TOPK/STAT3调控necroptosis恢复糖尿病心肌对七氟烷后处理敏感性的作用机制研究

Subjects with diabetes are more vulnerable to ischemic myocardial (IR) injury and less or not sensitive to sevoflurane postconditioning (Sevo-PostC) mediated cardioprotection, and the underlying mechanism is unclear. Our preliminary studies showed that post-ischemic myocardial injury was increased while Sevo-PostC cardioprotection was lost in hearts from diabetes, which was associated with T-lymphokine-activated killer cell-originated protein kinase (TOPK) inactivation and dephosphorylation of STAT3 as well as increased expression of RIP3 (induction of necroptosis). TOPK siRNA cancelled the cardioprotective effect of Sevo-PostC in cultured cardiomyocytes. Further, in cultured cardiomyocytes exposed to high glucose, post-hypoxic cellular injury was increased and the protective effects of Sevo-PostC were lost. All these can be reversed by TOPK overexpression. The above preliminary findings prompted us to postulate that hyperglycemia-induced inactivation in TOPK, leads to the inactivation of STAT3, resulting in induction of necroptosis, and leads to the loss of IPostC cardioprotection in diabetes. We, therefore, aim to examine the role of TOPK/STAT3 in Sevo-PostC cardioprotection against myocardial IR injury and to explore the underlying mechanism in relation to necroptosis in diabetic rodents. We will conduct series experiments using in vivo animal model, isolated heart model and in vitro cell models of myocardial IR injury in normal and diabetic mice with TOPK gene knock out (KO), RIP3 KO (necroptosis deficiency) mice, incorporating the use of TOPK and RIP3 adenoviruses to address mechanisms. This study will provide new insights regarding the molecular mechanism whereby TOPK/STAT3 restores diabetic hearts sensitivity to Sevo-PostC, and facilitate the development of effective therapy to combat ischemic heart diseases.

糖尿病（DM）心脏缺血再灌注（IR）损伤易损性增加，对七氟烷后处理（Sevo-PostC）不敏感，机制不明。申请者发现DM鼠心肌TOPK失活及后处理关键蛋白STAT3失活、necroptosis增加，心肌IR损伤加重且对Sevo-PostC不敏感。TOPK siRNA取消了Sevo-PostC对离体心肌细胞IR损伤的保护。高糖使心肌细胞IR损伤加重，且Sevo-PostC保护作用消失，TOPK过表达可激活STAT3，减少necroptosis且恢复Sevo-PostC对高糖心肌细胞的保护。我们推测DM心肌TOPK失活阻断STAT3，增加necroptosis，导致Sevo-PostC失效。我们将用TOPK和RIP3基因敲除鼠，从整体、离体器官及细胞水平研究高糖状态下TOPK/STAT3及其对necroptosis的调控对DM心肌Sevo-PostC的影响，为DM心肌缺血防治提供新靶点。

糖尿病（DM）心脏缺血再灌注（IR）损伤易损性增加，对七氟烷后处理（Sevo-PostC）不敏感，机制不明。申请者前期研究发现DM鼠心肌TOPK失活及后处理关键蛋白STAT3失活、necroptosis增加，心肌IR损伤加重且对Sevo-PostC不敏感。TOPK siRNA取消了Sevo-PostC对离体心肌细胞IR损伤的保护。高糖使心肌细胞IR损伤加重，且Sevo-PostC保护作用消失，TOPK过表达可激活STAT3，减少necroptosis且恢复Sevo-PostC对高糖心肌细胞的保护。我们推测DM心肌TOPK失活阻断STAT3，增加necroptosis，导致Sevo-PostC失效。本课题分别在糖尿病心肌IR模型（在体研究）及H9C2心肌细胞模拟的高糖与缺氧复氧模型（离体研究）进行了相关机制探讨。本研究中我们发现，非糖尿病状态下Sevo-PostC通过激活TOPK减轻氧化应激，从而减轻心肌IR损伤。但是，糖尿病心肌对Sevo-PostC失敏感，其分子机制可能与TOPK失活导致氧化应激损伤加重密切相关。并且，糖尿病小鼠TOPK过表达显著改善糖尿病小鼠心功能，减轻心肌IR损伤，伴随着心肌STAT3激活、necroptosis减少及氧化应激减轻。同样，在高糖状态下给予H9C2心肌细胞TOPK过表达激活缺氧复氧后心肌STAT3，抑制necroptosis，减轻细胞损伤。但是，TOPK过表达对糖尿病心肌保护作用被STAT3抑制剂消除，并加重necroptosis。因此，我们的研究结果表明：糖尿病心肌TOPK失活导致的氧化应激加重可能是Sevo-PostC对糖尿病心肌无效的重要机制。TOPK过表达减轻糖尿病心肌IR损伤，其分子机制与TOPK激活STAT3减轻necroptosis密切相关。本研究结果首次从TOPK介导的necroptosis角度阐明了TOPK/STAT3激活通过调控necroptosis对抗糖尿病心肌IR损伤的作用及分子机制，该研究的完成为治疗和逆转糖尿病性缺血性心脏病提供早期预防和治疗策略，以及为临床开发糖尿病心肌IR损伤保护药物或措施提供潜在的作用靶点，因而具有明确的理论与实用价值。