The Effects And Mechanism Of Urocortin On Myocardial Fibrosis In Diabetic Rats And Myocyte Hypertrophy Induced By High Glucose

ObjectiveFirstly, we used diabetic cardiomyopathy (DCM) rats model to observe the protective effects of endocrine vascular active peptide, Urocortin’s effects on serum biochemical index, haemodynamics, myocardial intracellular calcium levels, myocardial remodelling and myocardial injury in DCM rats. The aim of this study is to investigate the protective effects and possible mechanism of Urocortin myocardial fibrosis in diabetic cardiomyopathy rats. Urocortin exerted powerful protective effects on various cardiovascular disease models. Secondly, in this study, we also studied the effects of Urocortin on myocyte hypertrophy induced by high glucose, to observe the effects of Urocortin on fluorescent calcium content ([Ca2+]i) and the expression of calcineurin (CaN) in the cardio-myocytes to investigate the protective effects of Urocortin on myocyte hypertrophy induced by high glucose. Moreover, Urocortin could inhibit the myocyte hypertrophy induced by high glucose in dose dependent fashion, and possible mechanism were Urocortin inhibited the [Ca2+]i contents and the expression of CaN. These studies would provide a new evidence for clinical prevention and treatment on diabetic cardiomyopathy, and provide a new drug target for the treatment of DCM.Methods1. In vivo experiments:1.1 In this experiment, one hundred male Wistar healthy rats were used. eighty rats randomly were selected and injected streptozotocin (STZ) 55mg·kg-1 in the tail veins,3 days. AccuCheck type blood glucose meter were used to test the glucose level, fasting blood glucose level more than 16.7 mmol·L-1 72 h later were diabetic rat model. Another 20 Wistar rats were injected the same amount of citrate buffer as control group. These diabetic model rats were diabetic cardiomyopathy rats 12 weeks later, and randomly divided into 4 groups, includes:diabetic cardiomyopathy model group (DCM), Urocortin group (Urocortin 7μg·kg-1, UCN), Urocortin (7μg·kg-1)+Astressin (35μg·kg-1) group (UCN+AST) and Urocortin (7μg·kg-1)+Triciribine (0.5mg· kg-1) (UCN+TRI), in each group had 10 rats. The treatment groups were injected equal volumes of normal saline in DCM group. During the period of drug delivery observed the general state of experimental animal in each group.1.2 The subjects of experimental animals in each group were given different drugs, four weeks later, urethane was used to anesthesia experimental animal in each group. Cardiac function was observed in rats with BL-420F biomedica limage analysis system; Cardiac catheterizat ion was performed to evaluate cardiac function. Taken carotid artery blood 2ml to test the index as follow, include:the content of Opeptide, glycosylated hemoglobin (HbAlc). the levels of creatine phosphokinase isoenzyme (CK-MB), plasma brain natriuretic peptide (BNP). liLISA method was used to test the inflammatory factors (transforming growth factor beta 1, TGF-β1; connective tissue growth factor, CTGF).1.3 Half experimental animal were used to measure the heart weight index (HWI) and left ventricular mass index (LVMI) by balances after haemodynamics experiment; HE staining were used to detect myocardial histopathology changes; myocardial collagen volume fraction (CVF) were measured.1.4 After the above experiment, another half experimental animal were used to measure the level of calcium ions in the myocytes. RT-PCR method was used to test the expression of TGF-β1 and CTGF mRNA. Western blot method was used to detect the activation of signaling proteins, TGF-β1, CTGT, Akt, glycogen synthase kinase-3β (GSK-3β), p-Akt and p-GSK-3β in each group.2. In vitro experiment2.1 This study used new born Sprague-Dawley (SD) rats,2-3 days. Myocardial cells of neonatal rats were cultured in vitro. The hypertrophic myocytes were induced by high glucose (HG,25 mmol·L-1). Observe the inhibitory effects of different dosage Urocortin in HG-induced hypertrophic cardiac myocytes. Cultured neonatal rat cardiomyocyte cells were randomly divided into five groups, includes:the normal control group (Normal), the HG model group (HG), the Urocortin low dose group (1 μmol·L-1, UCN-L), the Urocortin middle dose group (5μmol·L-1, UCN-M) and the Urocortin low dose group (10μmol·L-1, UCN-H).2.2 Incubation the cardio-myocytes for 48 hours use different treatment factor. Total protein content were assayed by Lowry method. The diameter of the single cardio-myocyte and volume were measured by computer photogragh analysis system. The cardiomyocyte viability was analysed by MTT assay. [Ca2+]i transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of CaN was expressed by CaN/β-actin value, and determined by Western blot method and CAMIAS008 image analysis system for semi-quantitative analysis.Results1. In vivo experiments:1.1 In this experiment, compare with the control group, DCM rats had obviously symptoms in each group, activities to reduce and vulnerable to infections. Compare with DCM group, the state was improved, the weigh increased, the urine volume decreased and urine sugar level reduced in Urocortin groups (P<0.05).1.2 Compared with the Control group, blood sugar of the model group was significantly elevated (P<0.01). Left ventricular systolic pressure (LVSP) and left ventricular diastolic final pressure (LVEDP) were increased and ±dp/dtmax were obviously decreased in each DCM rats (P<0.01). The content of C-peptide was decreased, the level of HbAlc, CK-MB, BNP, TGF-β1 and CTGF were increased in DCM rats, compare with the Control group have significant difference (P<0.05). HWI, LVMI and the content of calcium were increased significantly (P<0.05). Compare with the DCM group, LVSP and LVEDP was significantly dropped (P<0.01), ±dp/dtmax were remarkably enhanced (P<0.05). Compared with the DCM model group, the level of HbAlc, CK-MB, BNP, TGF-β1 and CTGFwere decreased (P<0.01) in the UCN group. Moreover, HWI, LVMI and calciumion were decreased, compare with DCM group have significant difference (P<0.01). In addition, Astressin and Triciribine could inhibit the effects of Urocortin (P<0.05).1.3 Pathological results shows:The morphologically of myocardial cell were normal and muscle fibers were arranged regularly in the Control group; In the DCM model group, myocardial cell volume significantly increased, myocardial fibers were disorganized and collagen fibers elevated, the CVF was increased remarkably compared with the Control group (P<0.01); Compared with the DCM model group, The pathological changes were improved and CVF decreased in the UCN group (P<0.01). In the AST+UCN group and the TRI+UCN group, myocardial tissues were injuried, collagen fibersin increased and CVF elevated compared with the UCN group (P<0.05).1.4 Compared with the Control group, the content of calcium ion in each DCM model group was significantly elevated (P<0.01). Compare with the DCM group, the content of calcium ion in the UCN group was significantly dropped (P<0.05). However, Astressin and Triciribine could inhibit the effects of Urocortin had significant difference (P<0.05).1.5 Compared with the Control group, TGF-β1 and CTGF mRNA and protein levels in the diabetic group were higher than rats in the Control group (P<0.01), which were accompanied with disminished Akt and GSK-3β phosphorylation (P<0.01). Remarkably, the antagonist of CRF-2R, astressin could inhibit the effects of Urocortin, Triciribine could partial inhibit the urocortin’s effects (P<0.01). Urocortin attenuated TGF-β1 and CTGF mRNA (P<0.01). Compare with the Control group, phosphorylation of Akt and GSK-3β were reduced in the DCM group (P<0.01). However, compare with the DCM group, phosphorylation of Akt and GSK-3β were enhanced in the UCN group (P<0.01). All the effects of urocortin were abolished by the CRH receptor2’s antagonist, astressin. Triciribine, an Akt inhibitor, partially abolished effects of urocortin on myocardial dysfunction in diabetic rats.2. In vitro experiment2.1 HG induced the morphological changes of cardio-myocytes. The decrease in the number of cells, elongated, tended to fibroblasts. Urocortin could made cell morphology and growth state returned to normal.2.2 Compared with the Normal group (R<0.01), HG (25 mmol · L-1) significantly increased 46.2% the total protein content, enhanced 81.3% cardiomyocoytes cell volume, and decreased the cardiomyocyte viability by 36.1%(P<0.01). Different Urocotin group could decrease the total protein content, reduced cardiomyocoytes cell volume, and increased the cardiomyocyte viability (P<0.05). The total protein content and cardiomyocoytes cell volume were reduced by 6.8%,10.6%, and the cardiomyocyte viability increased 11.8% in the UCN-L group. Compare with the HG group, UCN-M and UCN-H could remarkably reduced the total protein content and cardiomyocoytes cell volume, and at same time enhanced the cardiomyocyte viability (P<0.01). Different dosage urocotin followed with dose-dependent fashion (r=0.94).2.3 Compared with the Normal group, the [Ca2+]i transient increased by 62.5%, the expression of CaN was significantly increased (P<0.01). Following treatment with different dosage Urocortin (UCN-L, UCN-M or UCN-H), the [Ca2+]i transient and the expression of CaN were reduced (P<0.05 or P<0.01) Urocortin (UCN-L, UCN-M or UCN-H) dose-dependently decreased [Ca2+]i transient and the expression of CaN (r=0.95).Conclusions1. Urocortin could improve the general state, cardiac function and hemodynamic indexes in DCM rats. Urocortin could reduce the level of HbAlc, CK-MB, BNP and have threapeutic effcts on DCM.2. Uocortin had a protective effect on diabetic myocardium, reduced myocardial injury, prevented myocardial fibrosis, inhibited calcium overload.3. Urocortin inhibited diabetic cardiomyopathy might be related to bind to CRII-R2, activated Akt/GSK-3β signaling pathway, and then inhibited the expression of TGF-β, and CTGF.4. These results suggested that Urocortin may have great therapeutic potential in the treatment of DCM by reducing total protein, cardiomyocoytes cell volume, and enhancing the cardiomyocyte viability.5. The protective effects of Urocortin on cardiomyocytes from HG-induced hypertrophy may be associated with the inhibition of increasing [Ca2+]i transient and decreasing expression of CaN.