沉默信息调节因子2相关酶1/3在休克线粒体损伤中的作用

Persistent or refractory hypotension following vascular hyporeactivity is one of the major causes of mortality during severe shock in clinic. As such the occurrence and development mechanisms need to be further elucidated. As has been shown, mitochondrial injury and dysfunction in arteriolar smooth muscle cells play important role in low vasoreactivity. Resveratrol, a SIRT1(Silent information regulator 2 homolog 1) activitor, ameliorates shock-induced mitochondrial injury in ASMCs (arteriolar smooth muscle cells) and contributes to restore the vasoreactivity. Our hypothesis is that modulation of mitochondrial ultrastructure and energy metabolism by SIRT1/3 underlies hyporeactivity of arteriolar smooth muscle in severe shock. In this study, we will investigate the role of SIRT1/3 mediating mitochondrial injury in the genesis of low vasoreactivity during severe shock in hemorrhagic shock rat model as well as in vitro cell model. This paper will focus on regulation of mitochondrial injury by SIRT1/3 following hemorrhagic shock in the following aspects: 1.To determine the changes of SIRT1/3 expression and activity in vascular smooth muscle during severe shock. 2. To demonstrate the mechanism of SIRT1/3 mediating mitochondrial injury in shock. 3. To explore the potential target of SIRT1/3 to effectively preserve ASMC mitochondrial integrity and to determine whether they have a significant therapeutic effect in severe shock. This study has very important significance with regard to further elucidation of mechanisms of the refractory hypotension during severe shock and highlighting the need to develop new treatment targets.

顽固性低血压是重症休克患者死亡重要原因，其发生、发展机制亟待进一步阐明。我们前期研究表明线粒体损伤介导休克血管低反应发生，沉默信息调节因子2 同源体1（Silent information regulator 2 homolog 1，SIRT1）的激动剂resveratrol可保护休克线粒体损伤和有利于恢复血管反应性。我们提出SIRT1/3 影响线粒体介导重症休克血管低反应作用的机制，即：SIRT1/3调控线粒体结构或能量代谢稳态。本研究拟采用重症休克动物模型，并结合体外细胞缺氧复氧模型，运用现代分子生物研究方法，完成以下研究：（1）查明重症休克血管平滑肌SIRT1/3的改变；（2）阐明SIRT1/3介导休克线粒体损伤的分子机制；（3）探讨SIRT1/3对抗线粒体损伤和恢复血管反应性的作用。这对于重症休克顽固性低血压发生机制的深入研究以及推动新的有效靶点药药物开发，具有十分重要的意义。

本项研究的目的是查明沉默信息调节因子沉默信息调节因子2 同源体（Silent information regulator 2 homolog，SIRT）1/3 在重症休克血管平滑肌线粒体损害发生中的作用及机制；在此基础上探索以SIRT1/3 为靶点保护休克线粒体，用于治疗休克低血管反应和顽固性低血压的可能性，为临床治疗提供新思路。本研究采用重症休克动物模型，并结合体外细胞缺氧复氧模型，运用近年发展的线粒体原位研究技术和现代分子生物研究方法，本研究联合采用在体复制大鼠重症失血性休克模型和体外小动脉平滑肌细胞缺氧复氧模型，探讨SIRT1调控血管平滑肌细胞线粒体通透性转变孔的信号通路及其在重症失血性休克线粒体损伤中的作用和机制。.结果查明：.1. 重症休克过程中SIRT1和SIRT3的去乙酰化酶活性的变化在不同时间点之间存在显著性差异。SIRT1和SIRT3的活性在休克60 min开始出现显著下降。回输血后120 min，与假手术组相比，SIRT1和SIRT3的蛋白表达分别下降了42.25±6.06%和40.67±4.08%。细胞免疫荧光实验也证实了在回输血后120 min SIRT1和SIRT3的蛋白表达下调，CypD乙酰化水平增加。.2. 与对照组相比，缺氧复氧时CypD的乙酰化水平显著增加；过表达SIRT1可降低CypD的乙酰化水平，而过表达SIRT1的同时下调SIRT3则可逆转CypD乙酰化水平的下降，过表达SIRT1的同时下调PGC1α的表达可部分抑制SIRT3的活性。SIRT1增加了缺氧复氧处理后ATP水平，下调SIRT3和PGC1α的表达抑制SIRT1的作用。.3. SIRT1激动剂和虎杖苷可显著抑制重症休克血管平滑肌SIRT1和SIRT3活性的下降。SIRT1激动剂和虎杖苷治疗后，血管平滑肌细胞的线粒体损伤、ATP的含量、线粒体通透性转变孔的开放和线粒体膜电位的下降，与休克组相比显著得到改善。动物平均动脉压较休克组显著改善。动物存活时间明显延长。. 总之，本文通过在体和离体实验证明，重症休克时血管平滑肌SIRT1/3活性下降引起CypD乙酰化水平增加，促进线粒体通透性转变孔开放，进而导致线粒体和细胞损伤；激活SIRT1可抑制重症休克时血管平滑肌细胞线粒体通透性转变孔的开放，保护线粒体的功能，改善血管反应性。