腺苷酸激活蛋白酶介导糖尿病大鼠心肌线粒体自噬的机制与TSC2的关系研究

Relationship between the mechanism of AMPK-mediated myocardial mitochondrial autophagy and TSC2 in diabetic rats

目的:探讨腺苷酸激活蛋白酶(AMP-activated protein kinase,AMPK)介导糖尿病大鼠心肌线粒体自噬的机制与结节性硬化复合物-2(tuberous sclerosis complex-2,TSC2)的关系。方法:成年雄性SD大鼠30只,随机分为对照组、模型组、模型+AMPK激动剂AICAR组(AICAR组)。采用腹腔注射链脲佐菌素建立大鼠糖尿病大鼠模型。AICAR组大鼠连续皮下注射AICAR 12周。采用超声诊断仪检测所有大鼠的心功能:左室舒张末期容积(left ventricular end-diastolic volume,LVEDV)、左室收缩末期容积(left ventricular end-systolic volume,LVESV)、左室射血分数(left ventricular ejection fraction,LVEF)、心率(heart rate,HR)、舒张早期血流峰值速度(peak velocity of early diastolic flow,E)、舒张晚期血流峰值速度(peak velocity of late diastolic flow,A)、E/A。取心肌组织,采用免疫共沉淀法检测AMPK和TSC2的结合水平;Western blot检测AMPK、TSC2、p-TSC2、LC3-Ⅱ、LC3-Ⅰ、Beclin-1和P62蛋白的表达水平。结果:AICAR组LVEDV、LVEF、E和E/A均高于模型组,而A低于模型组(P=0.004,P=0.001,P=0.000,P=0.011,P=0.027);AICAR组AMPK和TSC2蛋白表达水平高于模型组(0.71±0.06 vs.0.36±0.09,P=0.003;0.80±0.02 vs.0.40±0.05,P=0.017);AICAR组结合水平高于模型组(0.49±0.09 vs.0.23±0.03,P=0.002);AICAR组p-TSC2蛋白水平高于模型组(1.48±0.07 vs.0.92±0.07,P=0.029);AICAR组LC3-Ⅱ/LC3-Ⅰ、Beclin-1和P62均高于模型组(P<0.05)。结论:AMPK介导糖尿病大鼠心肌线粒体自噬的机制可能与其对TSC2的磷酸化作用有关。

Objective:To investigate the relationship between the mechanism of AMP-activated-protein-kinase(AMPK)-mediated myocardial mitochondrial autophagy and tuberous sclerosis complex-2(TSC2)in diabetic rats.Methods:Thirty adult male SD rats were randomly divided into control group,model group,and model group+AMPK agonist AICAR group(AICAR group).The diabetic rat model was established by intraperitoneal injection of streptozotocin.Rats in the AICAR group were subcutaneously injected with AICAR for 12 weeks.Cardiac function of all rats was measured by echocardiography,including left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),left ventricular ejection fraction(LVEF),heart rate(HR),peak velocity of early diastolic flow(E),peak velocity of late diastolic flow(A),and E/A.The myocardial tissue was taken to detect the binding levels of AMPK and TSC2 by immunoprecipitation and the expression levels of AMPK,TSC2,p-TSC2,LC3-Ⅱ,LC3-Ⅰ,Beclin-1 and P62 were detected by Western blot.Results:The levels of LVEDV,LVEF,E and E/A in AICAR group were higher than those in the model group,but A was lower than that in the model group(P=0.004,P=0.001,P=0.000,P=0.011,P=0.027).The levels of AMPK and TSC2 protein in AICAR group were higher than those in the model group(0.71±0.06 vs.0.36±0.09,P=0.003;0.80±0.02 vs.0.40±0.05,P=0.017).The binding level of AICAR group was higher than that in the model group(0.49±0.09 vs.0.23±0.03,P=0.002),and the level of p-TSC2 protein in AICAR group was higher than that in the model group(1.48±0.07 vs.0.92±0.07,P=0.029).The levels of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and P62 in AICAR group were higher than those in model group(P<0.05).Conclusion:The mechanism of AMPK-mediated myocardial mitochondrial autophagy in diabetic rats may be related to its phosphorylation to TSC2.