| Objective Diabetic cardiomyopathy (DCM) is a major part of diabetic cardiovascular complications, and is the main cause for death in patients with diabetes mellitus (DM). The aim of this research is to study the alterations of Wnt/β-catenin signaling pathway in the emergence and development of DCM and its regulatory mechanisms. This investigation will help to perfect the pathophysiological mechanisms for DCM, and to explore new therapeutic targets and new drug development goals for DCM treatment.Methods 1. Established model, grouping and sample collectionType 1 DM rat model was established by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The induction of DM was considered successful when fasting blood glucose (FBG) exceeded 16.7mmol/L 72h after STZ injection. The successful DM model rats also showing more to drink, eat, polyuria and weight decrease. The rats of Non-diabetic (ND) group and the successful DM model rats were respectively and randomly devided into 4 groups—2-week, 4-weeek,8-week and 12-week, greater than or equal 10 rats for each group with balancing body weight. On weeks 2,4,8 and 12, respectively, the rats’ FBG and their body weight (BW) were tested. The rats then were anesthetized, took blood from abdominal aorta, removed hearts quickly, eliminated blood vessels, fat and other non myocardial tissue, tested the heart weight (HW), kept portions of left ventricle (LV, with ventricular septal), tested the LV weight (LVW). The cardiac index (CI, HW/BW) and left ventricular weight index (LVWI, LVW/BW) were counted. The LV tissues were devided into 2 groups-some were fixed in 10% formaldehyde solution; the others were frozen in liquid nitrogen.2. The pathological observation and target detection for 2 weeks DM ratsHE staining was used to observe myocardial pathological changes; The alterations of DM myocardial Wnt2, β-catenin, c-Myc and DKK1 were detected by Western blotting (WB) and immunohistochemical (IHC) method.3. The pathological observation and target detection for 4,8,12 weeks DM ratsFasting plasma insulin (FINS) levels was measured using Enzyme-linked immunosorbant assay (ELISA) method. HE and Masson trichromatic staining were used to observe myocardial pathological changes; TdT-mediated dUTP nick end labeling (TUNEL) was used to determine the level of myocardial apoptosis; The alterations of Wnt/β-catenin signaling pathway components were detected using IHC and WB method; While real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was to detect myocardial Wnt2, GSK3β, β-catenin, TCF4, c-Myc and DKK1 mRNA expression changes.Results 1. The general characteristics of DM rats and the pathological changesCompared with ND group, DM group rats gradually showed more to drink, eat, polyuria and angular, listlessness, fur lackluster, smelling of urine ketone smell more aggravating, loose stools, etc; DM rats characterized by markedly lower FINS and higher FBG level compared with ND rats. There was decrease in HW and LVW due to BW loss in DM rats, but showed a significant increase in CI and LVWI compared with ND rats. HE results showed focal myocardial cell degeneration and necrosis in 2-week DM rats myocardium, but no significant myocardial hypertrophy was observed; HE and Masson trichromatic staining results showed that the myocardial collagen deposition area sustained increase, and the cross section area of myocardial cells were significantly higher than ND group, indicating the occurrence of myocardial fibrosis and cardiac hypertrophy; The myocardial cell apoptosis rate increased significantly with time dependence following 4-12 weeks DM according to the results of TUNEL.2. The expressions of Wnt/β-catenin signaling in 2-week DM rat myocardiumWB results showed that the expression of Wnt2, β-catenin and c-Myc in 2-week DM myocardium increased significantly compared with ND group (P<0.01), but the change of endogenous inhibitor DKK1 was not obviously. IHC results showed that, in 2-week DM myocardium, the expressions of Wnt2, P-catenin and c-Myc were significantly increased, while in ND group their expression was uniform distribution. We also found a small amount of nuclear expression of β-catenin while c-Myc was increased both in cytoplasm and nucleus.3. The protein and mRNA expressions of Wnt/p-catenin signaling in 4,8, 12 weeks DM rat myocardiumThe results showed that DM myocardial expression of Wnt2, β-catenin and c-Myc was gradually increased as the development of DCM (P<0.01), and their mRNA levels were also gradually increased (P<0.05), while TCF4 and its mRNA level incresed only in 8 and 12 weeks (P<0.01). P-catenin expression in 4-week DM myocardium was increased, and began to enter into nucleus, at 8 and 12 weeks, its nucleus expression was gradually increased with the decrease in cytoplasm (P<0.01); while the expression of c-Myc was gradually increased both in nucleus and cytoplasm as DCM developed (P<0.01). DKK1 and its mRNA levels were remarkably increased in 4-week DM heart, then it was decreased as DCM developed in 8 and 12 weeks, but the level was still higher than ND (P<0.01). In addition, P-GSK3β (Ser9) showed no obvious increase in 4-week DM myocardium, but it was gradually increased in 8 and 12 weeks, while accompanied by the progressive decrease of GSK3β mRNA (P<0.01).Conclusion 1. STZ-induced DM induced increase of myocardial cell apoptosis, along with significant myocardial necrosis, myocardial hypertrophy, myocardial fibrosis.2. Wnt/β-catenin signaling pathway components were increased in different degree along with the development of DCM, these results suggest that DM induced the activation of Wnt/β-catenin signaling pathway.3. Activation of Wnt/β-catenin signaling pathway is associated with the development of DCM, developing specific inhibitors for Wnt/β-catenin signaling pathway will bring gospel for DCM patients. |
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