实验性2型糖尿病心肌病大鼠模型的建立与评价

Establishment and Assessment of an Experimental Rat Model of Type 2 Diabetic Cardiomyopathy

目的建立和评价2型糖尿病心肌病(DC)大鼠模型,探究高糖脂饮食在模型建立中的作用。方法将雄性Wistar大鼠随机分成正常对照组、高糖脂饮食组和高糖脂负荷小剂量STZ组。高糖高脂膳食诱导11周负荷小剂量链脲佐菌素(STZ)(30 mg/kg)腹腔注射建立DC模型,并观察糖代谢、脂代谢和心功能的变化。结果①大鼠经高糖高脂饲料诱导4周后,与正常对照组相比,胆固醇(TCH)和甘油三酯(TG)均显著增高(P<0.05),血糖值没有明显变化(P>0.05)。②大鼠注射30 mg/kg STZ后72 h,血糖水平开始升高,继续以高糖高脂饲料喂养6周后,与正常对照组比较,高糖脂饮食组和高糖脂负荷小剂量STZ组大鼠TG、TCH维持高水平,差异有显著性(P<0.05);高糖脂负荷小剂量STZ组大鼠血糖值持续高水平,与正常对照组差异有显著性(P<0.001)。③心功能测量结果显示,高糖脂饮食组大鼠出现温和的心脏功能异常(左心室收缩压降低,左心室舒张末压升高);高糖脂负荷小剂量STZ组大鼠左心室收缩和舒张功能均出现异常(LVSP、每搏输出量、心排量降低,LVEDP、左心室最大舒张速率升高),但以舒张功能异常为主。结论大鼠高糖脂饮食诱导负荷小剂量STZ可建立类似临床症状的2型DC模型,高糖脂饮食在糖脂代谢紊乱和心脏功能损伤过程中有重要作用,结合糖、脂代谢指标和心脏功能指标可以有效简便评价糖尿病心肌病模型。

Objective Diabetic cardiomyopathy（DC） is an independent and specific diabetic complication occurred in the absence of coronary artery disease or systemic hypertension.Epidemiological and clinical studies have demonstrated the existence of diabetic cardiomyopathy in humans.As a clinical condition,DC diagnosed when ventricular dysfunction developed in patients with diabetes in the absence of coronary atherosclerosis and hypertension and often occurred as an unknown asymptomatic heart disease,which is a obstacle for clinical diagnosis and treatment.One major impediment to progress in this area is the lack of appropriate animal models,and a related difficulty is the lack of standardized measures for phenotypes associated with diabetes and its complications.It is important to bear in mind that the establishment of DC animal models must reflect the clinical features of DC patients and fulfill the definition of diabetic cardiomyopathy before the start of mechanism and medical studies.The aim of this study was to establish and assess an experimental rat model of type 2 diabetic cardiomyopathy（DC）,and to evaluate the contribution of high sucrose and high fat diet to the pathology and find easy and stable indexes of DC assessment.Methods 45 male Wistar rats（150 g～180 g） were randomly divided into 3 groups： the control group,high sucrose-high fat group（HSF） and high sucrose-high fat with low dose streptozotocin（STZ） group（HSF-STZ）.The rats in the control group were fed with normal food,the HSF and HSF-STZ rats were fed with high sucrose-high fat diet（consisted of 20% sucrose,10% lard,2.5% cholesterol and 67.5% normal food） for totally 11 weeks.Blood of all rats were collected after 12-hour fasting to measure fasting blood glucose（FBG）,total cholesterol（TCH） and triglyceride（TG） before high sucrose-high fat diet feeding and after 4 weeks（the fifth week） of feeding.Then the rats of the HSF-STZ group were injected intraperitoneally（ip） with a single dose of 30 mg/kg STZ（resolved in citric acid-sodium citrate buffer,pH=4.5） after 4 weeks＇ high sucrose-high fat diet feeding.FBG was tested after 72 hours and FBG ≥11.1 mmol/L was considered as the standard for the establishment of diabetes mellitus.Rats in control and HSF groups were injected i.p.with citric acid-sodium citrate buffer（pH = 4.5）.At the end of experiment（the eleventh week）,heart rate（HR）,stroke volume（SV） and cardiac output（CO） of the rats were measured by impedance plethysmography（IPG） and left ventricular systolic pressure（LVSP）,left ventricular end diastolic pressure（LVEDP）.The maximum rate of myocardial contraction（＋dP/dt max） and maximum rate of myocardial diastole（-dP/dt max） were measured by left ventricular catheterization using MP150 polygraph physiological signal recorder to evaluate the cardiac function.Then artery blood of the rats were collected to measure FBG,glycosylated hemoglobin（GHb）,glycosylated serum protein（GSP）,TCH,nonesterified fatty acid（NEFA） and high density lipoprotein（HDL）.Results 1.The effect of high sucrose and high fat diet on rats.Among the three groups,blood glucose and lipid were similar to each other at the beginning of the experiment.After 4 weeks of high sucrose-high fat diet feeding in rats,serum TCH and TG in HSF and HSF-STZ groups were significantly increased in comparrison with those of the control group（2.4±0.4,2.2±0.3 vs.1.9±0.2;0.75±0.17,0.89±0.28 vs.0.56±0.12;P0.05,P0.05）,but fasting blood glucose（FBG） didn＇t show a significant difference（5.0±1.2,4.3±0.8 vs. 4.8±1.8;P0.05）.2.The changes of glucose metabolism after high sucrose and high fat diet with STZ injection in rats.The FBG of rats in HSF-STZ group significantly increased at 72 hours after STZ injection as well as GHb and GSP of rats in HSF-STZ group were significantly higher than the values of the control group（18±8,35.5±9.7,1.8±0.4 vs.5.4±0.8,26.2±8.9,1.4±0.2;P0.001,P0.05,P0.05） indicating a sustained hyperglycemia level after STZ injection.But values of blood glucose were not significantly increased in the rats of HSF group（5.2±1.0,25.9±6.5,1.2±0.3 vs.5.4±0.8,26.2±8.9,1.4±0.2;P0.05）.3.The changes of lipid metabolism after high sucrose and high fat feeding with STZ injection in rats.The blood lipid maintained a high level after another 6 weeks of high sucrose-high fat diet feeding.Plasma NEFA in HSF and HSF-STZ groups were significantly increased in comparison with those of control group（0.74±0.33,0.64±0.24 vs.0.32±0.19;P0.05,P0.05）,and HDL was significantly lower（0.72±0.17,0.87±0.26 vs.1.18±0.29;P0.001,P0.05）.TCH of rats in HSF-STZ group was significantly higher than that of the control rats（2.6±1.0 vs.1.8±0.6;P0.05）,whereas the HSF rats didn＇t show significant difference（2.0±0.6 vs.1.8±0.6;P0.05）.4.The changes of cardiac function after high sucrose and high fat diet feeding with STZ injection in rats.After 11 weeks of high sucrose-high fat diet feeding,mild cardiac dysfunction was found in the rats of HSF group.The LVSP was lower than that of the control group（161±22 vs.184±22;P0.05） and LVEDP was higher（-8.6±1.8 vs.-15.2±6.8;P0.01）.But ±dP/dtmax,HR,SV and CO showed no significant differences（15460±5680,-12,110±5,170,426±47,0.035±0.010,10.9±2.8 vs.19 280±4170,-15 230±2 848,435±39,0.033±0.012,12±4;P0.05）.The rats of HSF-STZ group exhibited cardiac dysfunction,and diastolic dysfunction exhibited more significant.The LVSP, SV and CO in rats of HSF-STZ group were significantly lower（156±23,0.026±0.006,9.2±1.4 vs.184±22,0.033±0.012,12±4;P0.05） than those of the control group while LVEDP and-dP/dtmax were higher（-8.4±3.6,-10,801±2,783 vs.-15.2±6.8,-15,230±2,848;P0.05）.But HR didn＇t show a significant difference（396±44 vs.435±39,P0.05）.Discussion and conclusion The development of diabetic cardiomyopathy is multifactorial and its pathogenesis is still under controversial.Recent putative mechanisms include metabolic disturbances（depletion of glucose transporter,increased free fatty acids,carnitine deficiency）,abnormalities in ion homeostasis（changes in calcium homeostasis）,insulin resistance（hyperinsulinemia and reduced insulin sensitivity）,inner fibrosis（associated with increases in angiotensin Ⅱ,IGF-Ⅰ,and inflammatory cytokines）,small vessel disease（endothelial dysfunction and impaired coronary flow reserve）,and autonomic dysfunction.Recent studies have proposed that mechanisms involved in reduced myocardial contractility including impaired calcium homeostasis,increased oxidative stress,derrangement of substrate metabolism,mitochondrial dysfunction and upregulated renin-angiotensin system.Sustained hyperglycemia may increase glycation of interstitium such as collagen by various pathways,which also results in myocardial stiffness and impaired contractility.More and more recent experimental studies have focused on the animal model of DC and the present main DC models are as follows： 1.Transgenic animals,such as db/db diabetic mouse,ob/ob obesity mouse,Zucker diabetic fat rats,CIRKO mouse,IRS-1 KO mouse and so on.The heart of these animals exhibits increased rate of myocardial oxygen consumption,increased rate of fatty acid oxidation,decreased myocardial efficiency,mitochondrial dysfunction,myocardial insulin signaling impairment,lipotoxicity,increased oxidative stress and so on,which are similar to the clinical characteristics of DC patients.Although transgenic animals are excellent models for the study of non-insulin-dependent diabetes mellitus and its complications,their applications are limited because of high cost and strict experimental conditions.2.Simply high-fat feeding-induced animal model.A study has examined the temporal pattern of changes in insulin action and glucose metabolism in individual organs during chronic high-fat feeding in C57BL/6 mice.The mice showed cardiac dysfunction,abnormal metabolism,impaired insulin signaling and decreased cardiac efficiency.But cardiac dysfunction accompanied with mild hyperglycemia and hyperleptinemia occurred after 20 weeks of high-fat feeding.The experiment period of the model was too long and hyperglycemia was not significant,so it is also not a satisfactory DC model for related studies.Insulin resistance is a clear antecedent of type 2 diabetes on myocardial metabolism and function.Studies in humans with type 2 diabetes or obesity and insulin resistance have demonstrated significant increase in myocardial fatty acid uptake and oxidation as well as reduced concentrations of high-energy phosphates in individuals without coronary artery disease or heart failure.Recent studies have focused on the contribution of impaired insulin signaling or insulin action to the pathogenesis of contractile dysfunction in the diabetic heart.We induced insulin resistance in rats by feeding high sucrose and high fat diet.It showed that TCH and TG significantly increased after 4 weeks of high sucrose and high fat diet feeding which means the onset of dyslipidemia.As a kind of pancreatic beta-cell toxin,low dosage of STZ was administrated to induce partly pancreatic dysfunction simulating pancreatic islet cell hypofunction in type 2 DC patients.In this study rats in the HSF-STZ group were injected with 30 mg/kg STZ and fed with high sucrose and high fat diet.A rat model of DM was established because the rats in HSF-STZ group began to show an increase of blood glucose at 72 h after STZ injection and the blood lipid and glucose maintained a high level during the experiment time.High sucrose and high fat diet can induce dyslipidemia and combined with STZ injection may result in worsen disturbances of lipid and glucose metabolisms,which are similar to that observed in patients with type 2 DM.Metabolic disturbances play an important role in cardiac dysfunction and pathological changes in DM patients,and are closely related to heart diastolic dysfunction at the early age.Diastolic dysfunction is a characteritic cardiac dysfunction of diabetic cardiomyopathy that may precede the development of systolic dysfunction.LV diastolic dysfunction is characterized by impairment in early diastolic filling,an increase in atrial filling,a prolongation of isovolumetric relaxation and increased numbers of supraventricular premature beats.The measurement of cardiac function of rats in the HSF-STZ group showed that LVSP,SV and CO were significantly increased,and LVEDP and-dP/dtmax decreased in comparison with those in the control rats.It indicated that high sucrose and high fat diet with STZ could impair both diastolic and systolic function and diastolic dysfunction exhibited more significantly.While HR showing no significant difference compared with that in the normal rats indicated cardiac dysfunction was mainly induced by direct myocardial contractile dysfunction instead of autonomic neuropathy.The measurement of rats in HSF group which were fed with high sucrose and high fat diet for 11weeks showed mild cardiac dysfunction including decreased LVSP and increased LVEDP.So we got a conclusion that simply high sucrose and high fat diet induction played an important role in metabolic disturbances and cardiac dysfunction.In summary,experimental type 2 DC model can be established by high sucrose-high fat diet with single low dosage of STZ injection in rats.The rat model of DC can be evaluated effectively and simply by synthetic indexes of glucose and lipid metabolism and heart function.The model rats demonstrate glucose and lipid metabolism disturbances and cardiac dysfunction,especially the diastolic dysfunction is similar to that of clinical symptoms.Time of the establishment of this model is much shorter compared with simple high-fat feeding in rats（11 weeks vs.20 weeks） and the method is much easier to perform.