| 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 changes of SIRT1/Wnt/?-catenin signaling pathway in the development of DCM and its potential regulatory mechanisms, and to detect the protective effect of the ARB drug on myocardium via the intervention of OLM. 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.Methods1. DM model established Rats were randomly divided into non-diabetic group(ND) and diabetic model group(DM) according to the principle of weight balance. The DM group were fed with high fat liquid lavage while ND group always feeding with ordinary food. Five weeks later, the DM group rats were fasting for 12 h, carotid sinus blood detection of serum TC, TG and other biochemical indicators to establish a high-fat model. Type 2 DM rat model was established by intraperitoneal injection of streptozotocin(STZ, 30 mg/kg). The induction of DM was considered successful when fasting blood glucose(FBG) exceeded 11.1 mmol/L 72 h after STZ injection to indicate a successful DM model rat has been established.2. Groups and materials The successful DM model rats were respectively and randomly devided into 4groups—2-week, 4-weeek, 8-week and 12-week. Every time point, again divided into DM model group, Olmesartan Medoxomil(OLM) group, which set high, medium and low dose group, Diamicron(DIA) group and the combination of OLM and DIA group and each group with eight or more rats.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.3. Research process(1). FBG?TG?TC?HD-C?LDL-C were analyzed by biochemical analyzer.(2). The changes of heart function was detected by echocardiographic and analyzed the change of the following indexes: LVIDd,LVIDs,LVPWd,LVPWs,LVMass,FS,LVEF,LVMass/Body Weight..(3). Fasting plasma insulin(FINS) levels, CTn I levels and Ang II level were measured using Enzyme-linked immunosorbant assay(ELISA) method.(4). HE and Masson trichromatic staining were used to observe myocardial pathological changes.(5). Immunohistochemical(IHC) were used to detect the changes of the expression of the protein level and the distribution, namely Wnt2, ?-catenin, c-Myc, s FRP2, Dact1 and SIRT1.(6). The changes of SIRT1/Wnt/?-catenin signaling pathway components were detected using WB method;(7). Real-time fluorescent quantitative polymerase chain reaction(q RT-PCR) was to detect myocardial m RNA expression changes.Results1. The general characteristics of DM rats and the pathological changes Compared 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. A small part of established DM model was appeared self-healing and they were excluded out of experiment. The eventually rate of successfully established DM model is 66.7%. 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 BWI and LVWI compared with ND rats. The serum level of FBG, TG,TC and LDL-C were also increased. It also shows increasingly decrease in cardiac function via echocardiography. HE results showed focal myocardial cell degeneration and necrosis in4-week DM rats myocardium. HE result 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 in 8and 12-week DM groups. Whether in function or pathological of myocardial tissue indicators were better than DM model after the intervention of OLM.2. Expression of SIRT1/Wnt/?-catenin signaling in DM rat myocardium WB and PCR results showed that the expression of Wnt2 and ?-catenin in 2 aand 4-week DM myocardium increased significantly compared with ND group(P < 0.01), and the trend continued in a high level after 8-week time point. The expression of TCF4 and SIRT1 kept increasing during the whole duration compared with the ND group while the level of s FRP2 continuously decreased. IHC results showed that, in 2-week DM myocardium, the expressions of Wnt2, ?-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?-catenin.3. Research of SIRT1/Wnt/?-catenin signaling pathway on the intervention of OLM in DM model rats The results showed that DM myocardial expression of Wnt2, ?-catenin and SIRT1 was gradually decreased on the intervention of OLM, while the intervention of DIA didn't show a remark difference. The combination of OLM and DIA group didn't show significant difference with the OLM group(P>0.05). Compared with the decreased level of Ang II and the increased level of AT1-R in DM group, the level of Ang II increased and the AT1-R level decreased significantly on the intervention of OLM, while the DIA group didn't show any changes apparently.Conclusion1. The rats were induced by high-fat diet joint STZ accompanied by insulin resistance, and cardiac function had a tendency to reduce gradually, along with myocardial cell apoptosis and significant myocardial necrosis, myocardial hypertrophy, myocardial fibrosis.2. With the development of DCM, Wnt/?-catenin signaling pathway components were activated in different degree along with the expression of SIRT1 and the downstream factors regulated by SIRT1, which were just right the inhibitors of Wnt/?-catenin signaling.3. The SIRT1/Wnt/?-catenin signaling pathway is restrained under the intervention of OLM and the protection effect on myocardial tissue occurred. It suggests that ARB drugs have a certain meaning on the protection of myocardial and this also provides a specific inhibitors for targets on the signaling pathways. |
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