STAT3在抵抗心肌缺血再灌注损伤中调控钙，ROS和ATP的新机制

It is an important issue to reduce cardiac ischemia reperfusion (I/R) injury effectively in therapy of ischemia heart disease. However, the cardioprotection of therapeutic targets found right now is relatively simple. STAT3 is an important protein in signaling of cardioprotection, but the mechanism of cardioprotection-induced by STAT3 is not fully understood. Our previous work found that increased ROS during early reperfusion are key signaling inducing endogenous cardioprotection. ROS can activate STAT3 and lead the phosphorylated STAT3 into mitochondria and nucleus to reduce calcium overload during I/R, but the detail mechanism remained unclear. Besides, phosphorylated STAT3 is involved in regulation of production of ROS and ATP, but whether and how it regulates the homeostasis of ROS and ATP in cardioprotection is unveiled. Thus, we propose the new mechanism that STAT3 can regulate the homeostasis of calcium, ROS and ATP against cardiac I/R injury. With the model of hydrogen peroxide postconditioning (H2O2PoC), we proposed the main aims that are 1) defining phosphorylated STAT3 is involved in the cardioprotection induced by H2O2PoC; 2) clarifying the mechanism of phosphorylated STAT3 reducing the cytosolic calcium overload and mitochondrial calcium overload in H2O2PoC; 3) investigating the phenomenon and mechanism of phosphorylated STAT3 regulating the production and species of ROS in H2O2PoC; 4) explaining the the phenomenon and mechanism of phosphorylated STAT3 regulating the production of ATP in H2O2PoC.

减轻缺血再灌注（I/R）损伤是治疗缺血性心脏病的关键问题，但目前发现治疗靶点产生的保护效应较单一。STAT3是心肌保护中重要信号通路蛋白，但对其产生保护效应的机制尚未完全阐明。前期研究发现再灌注初期适度增加的活性氧（ROS）可以激活STAT3磷酸化进入线粒体和细胞核减轻I/R中胞浆钙超载，但具体机制尚不清楚。磷酸化STAT3还参与调控ROS和三磷酸腺苷（ATP）的生成，但其在心肌保护中如调控二者的稳态尚不清楚。因此，我们提出STAT3在抵抗心肌I/R损伤中对Ca2+，ROS和ATP稳态调控的新机制，运用过氧化氢后处理（H2O2PoC）模型，旨在1）明确STAT3参与H2O2PoC的心肌保护作用；2）阐明STAT3在H2O2PoC中减轻胞浆钙和线粒体钙超载的作用与机制；3）探讨STAT3在H2O2PoC中调控ROS的含量和种类；4）阐明STAT3在H2O2PoC中调控ATP含量的作用与机制。

STAT3是心肌保护中重要信号通路蛋白，但对其产生保护效应的机制尚未完全阐明。前期研究发现再灌注初期适度增加的活性氧（ROS）可以激活STAT3磷酸化进入线粒体和细胞核减轻缺血/再灌注（I/R）中胞浆钙超载，但具体机制尚不清楚。申请者的研究结果发现中等浓度的过氧化氢后处理（H2O2PoC）可以有效的模拟再灌注初期适度增加的ROS的现象，减轻I/R后的线粒体钙和胞浆钙超载，改善线粒体膜电位，保护线粒体的呼吸链复合物I和II的活性，保护ATP的生成。在成年大鼠的心肌细胞里下调STAT3和促进线粒体钙泵（MCU）开放可以取消中等浓度H2O2PoC的心肌保护作用以及减轻线粒体钙和胞浆钙超载的现象。过表达STAT3可以取消MCU开放所带来的心肌细胞损伤以及线粒体钙和胞浆钙超载的现象。免疫共沉淀和免疫荧光共定位的实验证明了H2O2PoC促进再灌注初期和末期STAT3与MCU的相互作用。在成年大鼠心肌细胞过表达N端缺失的MCU不能与STAT3蛋白产生相互作用。以上结果说明，STAT3通过与MCU的N端相互作用抑制MCU的开放从而减轻线粒体与胞浆钙超载。除此以外，我们的研究还发现H2O2PoC会增加I/R后ROS中H2O2的量，减少STAT3的磷酸化可以减少H2O2PoC促进H2O2的生成。筛选了相关氧化还原调控的基因的表达，发现H2O2PoC可以使得促进O2·-转变成H2O2生成的锰超氧歧化酶（MnSOD）、金属硫蛋白1（MT1）和金属硫蛋白2（MT2）mRNA表达升高而减少STAT3的磷酸化使得mRNA其表达下降。以上结果说明STAT3是心肌I/R中ROS里H2O2含量的调控者，深入的机制还需后续探讨。目前这些研究结果发表后已有一次药物化学相关期刊的引用，为药物开发设计提供了靶点机制的理论依据。