糖尿病心肌病大鼠动物模型的构建及评价指标的建立

Establishment of diabetic cardiomyopathy rat model and evaluation index

目的构建糖尿病心肌病大鼠动物模型并建立其评价指标。方法30只SPF级Wistar雄性大鼠按体重随机分为两组,正常组给予常规饲料喂养、模型组给予高糖高脂饲料喂养4周。模型组大鼠禁食12 h腹腔注射链脲佐菌素(STZ)25 mg/kg,尾尖取血检测随机血糖值≥16.7 mmol/L判定为2型糖尿病大鼠。实验期间每周记录一次大鼠的体重及血糖值,8周后计算心脏指数,检测各组大鼠的血脂四项指标以及心电图和标志性的心肌酶。Masson和HE染色观察大鼠心肌组织的纤维化程度和形态学变化,并计算心肌胶原容积分数。Western Blot检测大鼠心脏组织中Bax、Bcl-2、Caspase-3、TGF-β1、α-SMA、Collagen-Ⅰ、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)蛋白表达水平。结果相较于正常组,模型组大鼠血糖值在1~8周显著升高(P<0.05),体重从第3周起显著降低(P<0.05),心脏指数显著增大(P<0.05),心脏电生理指标表现为心率(HR)、QRS间期、ST段波幅和QT间期水平显著升高(P<0.05)。三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)和丙二醛(MDA)的含量显著升高(P<0.05),高密度脂蛋白胆固醇(HDL-C)和超氧化物歧化酶(SOD)的含量显著降低(P<0.05)。HE染色结果显示模型组大鼠心肌组织中心肌细胞排列紊乱且间隙增大,并伴有炎性细胞浸润。Masson染色显示与正常组相比,模型组大鼠的心肌胶原容积分数显著增大(P<0.05),纤维化严重。模型组大鼠心肌组织中Bax、Caspase-3、TGF-β1、α-SMA、Collagen-Ⅰ、TNF-α、IL-6蛋白的表达水平显著升高(P<0.05),Bcl-2蛋白水平显著降低(P<0.05)。结论高糖高脂饲料喂养4周后,大鼠腹腔注射小剂量STZ 25 mg/kg,继续喂养8周后的Wistar大鼠在一定程度上已出现了类似于人的糖尿病心肌病的临床表现,此法可用于构建糖尿病心肌病动物模型,上述检测指标可用于糖尿病心肌病大鼠模型的评价。

Objective To construct a rat model of diabetic cardiomyopathy and establish its evaluation index.Methods 30 SPF Wistar male rats were randomly divided into two groups according to body weight.The normal group was fed a conventional diet,and the model group was fed a high-sugar and high-fat diet for four weeks.Rats in the model group fasted for 12 hours and were intraperitoneally injected with streptozotocin(STZ)25 mg/kg.Blood samples at the tail tip were taken to detect random blood glucose after≥16.7 mmol/L and determined as type 2 diabetes rats.During the experiment,rats'body weight and blood glucose values were recorded once a week.After eight weeks,the heart index was calculated,and four indexes of blood lipid,electrocardiogram,and landmark myocardial enzymes were detected in each group.Masson and HE staining were used to observe the degree of fibrosis and morphological changes of rat myocardial tissue,and the volume fraction of myocardial Collagen was calculated.Western Blot detected the protein expression levels of Bax,Bcl-2,Caspase-3,TGF-β1,α-SMA,Collagen-ⅰ,TNF-α,and IL-6 in rat heart tissue.Results Compared with the normal group,the blood glucose value of the model group was significantly increased from week 1 to week 8(P<0.05),body weight was significantly decreased from week 3(P<0.05),Heart index was significantly increased(P<0.05),and Heart electrophysiological indexes were expressed as Heart rate(Heart rate,HR),QRS interval,ST segment amplitude,and QT interval level were significantly increased(P<0.05).The contents of triglyceride(TG),cholesterol(TC),low-density lipoprotein(LDL-C),lactate dehydrogenase(LDH),creatine kinase isoenzyme(CK-MB),and malondialdehyde(MDA)were significantly increased(P<0.05).The contents of high-density lipoprotein(HDL-C)and superoxide dismutase(SOD)were significantly decreased(P<0.05).HE staining showed that myocardial cells in the model group were disordered,and the space was enlarged,accompanied by inflammatory cell infiltration.Masson staining showed that compared with the normal group,myocardial collagen volume fraction in the model group was significantly increased(P<0.05),and fibrosis was severe.The protein expression levels of Bax,Caspase-3,TGF-β1,α-SMA,Collagen-ⅰ,TNF-α,and IL-6 in the myocardial tissue of the model group were significantly increased(P<0.05).In contrast,the protein level of Bcl-2 was significantly decreased(P<0.05).Conclusions High sugar and high-fat feed for four weeks intraperitoneal injection of low-dose STZ 25 mg/kg,continued to 8 weeks after feeding Wistar rats to a certain extent,has appeared similar to the clinical manifestations of diabetic cardiomyopathy.This method can be used to construct animal models of diabetic cardiomyopathy.These measurements can be used for diabetic cardiomyopathy in the rat evaluation model.