HSF1/AMPK信号通路在铁死亡参与糖尿病心肌病发病的机制研究

Role of the HSF1/AMPK signaling pathway in the pathogenesis of ferroptosis-involving diabetic cardiomyopathy

目的:探讨热休克因子1(heat shock factor 1,HSF1)/5′-单磷酸腺苷活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)信号通路调控铁死亡对糖尿病心肌病(diabetic cardiomyopathy,DCM)发病的影响。方法:本研究为实验研究,采用空白对照与多组实验对照。H9c2细胞随机分为4组:低葡萄糖组(control,Con)、高葡萄糖组(high glucose,HG)、HG+HSF1组、HG+HSF1+化合物C(compound C,CC)组。分别对细胞进行罗丹明胶质蛋白染色、细胞线粒体(reactive oxygen species,ROS)检测和细胞脂质ROS检测,并通过Western blot分析AMPK信号表达。雄性C57/BL6小鼠随机分为4组:NC组、NC+HSF1组、DM组和DM+HSF1组,每组12只。通过超声心动图评估了小鼠心血管功能参数。结果:与Con组相比,HG组HSF1、pAMPK/AMPK水平明显下调(P=0.005、0.002),和相对细胞表面积、线粒体Fe2+水平、线粒体ROS水平、细胞脂质ROS水平明显增加(P=0.001、0.003、0.006、0.002)。与HG组相比,HG+HSF1组明显逆转了这些变化(P=0.001、0.001、0.002、0.006、0.007、0.003),但加入CC时HSF1的逆转作用明显减弱(P<0.05)。与NC组相比,DM组EF%、FS%、E/A、E′/A′和心脏组织中HSF1、pAMPK/AMPK表达明显降低(均P<0.01),和心脏组织中Fe2+、ROS、丙二醛(Malondialdehyde,MDA)水平和4-羟基壬烯酸(4-Hydroxynonenal,4-HNE)蛋白水平明显增加(P=0.004、0.003、0.001、0.004),DM+HSF1组明显逆转了这些变化。结论:HSF1在DCM病理过程中发挥心脏保护作用,其抗铁死亡作用可能与AMPK依赖性的脂质代谢和线粒体稳态调节有关。

Objective:To investigate the effect of ferroptosis regulated by the heat shock factor 1(HSF1)/adenosine 5’-monophosphate-activated protein kinase(AMPK)signaling pathway on the pathogenesis of diabetic cardiomyopathy(DCM).Methods:This was an experimental study with a blank control and multiple experimental controls.H9c2 cells were randomly divided into 4 groups:low glucose group(control,Con),high glucose(HG)group,HG+HSF1 group,and HG+HSF1+compound C(CC)group.The cells were stained by rhodamine colloid protein,and tested for mitochondrial reactive oxygen species(ROS)and cellular lipid ROS.The AMPK signal expression was analyzed by Western blot.Male C57/BL6 mice were randomly divided into four groups:negative control(NC)group,NC+HSF1 group,DM group,and DM+HSF1 group,with 12 mice in each group.The cardiovascular function parameters of mice were evaluated by echocardiography.Results:Compared with the Con group,the levels of HSF1 and pAMPK/AMPK in the HG group decreased significantly(P=0.005,0.002),and the relative cell surface area,mitochondrial Fe2+level,mitochondrial ROS level,and cellular lipid ROS level increased significantly(P=0.001,0.003,0.006,0.002).Compared with the HG group,the HG+HSF1 group significantly reversed these changes(P=0.001,0.001,0.002,0.006,0.007,0.003),but the reversal effect of HSF1 was obviously weakened when CC was added(P<0.05).Compared with the NC group,the ejection fraction,fractional shortening,mitral ratio of peak early to late diastolic filling velocity(E/A),and E′/A′as well as the expression levels of HSF1 and pAMPK/AMPK in heart tissues in the DM group were significantly decreased(P<0.01),and the levels of Fe2+,ROS,malondialdehyde,and 4-hydroxynonenal protein in heart tissues were significantly increased(P=0.004,0.003,0.001,0.004);the DM+HSF1 group significantly reversed these changes.Conclusion:HSF1 plays a cardioprotective role in the pathological process of DCM,and its anti-ferroptosis effect may be related to AMPK-dependent lipid metabolism and mitochondrial homeostasis regulation.