Nox2调控Pyroptosis及Ferroptosis减轻糖尿病心肌缺血再灌注损伤的机制研究

Subjects with diabetes are susceptible to myocardial ischemia reperfusion injury (IRI). NADPH subunit Nox2-mediated increase of oxidative stress and inflammation has been suggested to play critical roles in this process. Oxidative stress-induced ferroptosis and inflammation-induced pyroptosis contribute are key players in many diseases including heart diseases, however, their roles in Nox2-mediated increased of myocardial IRI in diabetic hearts is unknown. We found that in cardiomyocytes incubated with high glucose, Nox2 was increased, which were associated with enhanced pyroptosis key protein NLRP3 and reduced ferroptosis marker GPX4, and decreased AMPK and increased post-hypoxic cell injury. Gene knockdown of Nox2 reduced NLRP3, increased GPX4, and decreased post-hypoxic cell injury in cardiomyocytes incubated with high glucose. Further, activation of AMPK reduced Nox2 and NLRP3, increased GPX4, and attenuated post-hypoxic cell injury in cardiomyocytes incubated with high glucose. The above preliminary findings promopted us to postulate that hyperglycemia-induced reduction of AMPK and enhancement of Nox2, may lead to the induction of pyroptosis and ferroptosis, resulting in increase of myocardial IRI in diabetes. We, therefore, aim to examine the role of AMPK and Nox2 in myocardial IRI in diabetes and their interplay with pyroptosis and ferroptosis. We will conduct series experiments using in vivo and in vitro cell models of myocardial IRI in normal and diabetic mice with Nox2 gene knockout, incorporating the use of Nox2 adenoviruses and AMPK or pyroptosis or ferroptosis inducers or inhibitors to address the mechanisms. This study will provide new insights regarding the molecular mechanism whereby AMPK/Nox2 attenuates diabetic myocardial IRI and facilitate the development of effective therapy to combat ischemic heart diseases.

糖尿病（DM）心肌对缺血再灌注损伤（IRI）耐受性降低，与Nox2诱导的氧化应激及炎症反应相关。氧化应激可诱导细胞铁死亡（ferroptosis）而炎症反应可诱导细胞焦亡（pyroptosis），我们前期发现心肌细胞高糖培养后Nox2升高，细胞焦亡关键蛋白 NLRP3上调，铁死亡标志物GPX4活性降低，伴随AMPK的降低及心肌细胞缺氧损伤加重。敲除Nox2降低NLRP3，上调GPX4并减轻高糖心肌细胞损伤。激活AMPK降低Nox2可减少NLRP3并上调GPX4，减轻高糖培养心肌细胞缺氧复氧损伤。我们推测DM心肌中AMPK下调，升高Nox2，进而增加NLRP3并下调GPX4活性，同时诱导细胞焦亡和铁死亡，导致DM心肌IRI。我们拟建立Nox2敲除鼠、心肌IRI模型，结合Nox2腺病毒转染技术，探讨AMPK/Nox2调控细胞焦亡和铁死亡在DM心肌IRI中的作用，为DM患心肌IRI防治提供依据。

缺血性心肌病仍是人类健康主要杀手，尤其在糖尿病病人中具有较高的发病率及致死率，而糖尿病（DM）心肌对缺血再灌注损伤（IRI）耐受性降低，与Nox2诱导的氧化应激及炎症反应相关。氧化应激可诱导细胞铁死亡（ferroptosis）而炎症反应可诱导细胞焦亡（pyroptosis）。我们利用链脲霉素导致的一型糖尿病大鼠，发现糖尿病状态下心肌缺血再灌注损伤更加严重，表现为：心肌梗死面积更大， SOD释放明显减少，MDA释放明显增加，蛋白Nox2表达明显增加，细胞焦亡关键蛋白 NLRP3上调，铁死亡标志物GPX4活性降低，同时蛋白AMPK表达显著减少。 心肌缺血再灌注前给与Nox2的抑制剂Vas2870及抗氧化剂N-乙酰-L-半胱氨酸(N-Acetyl-L-cysteine，NAC)， 能减少糖尿病心肌缺血再灌注导致的梗死面积增加， Nox2表达明显减少，同时蛋白AMPK表达显著增加， SOD释放明显增加， MDA释放减少， 减轻细胞焦亡及铁死亡， 从而减少糖尿病导致的心肌缺血再灌注损伤, 表明氧化应激水平在糖尿病心肌缺血再灌注导致的损伤中起重要作用。同时我们在H9C2细胞实验中发现，心肌细胞经高糖培养然后缺氧复氧损伤后蛋白Nox2表达升高，细胞焦亡关键蛋白 NLRP3上调，铁死亡标志物GPX4活性降低，伴随AMPK的降低及心肌细胞缺氧损伤加重。给与AMPK激动剂AICAR，可降低由高糖后缺氧复氧导致的Nox2表达增加， 同时可减少NLRP3并上调GPX4，减轻高糖培养心肌细胞缺氧复氧损伤。同时，我们利用siRNA敲除Nox2后， 能减少缺氧复氧导致的心肌LDH的释放，同时降低NLRP3，上调GPX4并减轻高糖心肌细胞损伤，但AMPK的表达未受影响，由此说明AMPK是可以通过调节Nox2发挥其在糖尿病心肌缺血再灌注损伤中的作用。根据以上实验结果，我们推测DM心肌中AMPK下调，升高Nox2，进而增加NLRP3并下调GPX4活性，进而诱导细胞焦亡和铁死亡，导致DM心肌IRI。本课题主要探讨了AMPK/Nox2调控细胞焦亡和铁死亡在DM心肌IRI中的作用，发现了AMPK能通过减少糖尿病状态导致的Nox2增加，从而减少细胞焦亡和铁死亡，进而减少糖尿病心肌IRI, 为DM患心肌IRI防治提供依据。