左归降糖舒心方对糖尿病心肌病MKR鼠心肌细胞损伤和凋亡的影响

Effects of Zuogui Jiangtang Shuxin Decoction on Cardiomyocyte Damage and Apoptosis in MKR Mice with Diabetic Cardiomyopathy

目的观察左归降糖舒心方对糖尿病心肌病(Diabetic cardiomyopathy,DCM)模型鼠心肌细胞损伤和凋亡的影响,并探讨其作用机制。方法以转基因2型糖尿病小鼠(MKR鼠)为实验对象,通过链脲菌素(streptozotocin,STZ)腹腔注射+持续高脂喂饲建立DCM模型,分为模型组、中药组、阳性药物组,另设C57小鼠为空白对照组。中药组给予左归降糖舒心方14.27 g生药/(kg·天)灌胃,阳性药物组给予二甲双胍250 mg/(kg·天)+依那普利4.5 mg/(kg·天)灌胃,连续给药4周。采用电化学法血糖仪测定空腹血糖(fasting blood glucose,FBG),光镜和透射电镜观察小鼠心肌组织形态学变化,ELISA法检测血清心肌肌钙蛋白Ⅰ(cardiac troponinⅠ,cTnⅠ)、乳酸脱氢酶(lactic dehydrogenase,LDH)水平,免疫组化法检测Ⅰ型胶原蛋白(Collagen-Ⅰ)、Ⅲ型胶原蛋白(Collagen-Ⅲ)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)的蛋白表达,TUNEL法检测心肌细胞凋亡。结果模型组FBG较空白对照组显著升高,经左归降糖舒心方干预后明显降低(P<0.01)。与模型组比较,中药组和阳性药物组的心肌组织病理形态明显改善,Collagen-Ⅰ、Collagen-Ⅲ蛋白表达减弱(P<0.01),血清cTnⅠ、LDH水平明显降低(P<0.01),细胞凋亡阳性表达减弱(P<0.01),Bax、Caspase-9表达减弱(P<0.01),Bcl-2表达增强(P<0.01)。中药组下调Collagen-Ⅰ和Collagen-Ⅲ表达、减少心肌细胞凋亡、下调Bax和Caspase-9表达等作用弱于阳性药物组(P<0.01),但上调Bcl-2表达的作用强于阳性药物组(P<0.01)。结论左归降糖舒心方对糖尿病心肌病MKR鼠的心肌细胞损伤和凋亡具有保护作用,其作用机制可能与降糖、改善心肌纤维化、抑制促凋亡因子Bax和Caspase-9表达、促进抗凋亡因子Bcl-2表达等有关。

Objective To observe the effects of Zuogui Jiangtang Shuxin Decoction on cardiomyocyte damage and apoptosis in MKR mice with diabetic cardiomyopathy(DCM), and to explore its mechanism of action. Methods The DCM models were established with MKR mice by intraperitoneal injection of streptozotocin(STZ) combined with continuous high-fat feeding, and then divided into model group, traditional Chinese medicine group, positive drug group,and C57 mice as blank control group. The traditional Chinese medicine group was given 14.27 g crude drug/(kg·d)Zuogui Jiangtang Shuxin Decoction by gavage, and the positive drug group was given 250 mg/(kg·d) metformin and 4.5mg/(kg·d) enalapril by gavage every day for 4 weeks. Fasting blood glucose(FBG) was measured, and the morphological changes of myocardial tissue in mice were observed under light microscope and transmission electron microscope. Serum cardiac troponin Ⅰ(cTnⅠ) and lactate dehydrogenase(LDH) were detected by ELISA. Immunohistochemical method was used to detect Collagen-Ⅰ, Collagen-Ⅲ, Bcl-2 associated X protein(Bax), Caspase-9, B-cell lymphoma-2(Bcl-2). The apoptosis of cardiomyocytes was detected by TUNEL method.Results The FBG of the model group was significantly higher than that of the blank control group, and it was significantly reduced after the intervention of Zuogui Jiangtang Shuxin Decoction(P<0.01). Compared with the model group, the pathological morphology of myocardial tissue in the Chinese medicine group and the positive medicine group was significantly improved, the expression of Collagen-Ⅰ and Collagen-Ⅲ was reduced(P<0.01), the serum cTnⅠ and LDH were significantly reduced(P<0.01), the apoptosis was reduced(P<0.01), the expression of Bax and Caspase-9 was weakened(P<0.01), and the expression of Bcl-2 was enhanced(P<0.01). The effect of down-regulating Collagen-Ⅰ and Collagen-Ⅲ expression, reducing myocardial cell apoptosis, down-regulating the expression of Bax and Caspase-9 in the traditional Chinese medicine group was weaker than that of the positive drug group(P<0.01), but the effect of up-regulating the expression of Bcl-2 was stronger than that of the positive drug group(P<0.01). Conclusion Zuogui Jiangtang Shuxin Decoction has protective effects on cardiomyocyte damage and apoptosis in MKR mice with diabetic cardiomyopathy. Its mechanism may be related to lowering blood glucose, improving myocardial fibrosis, inhibiting the expression of pro-apoptotic factors Bax and Caspase-9, and promoting the expression of anti-apoptosis factor Bcl-2.