【作者】 袁勇；

【导师】 刘季春；

【作者基本信息】 南昌大学 ， 临床医学（专业学位）， 2025， 博士

【摘要】 背景:急性心肌梗死(acute myocardial infarction,AMI)作为严重致残和危及生命的心血管疾病,是造成全球人员死亡的主要元凶。早期恢复血运是降低AMI患者猝死率和改善其预后的首选治疗策略,但再灌注会进一步诱发心肌损伤和功能障碍,谓之心肌缺血再灌注损伤(Myocardial ischemia reperfusion injury,MIRI)。MIRI的病理生理过程错综复杂,涉及多方因素,包括氧化应激、凋亡、炎症、自噬、铁死亡等在内的多种生物过程。因此,防治MIRI成为目前亟待解决的重要命题。姜黄中提取的姜黄素(Curcumin,Cur)具有强大的抗凋亡、抗氧化应激、抗自噬及抗铁死亡等药理学作用,尤其在心脑血管方面。然而,Cur在MIRI中确切的作用及机制尚未明确。目的:本文旨在通过采用体外缺/复氧(Anoxia/reoxygenation,A/R)和体内MIRI模型,分别从细胞和动物层面探索Cur在MIRI中的药理作用及机制。方法:采用H9c2心肌样细胞构建A/R模型和缝扎大鼠冠状动脉左前降支(left anterior descending coronary artery,LAD)构建MIRI模型,探索Cur在MIRI中的保护作用机制。(1)通过检测细胞活力和LDH活性评估Cur对A/R的量效关系,并筛选Cur最佳保护浓度;(2)采用流式细胞术、免疫荧光、Western blot、透射电镜及生化检测评估Cur对A/R损伤中氧化应激、自噬、铁死亡、凋亡及线粒体功能的影响;(3)通过腺病毒p AD/HES1-sh RNA沉默HES1和Compound C抑制AMPK活性,明确HES1及AMPK对Cur抑制A/R损伤中氧化应激、自噬、铁死亡、凋亡及线粒体功能的重要作用;(4)通过免疫共沉淀技术验证HES1与AMPK是否存在相互作用;(5)进一步通过心脏超声、大鼠血清和心肌组织生化检测评估大鼠心功能损伤情况;(6)采用伊文思蓝/TTC双染色观察心肌梗死范围;(7)TUNEL染色测定心肌细胞阳性凋亡数量;(8)HE染色观察心肌形态结构;(9)DHE染色测定心肌组织ROS水平;(10)Western blot检测心肌组织蛋白和透射电镜评估心肌组织线粒体损伤情况等功能实验进一步验证Cur在体内是否通过调控HES1/AMPK轴影响MIRI中自噬、铁死亡、凋亡及线粒体功能。结果:1.在A/R损伤模拟的体外MIRI模型中,A/R损伤时涉及多种调节性细胞死亡(自噬、铁死亡、凋亡),且抑制自噬、铁死亡和凋亡是减轻A/R损伤的有效手段。暴露A/R后,细胞活力降低,LDH水平升高;心肌细胞内SOD活性降低、ROS和溶酶体的水平增加、自噬蛋白P62的表达降低及LC3II/LC3I的比值升高;结果也显示细胞内铁离子、MDA和LIPROS的含量堆积,且PTGS2表达水平上调而GPX4下调;此外Caspase-3活性升高,细胞凋亡率增加及BCL2/BAX比值下降;与此同时,A/R处理后导致线粒体功能障碍,表现为线粒体形态结构异常、m PTP过度开放、MMP降低、HES1、UQCRC2、NDUFB8表达和p-AMPK/AMPK的比值下调且ATP水平降低。Cur预处理通过抑制凋亡、抑制自噬、抑制铁死亡、改善能量代谢失衡和线粒体损伤来减轻A/R触发的心肌损伤,而p AD/HES1-sh RNA或Compound C的干预则显著削弱了Cur的上述保护效应。另外,Cur可增强HES1与AMPK复合物在细胞中形成。2.在SD大鼠构建的体内MIRI模型中,I/R处理后,超声心动图提示LVEF和LVFS值降低,血清学指标CK-MB和LDH升高,心梗范围增大;TUNEL阳性细胞数量增多及BCL2/BAX值降低;心肌形态结构排列紊乱,且组织中SOD活性、P62蛋白的表达水平降低及LC3II/LC3I的比值升高;心肌组织中的铁、MDA和ROS含量的堆积,且PTGS2表达水平上调而GPX4下调;此外,在线粒体形态和功能方面,我们发现与Sham组对比,MIRI组心肌中UQCRC2、NDUFB8的表达显著下降,p-AMPK/AMPK的比值明显降低且ATP水平降低;同时,线粒体结构出现明显损伤,表现为形态扭曲,嵴减少或膜断裂等。Cur预处理能有效逆转MIRI导致的这些指标改变发挥心脏保护,而p AD/HES1-sh RNA或Compound C的干预则显著抑制了Cur通过抗凋亡、抑制自噬、抗铁死亡、改善能量代谢失衡和线粒体损伤来减轻MIRI的保护效应。结论:Cur通过调控HES1/AMPK轴减少心肌梗死范围、抑制凋亡、拮抗铁死亡、抑制自噬、改善能量代谢失衡和线粒体功能障碍来减轻MIRI。更多还原

【Abstract】 Background:Acute myocardial infarction(AMI)is the foremost cause of mortality on a global scale,representing the most severe disabling and life-threatening cardiovascular disease.Early restoration of blood flow is the preferred therapeutic strategy to reduce the rate of sudden death and improve the prognosis of AMI patients,but reperfusion induces further myocardial injury and dysfunction,termed myocardial ischemia-reperfusion injury(MIRI).The pathophysiological process of MIRI is complex and involves multiple factors,including oxidative stress,apoptosis,inflammation,autophagy,ferroptosis,and other biological processes.Consequently,the prevention and treatment of MIRI have become an important proposition that needs to be addressed urgently.Curcumin(Cur),a bioactive compound from turmeric,exhibits anti-apoptotic,antioxidant,anti-autophagic,and anti-ferroptosis properties,particularly in cardiovascular and cerebrovascular contexts.However,the precise role and underlying mechanisms of Cur in MIRI remain to be fully elucidated.Objective:This study aims to investigate the pharmacological effects and mechanisms of Cur in MIRI using in vitro anoxia/reoxygenation(A/R)models and in vivo MIRI models at cellular and animal levels.Methods:H9c2 cardiomyocyte-like cells were used to construct an A/R model,and sutured rat LAD was used to build a MIRI model to explore the mechanism of Cur’s protective effect in MIRI.(1)The quantitative-effect relationship of Cur on A/R was assessed by detecting cell viability and LDH activity,and the optimal protective concentration of Cur was screened;(2)the effects of Cur on oxidative stress,autophagy,ferroptosis,apoptosis,and mitochondrial function in A/R injury were evaluated by flow cytometry,immunofluorescence,Western blot,transmission electron microscopy,and biochemical assays;(3)Silencing of HES1 by adenoviral p AD/HES1-sh RNA and inhibition of AMPK activity by Compound C clarified the important roles of HES1 and AMPK on oxidative stress,autophagy,ferroptosis,apoptosis,and mitochondrial function in Cur inhibition of A/R injury;(4)Verified whether HES1 interacts with AMPK by immunoprecipitation technique;(5)Further assessment of cardiac function injury in rats by cardiac ultrasound,serum and myocardial tissue biochemical assays;(6)Observation of the extent of myocardial infarction by Evans blue/TTC double staining;(7)Determination of the number of positive apoptosis in cardiomyocytes by TUNEL staining;(8)Observation of myocardial morphology and structure by HE staining;(9)Determination of the ROS level in myocardial tissue by DHE staining;and(10)Functional experiments such as Western blot to detect myocardial tissue proteins and transmission electron microscopy to assess mitochondrial damage in myocardial tissues further verified whether Cur affects autophagy,ferroptosis,apoptosis,and mitochondrial function in MIRI in vivo by regulating the HES1/AMPK axis.Results:1.In the in vitro MIRI model simulated by A/R injury,multiple roles of regulated cell death(autophagy,ferroptosis,apoptosis)were found to be involved,and the inhibition of autophagy,ferroptosis,and apoptosis was found to be an effective means of alleviating A/R injury.Following A/R exposure,a decline in cell viability and an increase in LDH levels were observed.Additionally,a decrease in SOD activity,an elevation in ROS and lysosome levels,a reduction in autophagy protein P62 expression,and an elevation in the ratio of LC3II/LC3I in cardiomyocytes were detected.Furthermore,an accumulation of intracellular iron ions,MDA,and LIPROS content,an up-regulation of PTGS2,and a down-regulation of GPX4 expression were noted.The present study investigates the effects of Cur on the regulation of apoptosis in H9c2 cells.The results demonstrate that A/R treatment led to an elevation in Caspase-3 activity,an increase in the apoptosis rate,and a decrease in the BCL2/BAX ratio.In addition,A/R treatment resulted in mitochondrial dysfunction,characterized by mitochondrial morphological structural abnormalities,over-opening of m PTP,reduction of MMP,down-regulation of the expression of HES1,UQCRC2,NDUFB8,and the ratio of p-AMPK/AMPK,and reduction of the ATP level.Cur Preconditioning attenuated A/R-triggered myocardial injury by inhibiting apoptosis,suppressing autophagy,inhibiting ferroptosis,and ameliorating energy metabolism imbalance and mitochondrial damage,whereas intervention with p AD/HES1-sh RNA or Compound C significantly attenuated the above protective effects of Cur.Furthermore,Cur enhanced HES1 and AMPK complex formation in cells.2.In the in vivo MIRI model established in SD rats,I/R treatment induced significant cardiac dysfunction,as evidenced by reduced LVEF and LVFS values,elevated serum CK-MB and LDH levels,and enlarged infarct size.Pathological analysis revealed disrupted myocardial architecture,increased TUNEL-positive apoptotic cells,and decreased BCL2/BAX ratio.I/R injury suppressed SOD activity and P62 expression,elevated the LC3II/LC3I ratio,and triggered iron overload,MDA accumulation,and ROS overproduction.Concurrently,PTGS2 expression was upregulated,while GPX4 was downregulated.Mitochondrial dysfunction was prominent in the MIRI group,characterized by ultrastructural damage(e.g.,distorted morphology,cristae loss,membrane rupture),reduced expression of UQCRC2 and NDUFB8,diminished p-AMPK/AMPK ratio,and depleted ATP levels.Cur pretreatment effectively counteracted these alterations,demonstrating cardioprotection via attenuating apoptosis,suppressing autophagy,inhibiting ferroptosis,restoring energy metabolism,and preserving mitochondrial integrity.Notably,the therapeutic benefits of Cur were significantly abrogated by either p AD/HES1-sh RNA or Compound C.Conclusions:Cur attenuates MIRI by modulating the HES1/AMPK axis to reduce the extent of myocardial infarction,inhibiting apoptosis,antagonizing ferroptosis,inhibiting autophagy,and ameliorating energy metabolism imbalances and mitochondrial dysfunction.更多还原