Protective Effects Of Grape Seed Proanthocyanidin Extracts On Myocardium In Diabetic Rats

Background:Diabetes mellitus (DM) is an endocrine and metabolic disease caused by multiple etiological factors. Because of insulin secretion and /or effect discrepancy, it cause metabolic disorders of glucoses, fats and proteins. Its characteristic is chronic hyperglycemia . The original damage caused by diabetes mellitus is due to all kinds of diabetic complications. The diabetic cardiomyopathy (DCM) is one of the important complications. It is the major cause of death of advanced stage of diabetes mellitus. So to study the pathogenesis of diabetic cardiomyopathy and to investigate the protecting mechanisms of grape seed proanthocyanidin extracts (GSPE) on myocardium of diabetic rats are of important clinical significance. It can postpone pathologic process of DCM and to improve its prognosis, and to cut down its mortality.The mechanisms of diabetic complications include: increased intracellular formation of AGEs, increased polyol pathway flux, activation of protein kinase C, increased flux through the hexosamine pathway and oxidative stress. Under the high glucose circumstances, all mechanisms affect each other, they cause a series of pathological changes. Among them, the pathway of AGEs is still a focal point of researching. Along with the discovering of AGEs receptor for advanced glycation end products (RAGE) , nuclear factor kappa B (NF-κB) , many kinds cytokines and inflammatory factors, the complicated relationship among them are thought highly by people more and more, they can induce and promote diabetic complications, as DCM. An important pathway of producing a marked effect of AGEs is that they can combine with idio-acceptor RAGE on cellular membranes of endothelial cells and cause the changes of contents of the dissolubility materials, such as cytokines, hormones, oxygen free radical and so on . They induce changes of the level of proteins genic expression and participate the process of apoptosis. They can initiate a series of metabolic disorders. They may be the key pathway of inducing the diabetic complications. The blockage of AGEs-RAGE interaction by administration of anti-RAGE IgG or sRAGE reversed the progresses of cardiovascular complications. GSPE, a naturally polyphenolic compounds obtained from grape seeds, have been reported to posses potent radical-scavenging and antioxidant properties, they also have effects of anti-arteriosclerosis, adjust blood lipids and so on. There is just a few researches about this in abroad or in China. So, based on the diabetic rats induced by the injection of STZ and apply GSPE to intervene them , we utilized the methods of transmission electron microscope , dyeing of HE, PAS and MASSON , immunochemistry and RT-PCR, meanwhile we determine AGEs in plasma, in order to study the pathogenesis of DCM and observed the therapeutic efficacy of GSPE.Objective: Wistar rats were injected with 0.1% STZ by tail vein to establish diabetic model and apply GSPE was given to intervene them. We study the mechanisms of DCM and investigate on the treatments of DCM by GSPE.Methods: The male Wistar rats were subjected to 0.1% STZ through the tail vein to induce diabetic mode. Then, they were randomly divided into two groups: diabetic model group (DM1, n=8) and GSPE-treatment diabetic group (DM2, n=12, 250mg.kg^-1.d^-1). Non-diabetic Wistar rats were recruited in other two groups: the control group (C1,n=10) and the GSPE-treatment group 250mg.kg^-1.d^-1(C2,n =10, 250mg.kg^-1.d^-1) .After 24 weeks, all rats were killed and blood were collected to measure fasting plasma glucose (FPG), glycosylated hemoglobin (HbA_1C) and AGEs. Electronic microscope was used to observe the changes of myocardial ultrastructure. The pathologic changes of rat myocardial tissue were studied by HE, PAS and MASSON staining. The RAGE, NF-κB, TGF-β₂1 and CTGF were measured by immunohistochemistry. RT-PCR were performed to detect the expression of RAGE, NF-κB , TGF-β₁ and CTGF mRNA in the myocardial tissue of all rats.Results:1. General data: In the course of experiment, the rats of DM1 group showed hyperdiuresis, polydipsia, polyphagia and emaciation markedly, furs were filthy reluster and growth slowly. Above-mentioned symptoms decreased of the rats in DM2 group. The rats in C1 and C2 group had no diabetic symptoms, their growth and nurtures were well. The body weight of the rats had no difference among 4 groups. After 24 weeks, there was no difference between C1 group and C2 group, there was no difference between DM1 group and DM2 group too, however, the body weight of the rats in DM1 were decreased obviously than that in C1 group( P< 0.001). The functions of liver and renal had no statistic difference between C1 group and C2 group. Some items including FPG, HbA_1C increased significantly in DM1 group than that in C1 group( P<0.001).2. Plasma AGEs: After 24 weeks, AGEs increased significantly in DM1 group than that in C1 group or DM2 group.3. The pathologic changes in myocardial tissue of rats:(1) The myocardial tissues of rats staining by HE, PAS and MASSON showed : In C1 group and C2 group, the cardiac muscle fibers had no hypertrophy, caryon of cadiocyte were round and ellipse, they were in the centre of the cadiocytes, the myocardial mesenchymals had normally structure. There were no PAS positive material could be found in cardiac muscle fibers; the myocardial collagen fibers distributed uniformly. In DM1 group , the cadiocyte were hypertrophy and anachromasis, they were obscure boundary and their caryons were nonuniform. Sometimes even focal necrosis of cadiocytes and inflammatory cells cound be found. There were many PAS positive materials could be found in cardiac muscle fibers. The myocardial collagen fibers were misdistribution abnormally and ranked disordered, they connected to nets each other .These changes were seldom seen in DM2 group. (2 ) The immunohistochemistry showed that the expression of RAGE, NF-κB , TGF-β₁ and CTGF were strong in DM1 group , and they were less expressed in DM2 group, and they were almost not expressed in C1 group and C2 group.4. The changes of myocardial ultrastructures: When the experiment ended, we observed tissue slice of myocardial tissue in each group by electron microscope. In C1 group, the karyomorphism of cadiocytes were regular, there were less heterochromatin; the muscle fibrils and chondriosomes ranked regular ; the crista mitochondriales were plentiful and the structures of intercalary disc were normally; the basilemma and cellular membranes of cadiocytes were complete. In C2 group, the muscle fibrils and chondriosomes ranked regulation; the crista mitochondriales were plentiful; T tubules and sarcoplasmic reticulums had no swelling. In DM1 group, the perinuclear cisterna of cadiocytes were swollen. There were lipid granules of electron dense appeared in the kytoplasms and ambi-caryons; the muscle fibrils and chondriosomes ranked disorderly; the size and shape of chondriosomes were vary; the sarcoplasmic reticulums swell slightly and the structures of intercalary discs were abnormal slightly. In the DM2 group, the karyomorphism of cadiocytes were regular relatively; the chromatins distributed were normal; the ambi-caryons, had no swelling and the intercalary discs were normally; the muscle fibrils ranked regular; the basilemma were intact; the size of some chondriosomes were vary and swollen. All abnormal structures were improved obviously in DM2 group than that in DM1 group.5. The PT-PCR showed that the gene expression of RAGE, NF-κB, TGF-β₁ and CTGF mRNA in DM1 group were more stronger than that in DM2 group or in C1 group.Conclusions:1. AGEs/RAGE and NF-κB participate the development of DCM;2. GSPE had protective effect on myocardium of diabetic rats by blocking the combing effect of AGEs/RAGE:(1) GSPE could improve the cardiopathology of diabetic rats;(2) GSPE could improve the myocardial ultrastructure of diabetic rats; (3 ) GSPE could down-regulate the gene expression of TGF-β₁ and CTGF, so it could participate the process of inhibiting myocardial fibrosis of diabetic rats.3. GSPE is a naturally polyphenolic compounds obtained from grape seeds, our results will be important reference basis of theory which contribute to research and development of new medicine.Significance:This research found out in STZ-induced diabetic rats, hyperglycemia could increase plasma AGEs. An important pathway is that they can combine with idio-receptor RAGE on cadiocytes and induced oxidative stress, activated NF-κB, so they can cause the gene over expression of cytokines such as TGF-β₁ and CTGF, by which they induced the damages of cadiocytes and encourage the development of DCM. GSPE can inhibite non-enzymatic conversion, blocke the combination of AGEs/RAGE, and blocke the signal transduction pathway mediating by NF-κB and RAGE. So they could relieve the damage of myocardium in diabetic rats. GSPE will be important reference basis of theory which contribute to research and development of new medicine.