The Expression Of NF-κB,MMP-9 And Oxidative Stress In Myocardium Of T2DM Rats And The Therapeutical Effect Of Rosiglitazone

Objective: Diabetic cardiomyopathy (DC) is the one of the most common cardiovascular complications of diabetes mellitus and the leading cause of heart dysfunction and congestive heart failure. Different from coronary heart disease, hypertensive heart disease and primary myocardial disease, diabetic cardio- myopathy is a unique entity, and its pathogenesy is not comp- letely clear yet. Recent years, It has been revealed that cardiovascular diease of diabetes is an chronic inflammatory disease, and lots of inflammatory factors such as TNF-α, IL-1β, IL-6 and so on play an important role in the genesis and development of cardiovascular disease in T2DM. Now it has been confirmed that there are binding sites of NF-κB among these genes of inflammatory factors, activation of NF-κB leads to proinflammatory genetic transcription. Through oxidative stress and other inflammatory reactions, they damage myocard- ial cells and microvascular endothelial cells, et al. Presently the studys in the impact of NF-κB on myocardium focus on ischemical reperfusion injury. Meanwhile interstitial collagen metabolism in myocardium is regulated by MMPs and their inhibition factors. Among them, MMP-9 is the key enzyme for the degradation of interstitial substance, and its substrates include collogen, gelatine, elastin, et al. Over expression of MMP-9 can induce the destruction of collogen network and increase of soluble degenerative collagenous fibrils, thus cause cardiac hypertrophy, myocardial fibrosis, ventricular dilatation and heart failure. The level of MMP-9 of plasma and tissue raises up in artherosclerosis and acute myocardial infarction, nevertheless the research on its expression in DC is less.Rosiglitazone(RSG) which belongs to thiazolidinediones (TZDs), is a kind of new drug treating T2DM through activating PPARγ. In diabetic myocardium, the effect of rosiglitazine on the expression of NF-κB and MMP-9 is not reported yet. In the present study, we investigate myocardial expression of NF-κB and MMP-9 in rat DC model induced by long-term high-fat feeding accompanied with intraperitonealinjection of streptozotocin (STZ), and also research the effect of rosig- litazine on myocardial expression of NF-κB and MMP-9, explored the mechanisms of DC treatment by PPARγagonist.Methods: After breeded adaptively for 2 weeks,a total of 45 healthy male Whistar rats were divided randomly into 2 groups: control group (Group A,n=15) received a regular diet and experimental model group (n =30) was fed for 2 months. When the high-fat diet animals appeared the insulin resistance evaluated by Li Guangwei HOMA index formula, STZ was administered via intraperitoneal injection at 30mg/kg to make model. Then the modeling rats were further divided randomly into 2 groups: non-intervention of T2DM group (Group B) was fed the high-fat diet continuously for 2 months, and rosiglitazone-treated group (Group C) was fed a high-fat diet meanwhile rosiglitazone was administered by oral gavage (3mg/kg) once daily. Except for accidental death and abortive sample, there remain 12 rats each group at the end of experiment.All the rats were weighed and their blood was taken at the beginning and end of the experiment. The rats were fasted at 20:00 in the day before experiment, blood was taken from the angular vein at 8:00 of experimental day, and the blood serum was separated and stored at -20℃for all the indexes'detection (the mensuration of the indexes were performed with the same batch of kits). At the end of experiment, the heart tissues were taken and fixed for histological analysis by neutral paraform. The others were stored at -85℃for dectecting the contents of SOD, MDA and NOS from homogenate extracts of cardiac muscle tissues.1 Assays blood glucose and insulinWe measured fasting glucose by glucose oxidase method, and insulin by radioimmunoassay on serum samples. The level of insulin resistance was evaluated according to Homeoestasis Model Assessment (HOMA-IR) and Insulin Sensitivity Index (ISI).2 Assays of blood fatTriglyceride(TG), total cholesterol(TC) were measured by chromometry. FFA was measured by Cu²⁺ chromatometry.3 Assays the factors correlated with oxidative stressThe indexes were performed with kits operated by instruction. SOD, MDA and NOS content in myocardium were measured by chromatometry. The absorbance of each index was put in formula to get final data.4 Cell morphology examination of myocardiumThe tissue section was made by routine method for light microscope examination, and proceed with HBFP specific staining, NF-κB and MMP-9 immunohistochemistry staining.5 Data analysis and statistical evaluationAll data were treated with SPSS11.0. Measurement data were expressed as mean values±standard deviation. The difference between means was compared by the Student's t-test, and analysis of variance for one factors (ANOVA) was used to compare continuous variables among groups. In pykno-ranked data, the difference between two groups was compared by the Wilcoxon's rank test, and Kruskal-Wallis H's test was used to compare the difference among groups. The degree of significance was judged by P values when necessary. P<0.05 means the difference is significant, P<0.01 means the difference is highly significant.Results:1 Body weight: There was no statistically significant difference in weight among each group of the rats at the beginning of the experiment. At the end of the experiment, the weight of rats in group B and group C was significantly higher than that in group A (P<0.05), and there was no significant difference between group C and group B (P>0.05).2 Blood glucose and insulin: There was no significant difference in fasting glucose among the rats of each group at the beginning of the experiment. After the injection of STZ, the blood glucose level of model rats and the rats treated with rosiglitazone significantly raised up from normal baseline (P<0.01). The blood glucose level of rats treated with rosiglitazone was lower than that of model rats (P<0.01), but it was still hyperglycemia. The insulin level of model rats was markedly higher than that of rats treated with rosiglitazone and control rats (P<0.01), The insulin level of control rats lower than that of rats treated with rosiglitazone (P<0.05).3 Blood lipid: The plasma levels of TG, TC and FFA in rats with T2DM were obviously higher than those in control rats (P<0.01). After treated with rosiglitazone, their plasma levels decreased to some extent, but they were still higher than those in control rats.4 Comparison of the factors correlated with oxidative stress among groups: The content of MDA in model rats and rats treated with rosiglitazone notablely increased (P<0.01), con- trasted with normal baseline. But the content in rats treated with rosiglitazone was lower than that in model rats (P<0.05). The activity of SOD in rats treated with rosiglitazone was markedly higher than that in model rats (P<0.01). But it was still lower than that in control rats (P<0.05). The activity iNOS of in rats treated with rosiglitazone was significantly higher than that in control rats (P<0.01). But it was still lower than that in model rats (P<0.01).5 Immunohistochemistry: The NF-κB expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than group B (P<0.01). The MMP-9 expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than that in group B (P<0.01).6 The pathological morphological change of myocardium (HBFP-staining method): Under light microscope, most of cardiac muscle fibers were stained with yellow, only few showed red in control rats. The majority of cardiac muscle fibers were stained with red and the area liked mass in model rats. Some cardiac muscle fibers showed red in rats treated with rosiglitazone, but the area was smaller and the coloration was lighter than those in model rats. Yellow staining signified there was no ischemia in cardiac muscle. Red staining signified there was ischemia in cardiac muscle, and the colourity of staining was positively correlated with the degree of myocardial ischemia.Conclusions:1 It shows that there exists early myocardial ischemia in the model of T2DM rats. The expression of NF-κB and MMP-9 increases, and the reaction of oxidative stress enhances in cardiac muscle. Owing to promoting the development of diabetic cardiomyopathy, they are importantly influential factors for the myocardial abnormality of structure and function in T2DM.2 By activating PPARγ, RSG plays an important role in improving glucolipid metabolism, increasing the sensitivity of insulin and reducing the insulin resistance. Moreover, it can lower the expression of NF-κB and MMP-9 in cardiac muscle, increase the activity of SOD, reduce the content of MDA and the activity of iNOS. Thus it exerts the action against inflammatory reaction and oxidative stress, protects for cardiac muscle in T2DM, and slows the process of diabetic cardio- myopathy.