降糖舒心方抑制内质网应激-过度自噬对糖尿病大鼠心肌病变的影响

Effect of Jiangtang Shuxin Recipe(降糖舒心方)on Cardiomyopathy in Diabetes Rats by Inhibiting Endoplasmic Reticulum Stress-Hyperautophagy

目的:探讨降糖舒心方对自噬通路过度激活反应的影响及其防止糖尿病合并心力衰竭心肌重构的机制。方法:100只大鼠按随机数字表法分成模型组、西药组、降糖舒心方低剂量组、降糖舒心方高剂量组、假手术组,每组20只。先对大鼠腹腔单次注射链脲佐菌素(STZ)进行糖尿病造模,再采用腹主动脉缩窄手术方法建立慢性心力衰竭动物模型。各给药组分别灌胃给予相应药物,假手术组和模型组大鼠灌胃给予等容积蒸馏水,1次/d,连续2个月。检测心脏左室射血分数(LVEF)、舒张末期室间隔厚度(IVSd)、收缩末期室间隔厚度(IVSs)、收缩末期左心室后壁厚度(LVPWs)与舒张末期左心室后壁厚度(LVPWd);HE染色检测大鼠心肌组织病理学改变情况;透射电镜观察心肌组织自噬超微结构变化;RT-PCR检测内质网应激信号分子GRP78 mRNA、IRE-1 mRNA、TRAF2 mRNA相对表达量;Western blotting检测心肌组织中自噬蛋白LC3-Ⅱ、Beclin 1表达情况。结果:与模型组比较,各给药组大鼠LVPWs、LVPWd、IVSs、IVSd均明显降低(P<0.05),LVEF均明显升高(P<0.05);心肌组织病理学积分均明显降低(P<0.05);大鼠心肌细胞损害程度有所减轻,自噬小体和空泡有所减少,纤体化程度减轻;心肌组织GRP78 mRNA、IRE-1 mRNA、TRAF2 mRNA相对表达量均明显降低(P<0.05);LC3-Ⅱ、Beclin 1蛋白相对表达量均明显降低(P<0.05)。与西药组比较,降糖舒心方高剂量组大鼠LVEF明显升高(P<0.05);降糖舒心方低、高剂量组大鼠心肌组织病理学积分均明显降低(P<0.05);大鼠心肌细胞数量较多,自噬小体和空泡减少,纤体增生减轻;心肌组织GRP78 mRNA、IRE-1 mRNA、TRAF2 mRNA相对表达量均明显降低(P<0.05);LC3-Ⅱ、Beclin 1蛋白相对表达量均明显降低(P<0.05)。与降糖舒心方低剂量组比较,降糖舒心方高剂量组大鼠心肌肌原纤维排列整齐,间质增生少,胞体大部分光滑完整,胞内有少量自噬小体和空泡,各项指标均有剂量依赖性。结论:内质网应激-自噬在心力衰竭的发生发展过程中发挥了重要的作用。糖尿病状态过度自噬可导致心肌细胞数量降低,使心室壁变薄。降糖舒心方能减轻过度自噬对心肌的损害,抑制心肌重构,改善心功能。

Objective:To explore the mechanism of Jiangtang Shuxin(JTSX)recipe in regulating the hyperactivation of autophagy,and reveal its mechanism of preventing myocardial remodeling in diabetic heart failure.Methods:The 100 rats were randomly divided into model group,Western medicine group,low-dose JTSX group(JTSX1),high-dose JTSX group(JTSX2),and sham-operation group using a random number table method,with 20 rats in each group.The diabetic model was first established by intraperitoneal injection of streptozotocin in rats,and then the chronic heart failure was modeled by abdominal aortic coarctation.Each medication group was given corresponding drugs by gavage,while rats in sham-operation group and model group were given equal volume distilled water by gavage once a day for two consecutive months.The 1eft ventricular ejection fraction(LVEF),interventricular septal depth(IVSd),interventricular septal thickness at end-systole(IVSs),left ventricular posterior wall systole(LVPWs)and 1eft ventricular posterior wall diastole(LVPWd)were tested.The HE staining was used to detect myocardial pathology,and the ultrastructural changes of myocardial autophagy were observed by transmission electron microscopy.The transcription of glucose-regulated protein78(GRP78)mRNA,Inositol-requiring enzyme 1(IRE-l)mRNA and tumor necrosis factor receptor-associated factor 2(TRAF2)mRNA were tested by reverse-transcription polymerase chain reaction(RT-PCR),and the autophagy protein of microtubule-associated protein light chain-Ⅱ(LC3-Ⅱ)and Beclin1 were detected by Western blotting.Results:Compared with the model group,the LVPWs,LVPWd,IVSs and IVSd decreased in medication groups,while the LVEF increased in medication groups,with statistically significant difference(P<0.05);The scores of pathological changes in myocardial tissue were significantly reduced in medication groups(P<0.05);The degree of myocardial cell damage has been reduced in medication groups,with autophagosomes,vacuoles and fibrosis decreased;The relative transcription levels of GRP78 mRNA,IRE-1 mRNA and TRAF2 mRNA in myocardial tissue declined significantly in medication groups(P<0.05);The LC3-Ⅱand Beclin 1 were decreased in medication groups(P<0.05).Compared with the Western medicine group,LVEF significantly increased in JTSX2 group(P<0.05);The scores of pathological changes in myocardial tissue were significantly reduced in JTSX1 group and JTSX2 group(P<0.05);The number of myocardial cells is relatively large,with a decrease in autophagosomes and vacuoles,and a reduction in fibrous proliferation;The relative expression levels of GRP78 mRNA,IRE-1 mRNA,and TRAF2 mRNA in myocardial tissue were significantly reduced in JTSX1 group and JTSX2 group(P<0.05);The relative expression levels of LC3-Ⅱand Beclin 1 proteins were significantly reduced JTSX1 group and JTSX2 group(P<0.05).Compared with JTSX1 group,JTSX2 group have orderly arrangement of myocardial myofibrils,with less interstitial hyperplasia,mostly smooth and intact cell bodies,and fewer of autophagosomes and vacuoles in cells.All indicators were dose-dependent.Conclusion:Endoplasmic reticulum stress autophagy plays an important role in the occurrence and development of heart failure.Excessive autophagy in diabetes can reduce the number of myocardial cells and damage the ventricular wall.JTSX can alleviate excessive autophagy in myocardial cells,inhibit myocardial remodeling,and improve cardiac function.