从AMPK/mTOR通路探讨人参-三七-川芎提取物对糖尿病小鼠心脏老化的保护作用机制

Explore Protective Mechanism of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma Extract on Cardiac Aging in Diabetic Mice Through AMPK/mTOR Pathway

目的:从AMP激活的蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路的活化及心脏病理形态学变化、相关衰老蛋白的改变,探讨人参-三七-川芎提取物(GNC)对糖尿病小鼠心脏老化的保护作用机制。方法:C57BL/6雄性小鼠,SPF级,随机分为正常组和高糖组。高糖组腹腔注射链脲佐菌素(STZ)联合喂食高脂饲料,造模成功后再次随机分为模型组,GNC低剂量组(0.819 g·kg^-1),GNC高剂量组(1.638 g·kg^-1)及二甲双胍组(150 mg·kg^-1)。每天1次,灌胃给药,连续9周,监测血糖变化。4周龄雄性C57BL/6小鼠正常喂养1周,为青年组。观察小鼠一般情况,苏木素-伊红(HE)染色结合透射电镜(TEM)观察小鼠心脏病理形态学变化,冯库萨(Von Kossa)染色判断小鼠心脏微血管钙盐沉积程度,蛋白免疫印迹法(Western blot)检测小鼠心脏组织中AMPK/mTOR信号通路活化状态,以及衰老相关蛋白基质金属蛋白酶-2(MMP-2),抑癌基因p53(p53),磷酸化抑癌基因p53(p-p53)表达。结果:与正常组比较,模型组血糖显著升高(P<0.01);与模型组比较,各给药组血糖明显下降(P<0.05,P<0.01)。HE,TEM,Von Kossa 3个病理形态学实验结果显示:与正常组比较,模型组小鼠心肌细胞肥大,心肌纤维排列紊乱、灶性溶解坏死,线粒体肿胀、变性、嵴断裂、空泡变,心脏微血管结构紊乱、染色不均匀、中内膜有大量钙盐沉积;与模型组比较,各给药组小鼠病理形态学变化均有不同程度的改善。与正常组比较,模型组MMP-2,p53和p-p53蛋白表达明显升高(P<0.05,P<0.01),p-肝激酶B1(LKB1)/LKB1,p-AMPK/AMPK蛋白明显降低(P<0.05,P<0.01),p-mTOR/mTOR,p-核糖体蛋白S6激酶p70S6K/p70S6k蛋白显著升高(P<0.01);与模型组比较,各给药组MMP-2,p53和p-p53蛋白表达明显降低(P<0.05,P<0.01),p-LKB1/LKB1,p-AMPK/AMPK蛋白明显升高(P<0.05,P<0.01),p-mTOR/mTOR,p-p70S6k/p70S6k蛋白明显降低(P<0.05,P<0.01)。结论:STZ联合高脂饲料能够诱导小鼠心脏老化,GNC能够改善糖尿病小鼠心脏老化,其作用机制可能与调控AMPK/mTOR通路相关蛋白表达有关。

Objective:To explore the protective mechanism of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma(GNC)extracts on cardiac aging in diabetic mice by observing the activation of AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway,changes of cardiac pathomorphological and related senescent proteins.Method:C57BL/6 male mice,SPF level,were randomly divided into normal control group and high-glucose group.The mice in high-glucose group were intraperitoneally injected with streptozotocin(STZ)and fed with high-fat diet.After successful modeling,they were randomly divided into model group,low-dose GNC group(0.819 g·kg^-1),high-dose GNC group(1.638 g·kg^-1)and metformin group(150 mg·kg^-1).The drug was administered by gavage once a day for a continuous period of 9 weeks.4-week-old male C57BL/6 mice were normally fed for 1 week as a youth group.General conditions of mice were observed.Hematoxylin-eosin(HE)staining combined with transmission electron microscope(TEM)was used to observe the cardiac pathomorphology in mice.Von Kossa staining was used to determine the degree of calcium salt deposition in cardiac micro vessels.Western blot was used to detect the activation of signaling pathways in myocardial tissue of mice,as well as the expression levels of matrix metalloproteinases-2(MMP-2),tumor suppressor p53(p53),and phospho-tumor suppressor p53(p-p53).Result:As compared with the normal group,the blood glucose in the model group increased(P<0.01),as compared with the model group,the blood glucose in each administration group decreased significantly(P<0.05,P<0.01).The results of three pathological morphology experiments(HE,TEM,and Von Kossa)showed that as compared with the normal control group,the mice in model group showed cardiomyocytes hypertrophy,disordered arrangement of myocardial fibers,focal dissolving and necrosis,mitochondria swelling,degeneration,crest fracture,vacuolar alteration,disordered microvascular structure of the heart,uneven staining,and a large amount of calcium deposition in tunica media and intima.As compared with the model group,the pathomorphological changes of mice in each administration group were improved in varying degrees.Compared with the normal group,the expression levels of MMP-2,p53 and p-p53 protein in the model group were significantly increased(P<0.05,P<0.01),the protein ratios of p-liver kinase B2(LKB1)/LKB1,p-AMPK/AMPK were significantly decreased(P<0.05,P<0.01),and the average gray level of p-mTOR/mTOR and p-p70S6k inase(p70S6k)/p70S6k protein was significantly increased(P<0.05,P<0.01),while the protein ratios of p-mTOR/mTOR,p-p70S6k/p70S6k were increased(P<0.01).As compared with the model group,the expression levels of MMP-2,p53 and p-p53 protein in each administration group were significantly decreased(P<0.05,P<0.01),the protein ratios of p-LKB1/LKB1,pAMPK/AMPK were significantly increased(P<0.05,P<0.01),while the protein ratios of p-mTOR/mTOR and p-p70S6k/p70S6k were decreased(P<0.05,P<0.01).Conclusion:STZ combined with high-fat diet can induce cardiac aging in mice,and GNC can improve cardiac aging in diabetic mice,which may be related to the inhibition of AMPK/mTOR pathway related protein expression.