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

RelationshipbetweenthemechanismofAMPK-mediatedmyocardialmitochondrialautophagyandTSC2indiabeticrats

摘要:

目的：探讨腺苷酸激活蛋白酶（AMP-activatedproteinkinase，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（tuberoussclerosiscomplex-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AICAR12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（leftventricularend-diastolicvolume，LVEDV）、左室收缩末期容积（leftventricularend-systolicvolume，LVESV）、左室射血分数（leftventricularejectionfraction，LVEF）、心率（heartrate，HR）、舒张早期血流峰值速度（peakvelocityofearlydiastolicflow，E）、舒张晚期血流峰值速度（peakvelocityoflatediastolicflow，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；Westernblot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±0.06vs.0.36±0.09，P=0.003；0.80±0.02vs.0.40±0.05，P=0.017）；AICAR组结合水平高于模型组（0.49±0.09vs.0.23±0.03，P=0.002）；AICAR组p-TSC2蛋白水平高于模型组（1.48±0.07vs.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。

Abstract:

Objective：ToinvestigatetherelationshipbetweenthemechanismofAMP-activated-protein-kinase（AMPK）-mediatedmyocardialmitochondrialautophagyandtuberoussclerosiscomplex-2（TSC2）indiabeticrats.Methods：ThirtyadultmaleSDratswererandomlydividedintocontrolgroup，modelgroup，andmodelgroup+AMPKagonistAICARgroup（AICARgroup）.Thediabeticratmodelwasestablishedbyintraperitonealinjectionofstreptozotocin.RatsintheAICARgroupweresubcutaneouslyinjectedwithAICARfor12weeks.Cardiacfunctionofallratswasmeasuredbyechocardiography，includingleftventricularend-diastolicvolume（LVEDV），leftventricularend-systolicvolume（LVESV），leftventricularejectionfraction（LVEF），heartrate（HR），peakvelocityofearlydiastolicflow（E），peakvelocityoflatediastolicflow（A），andE/A.ThemyocardialtissuewastakentodetectthebindinglevelsofAMPKandTSC2byimmunoprecipitationandtheexpressionlevelsofAMPK，TSC2，p-TSC2，LC3-Ⅱ，LC3-Ⅰ，Beclin-1andP62weredetectedbyWesternblot.Results：ThelevelsofLVEDV，LVEF，EandE/AinAICARgroupwerehigherthanthoseinthemodelgroup，butAwaslowerthanthatinthemodelgroup（P=0.004，P=0.001，P=0.000，P=0.011，P=0.027）.ThelevelsofAMPKandTSC2proteininAICARgroupwerehigherthanthoseinthemodelgroup（0.71±0.06vs.0.36±0.09，P=0.003；0.80±0.02vs.0.40±0.05，P=0.017）.ThebindinglevelofAICARgroupwashigherthanthatinthemodelgroup（0.49±0.09vs.0.23±0.03，P=0.002），andthelevelofp-TSC2proteininAICARgroupwashigherthanthatinthemodelgroup（1.48±0.07vs.0.92±0.07，P=0.029）.ThelevelsofLC3-Ⅱ/LC3-Ⅰ，Beclin-1andP62inAICARgroupwerehigherthanthoseinmodelgroup（P<0.05）.Conclusion：ThemechanismofAMPK-mediatedmyocardialmitochondrialautophagyindiabeticratsmayberelatedtoitsphos－phorylationtoTSC2.