MiR-34a调控SIRT1-p66shc通路在肠缺血再灌注多器官损伤中的作用及防治靶点研究

Intestinal ischemia/reperfusion (II/R) is the initiating factor of multiple organ dysfunction syndrome (MODS) with complex pathogenesis. Our previous studies suggested that the p66shc signaling pathway is involved in the pathogenesis of II/R and subsequent liver and lung damage, while p66shc suppression is an effective means to alleviate such damage. Silent information regulator type 1 (SIRT1) plays a crucial role in regulating oxidative stress and apoptosis, and we further found that: 1) Activation of SIRT1 inhibited p66shc level significantly and reduced the multiple organ injury induced by II/R; 2) MiR-34a up-regulated in the intestine, liver and lung after II/R, and were negatively correlated with SIRT1 expression. 3) Bioinformatics predicted that SIRT1 is a target gene of miR-34a. Accordingly, we firstly point out that regulating SIRT1-p66shc signaling pathway by miR-34a inhibition exert as an important control strategy against multiple organ injury induced by II/R. Here, we plan to use an in vitro model of hypoxia/reoxygenation in intestinal epithelial cells and an in vivo ischemia/reperfusion model in SIRT1 gene knockout mice to investigate the mechanism of SIRT1-p66shc signaling pathway on II/R injury, and a variety of advanced molecular biology methods will be used; Moreover, we also focus on the targeting effect of miR-34a suppression which regulates SIRT1-p66shc pathway and protects against II/R induced multiple organ injury.

肠缺血再灌注（II/R）是多器官功能障碍综合征（MODS）的始动因素，发病机制复杂。我们的研究显示，抑制衔接蛋白p66shc是减轻II/R肝肺损伤的有效手段；沉默信息调节因子1（SIRT1）是调节氧化应激及凋亡的重要因子。进一步研究发现：1）活化SIRT1可明显抑制p66shc水平，减轻II/R多器官损伤；2）MiR-34a在II/R肠、肝等组织中上调且与SIRT1负相关；3）生物信息学预测SIRT1是miR-34a的靶基因。据此，首次提出下调miR-34a调控SIRT1-p66shc信号通路作为II/R多器官损伤防治策略的假说。本研究利用SIRT1基因敲除鼠II/R多器官损伤模型和肠上皮细胞缺氧复氧模型，采用先进的分子生物学方法，探讨SIRT1-p66shc信号通路在II/R多器官损伤中的机制；研究下调miR-34a靶向SIRT1-p66shc通路在II/R多器官损伤中的防治策略。

肠缺血再灌注（I/R）是临床常见的病理生理过程，是引发全身炎症反应综合征（ SIRS）或多器官功能障碍综合征（MODS）的重要始动因素。肠I/R损伤发病机制复杂，近年来研究认为肠上皮细胞内活性氧（ROS）的过度产生及大量上皮细胞凋亡的发生是肠I/R损伤过程中重要的发病机制。我们前期研究发现，沉默信息调节因子1（SIRT1）是细胞内调节氧化应激及凋亡的重要因子，其介导的抗氧化信号通路在肠I/R后明显受损。本研究利用C57BL/6小鼠夹闭肠系膜上动脉法构建体内肠I/R损伤模型，体外采用人源Caco-2细胞或大鼠IEC-6细胞缺氧复氧（H/R）模型模拟体内I/R损伤，采用多种分子生物学实验手段，探讨SIRT1信号通路在肠I/R多器官损伤中的作用，并阐明了靶向SIRT1的微小RNA（miRNA）在调节肠I/R后ROS蓄积和细胞凋亡中的作用及机制。结果发现：SIRT1在缺血小肠的再灌注早期表达逐渐降低，而后表达逐渐恢复，再灌注早期SIRT1的表达抑制可引起I/R后小肠组织ROS含量显著升高和大规模细胞凋亡。随后通过生物信息学预测结合实时定量PCR，我们证实了在可能靶向SIRT1的41种miRNAs中，只有miR-34a-5p和miR-495-3p在肠I/R后显著上调。miR-34a-5p可直接靶向结合作用SIRT1并在肠上皮细胞中负向调控SIRT1表达。抑制miR-34a-5p而非miR-495-3p可在体内外减轻肠I/R损伤，其保护作用可能与下调p66Shc的表达、升高抗氧化酶MnSOD的表达、抑制caspase-3的激活有关。此外，下调miR-34a-5p还可减轻小肠组织病理学损伤，减少肠上皮细胞ROS生成和细胞凋亡，减轻远隔肺肝损伤，减轻全身炎症反应，改善动物生存率。有趣地是，抑制miR-34a-5p对肠I/R的保护作用与其对SIRT1的调控紧密相关。该研究首次证实miRNA在肠I/R损伤过程中对肠上皮细胞氧化应激和凋亡的调控作用，抑制miR-34a-5p通过上调SIRT1表达减轻肠上皮ROS的过度蓄积与细胞凋亡，进而缓解肠I/R多器官损伤。本研究为干预肠I/R损伤提供了新的作用靶点，同时也为开发核酸类药物以预防肠I/R损伤提供了重要证据。另外，我们同时开展多项工作围绕SIRT1或p66Shc核心通路鉴定出多个上下游因子，有助于深刻理解肠I/R发病分子调控网络。