstitial fibrosis is an important factor in the development of DCM.But the mechanisms leading to diabetic cardiomyopathy remain uncertain.Recently,Research indicated that fibrosis may in part be due to activation of CTGF activity.CTGF can induce extracellular matrix,regulate cytodieresis,mediate cell adhension stimulate cell migration,promote neovascularization,and so on.It can play significant role in fibrosis,cell proliferation,angiogene- sis,wound healing.The activation of Diacylglycerol(DAG)- Protein Kinase C(PKC) pathway is a considerable element in DCM.DAG-PKC constitutes information network system and is the center of signal pathway.It participates many pathophysiology processes,such as cell proliferation and collagen synthesis.In order to evaluate the role of CTGF in the pathogenesis of diabetic cardiomyopathy and the relation of PKC,CTGF and fibronectin(FN),and to provide the evidence of the treatment in DCM state,this study was designed:1 under the standard environment,Streptozotocin(STZ) was injected into the rats, the model was consisted successful through the clinical symptom,vein glucose and urine glucose. 2 to investigate the expression of CTGF,PKC and FN in myocardium of diabetic rats with the method of immunohistochemistry and analyse the change of protein content. 3 to observe the gene expression of CTGFmRNA.This will produce more meaningful findings on the cause of diabetic cardiomyopathy.Methods:1 Experimental rats groups and reproduced of diabetic model:sixty 8-week-old male Sprague-Dawley(SD) rats were divided into two groups(30 rats in each group) randomly:the normal control group and the diabetic group.Diabetes was induced by Streptozotocin(STZ) intraperitoneal injection in SD rats of diabetic group.The normal control rats were injected by the same volume of citric acid buffer.After 72h,we determined fasting blood glucose and urine glucose.Fasting blood glucose≥16.7mmol/L,urine glucose≥+++, animals appeared polydipsia,polyphagia and hyperdiuresis.Then, the model was definited Diabetes.In the period of breed,the normal control group and diabetic group were administrated the identical food and water.Ten rats were selected at random from the two groups respectively at 4 and 8 weeks of duration,and killed them.In the diabetic duration,we did not treat.2 The measurement of blood glucose and body weight.We measured blood glucose after fasting 12h, Blood glucose and weight of all rats were accessed once week.3 The measurement of the whole heart and left ventricle.After rats were killed,the weights of the whole heart and left ventricle were measured,which were used to calculated the ratio of heart weight to body weight(H/B) and the left ventricle mass index(LVMI).4 The analysis of immunohistochemistry.The protein expression of CTGF, PKC and FN were compared between the two groups of various duration by immunohistochemistry method.5 The gene expression of CTGF in diabetic rats:Total RNA from fresh cardiac muscle using Trizol one-step-method were reversed transcribed to be cDNA.cDNA and differert prime were mixed in PCR to amplificate target gene(CTGF) and internal controlβ-actin gene.The product were fractionated on 1% agarose gels.The ratio of the intension of target gene to internal control were calculated.Results:1 Compare with concurrent control groups,blood glucose were significantly increased(p<0.01),and body weight were significantly decreased(p<0.01) in the diabetic groups;2 Compare with concurrent control groups, H/B and LVMI were raised in the diabetic group, which were aggravated with the development of diabetes;3 Notable upregradulation of CTGF protein coincide with an apparent increase of PKC and FN expression in diabetic myocardium.These alterations were enhanced gradually along with longer diabetic duration; 4 The expression of CTGF mRNA was enhanced gradually along with longer diabetic duration.Conclusions:1 H/B,LVMI and body weight were raised in the early stage of diabetes;2 Notable upregradulation of CTGF protein coincide with an apparent increase of PKC and FN expression in diabetic myocardium.It indicates the changes of interstitial fibrosis in myocardium of diabetes;3 Upregulation of CTGF causes the concomitant accumulation of FN.CTGF plays an important role in the development of fibrotic lesions,and is involved in the onset and progression of diabetic cardiomyopathy;4 In diabetic myocardium, PKC is enhanced gradually along with longer diabetic duration.It promotes the expression of FN by CTGF, and causes myocardium fibrosis.