丹蛭降糖胶囊通过上调Nrf2/HO-1信号通路减轻糖尿病心肌病大鼠心肌纤维化

Danzhi Jiangtang capsule alleviates myocardial fibrosis in diabetic cardiomyopathy rats by up-regulating Nrf2/HO-1 signaling pathway

目的基于核转录因子E2相关因子(Nrf2)/血红素加氧酶(HO)-1信号通路观察丹蛭降糖胶囊(DJC)对糖尿病心肌病(DCM)大鼠心肌纤维化的影响。方法80只雄性SD大鼠随机选取10只作为空白对照组,其余70只采用高糖高脂饮食联合腹腔注射链脲佐菌素(STZ)55 mg/kg建立2型DCM模型,最后65只大鼠造模成功,随机分为DJC低、中、高剂量组、二甲双胍组和模型组,DJC低、中、高剂量组按成人6 g/d等效剂量的0.5、1.0、2.0倍(270、540、1080 mg/kg)灌胃处理;二甲双胍组给予二甲双胍150 mg/(kg·d)剂量灌胃处理;模型组给予等容量蒸馏水灌胃处理,每组各13只,空白对照组给予等量蒸馏水,干预8 w后,麻醉状态下取材。取血清和心肌组织,检测血清空腹血糖(FPG)、糖化血红蛋白(HbA1c)、总胆固醇(TC)、三酰甘油(TG)、心肌谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧(ROS);采用苏木素-伊红(HE)染色法观察心肌组织病理学变化;用酶联免疫吸附实验(ELISA)检测心肌GSH-Px、SOD、MDA、ROS的表达;Western印迹法和实时荧光定量聚合酶链反应(PCR)法检测心肌组织Nrf2、HO-1蛋白和基因表达水平。结果与空白对照组比较,模型组光镜下心肌纤维走行较为紊乱,部分溶解、断裂,胞核位置不一,细胞间界限不清,间质出现纤维细胞增生。与模型组比较,DJC各剂量组和二甲双胍组光镜下心肌细胞病变明显减轻。与空白对照组比较,模型组血清FPG、HbAlc、TC、TG及心肌MDA、ROS含量显著升高,心肌GSH-Px、SOD含量及Nrf2、HO-1 mRNA及蛋白相对表达水平显著下降(P<0.01,P<0.05)。与模型组比较,DJC各剂量组和二甲双胍组血清FPG、HbA1c、TC、TG及心肌MDA、ROS含量明显下降,心肌GSH-Px、SOD含量及Nrf2 mRNA及蛋白相对表达水平显著升高,DJC中、高剂量组和二甲双胍组心肌HO-1 mRNA及蛋白相对表达水平显著升高(P<0.01,P<0.05)。结论DJC可能通过上调Nrf2/HO-1信号通路,减轻氧化应激损伤,改善糖脂代谢水平,延缓DCM大鼠心肌纤维化,发挥心肌保护作用。

Objective To observe the effect of Danzhi Jiangtang capsule(DJC)on myocardial fibrosis in diabetic cardiomyopathy(DCM)rats based on nuclear transcription factor E2-related factor(Nrf2)/heme oxygenase(HO)-1 signaling pathway.Methods Ten of 80 male SD rats were randomly selected as blank control group,and the other 70 rats were treated with high-sugar and high-fat diet combined with intrapitoneal injection of streptozotocin(STZ)55 mg/kg to establish type 2 DCM model.Finally,65 rats were successfully established and randomly divided into low,medium and high dose DJC groups,metformin group and model group.Low,medium and high dose DJC groups were treated by gavage 0.5,1.0,2.0 times(270,540,1080 mg/kg)of adult equivalent dose of 6 g/d by gavage;metformin group was given 150 mg/(kg•d)metformin by gavage.Model group was given equal volume of distilled water by gavage,with 13 rats in each group,blank control group was given the same amount of distilled water,8 w after intervention,sampling under anesthesia.Serum and myocardial tissues were collected,and serum fasting blood glucose(FPG),glycosylated hemoglobin(HbA1c),total cholesterol(TC),triacylglycerol(TG),myocardium glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),malondialdehyde(MDA)and reactive oxygen species(ROS)were detected.Hematoxylin-eosin(HE)staining was used to observe the myocardial histopathological changes.The expressions of myocardium GSH-Px,SOD,MDA and ROS were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of Nrf2 and HO-1 protein and gene in myocardial tissue were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction(PCR).Results Compared with blank control group,the myocardium fibers in the model group were disordered,partially dissolved and broken,with different nuclei,unclear boundaries between cells,and fibrocyte hyperplasia in the interstitium.Compared with model group,cardiomyocyte lesion were significantly reduced in each dose group of DJC and metformin group under light microscopy.Compared with blank control group,the contents of serum FPG,HbAlc,TC,TG and myocardial MDA and ROS were significantly increased in model group,while the contents of myocardial GSH-Px,SOD and the relative expression levels of Nrf2 and HO-1 mRNA and protein were significantly decreased in model group(P<0.01,P<0.05).Compared with model group,the contents of serum FPG,HbA1c,TC,TG and myocardial MDA and ROS in each dose group of DJC and metformin group were significantly decreased,while the contents of myocardial GSH-Px and SOD and the relative expression levels of Nrf2 mRNA and protein were significantly increased,the relative expression levels of myocardial HO-1 mRNA and protein in medium and high dose DJC groups and metformin groups were significantly increased(P<0.01,P<0.05).Conclusions DJC might play a role in myocardial protection by up-regulating Nrf2/HO-1 signaling pathway,reducing oxidative stress damage,improving glucose and lipid metabolism levels,delaying myocardial fibrosis in DCM rats.