基于PLC-γ1/ROS馈路的外源性ANP抗创伤失血性休克诱发肠道屏障损伤研究

Intestinal barrier injury plays a pivotal role in the development of multiple organ dysfunction syndrome induced by traumatic hemorrhagic shock. Atrial natriuretic peptide (ANP), which possesses a short half-life, has proven to be protective for the intestinal epithelium. However, its role and mechanism of action in traumatic hemorrhagic shock have not been understood. The project applicant has recently found that intravenous administration of exogenous ANP during the shock stage of traumatic hemorrhagic shock can significantly increase serum ANP level after 24 h, this was associated with increased ROS activity in the intestinal epithelium and correlated with multiple organ protection. The applicant proposes a hypothesis that there may exist a PLC-γ1/ROS feedback circuit within intestinal epithelial cells which results in overexpression of serum ANP. This project will explore intermediate molecular events within the circuit involved in ANP overexpression by a series of in vivo/vitro inhibition and overexpression tests. Moreover, it will try hard to illuminate that the PLC-γ1/ROS feedback circuit, which can be activated by exogenous ANP concentration triggering mechanism, results in increased serum ANP. This project will lay the basis for the potential use of ANP in early treatment of traumatic hemorrhagic shock.

肠道屏障损伤是创伤失血性休克诱发多器官功能障碍综合征的重要原因。半衰期较短的心房钠尿肽（atrial natriuretic peptide，ANP）被发现具有肠道上皮保护作用，但其在创伤失血性休克中的作用及其机制尚未清楚。申请人最近研究发现，大鼠创伤失血性休克后在休克期静脉给予外源性ANP可显著提高24 h后血清ANP水平，并发现其与小肠上皮ROS活性增加及多器官保护作用有关。申请人据此提出肠上皮细胞内的 PLC-γ1/ROS馈路形成，可促使血清ANP过表达主要机制的假说。本项目拟进一步通过体内及体外一系列抑制和过表达试验，探索血清ANP过表达机制的馈路内分子事件，力图阐明外源性ANP的浓度依赖性触发作用激活肠上皮细胞PLC-γ1/ROS馈路，最终引起内源性ANP过表达。该项目研究将为ANP在创伤失血性休克早期治疗的潜在应用奠定理论基础。

背景：申请人前期发现短期应用短半衰期心房钠尿肽（ANP）可提升创伤失血性休克（THS）24 h后ANP水平（本已正常），该机制尚未清楚。本项目拟结合体内、外实验验证内源性ANP过表达机制。.主要研究内容：首先，建立大鼠THS模型，休克期给予若干剂量静脉外源性ANP，以掌握血清及小肠上皮ROS活性，NF-KB、ANP与紧密连接蛋白表达及IECs凋亡变化，目的是摸索适宜药物剂量。其次，在适宜ANP给药剂量时腹腔注射NOXs或p38 MAPK抑制剂阻断假定ANP生成的通路，掌握ROS和p38 MAPK参与情况。最后，通过对PLC过表达或抑制判断ANP过表达是否经PLC介导，并利用PI3K抑制剂实验知晓PLC是否经PK3K途径激活。.重要结果：休克期以ANP 0.025 ug/kg/min剂量给药显著增加大鼠THS 24 h后血清和小肠ROS活性，增加小肠claudin-1与ZO-1表达，增加小肠Bcl-2表达而降低Bax表达。在此适宜剂量之上，THS休克期腹腔注射NOXs抑制剂或p38 MAPK抑制剂降低ROS活性，同时降低血清和小肠ANP，减少NF-kB mRNA、NPR-C mRNA表达，但不降低NPR-A mRNA表达。IEC-6细胞经缺氧/复氧（H/R）后，在ANP干预的基础上使用U73122显著降低细胞ROS活性及ANP表达，同时显著降低Bcl-2和增加Bax表达。在过表达PLC的H/R IECs中，加用ANP对ROS活性无显著影响。ANP或PLC过表达显著上调细胞Bcl-2和下调Bax。但ANP和PLC过表达处理的IECs在Bcl-2和Bax蛋白表达之间差异无统计学意义。过表达PLC显著降低H/R诱导的IECs细胞凋亡。PI3K抑制剂显著下调有或无ANP处理的H/R IECs的PLC表达。.关键数据：获得了THS后外源性ANP干预的适宜剂量（0.025 ug/kg/min）；外源性ANP在适宜剂量激活PLC/ROS馈路后促使内源性ANP过表达，该过程可受p38 MAPK和PI3K调控。IECs本身不具备有效PLC/ROS馈路，该机制估计来源于巨噬细胞。.科学意义：ANP在保护THS肠道屏障方面具有潜在应用价值。THS后早期使用ANP有助于抑制肠道屏障损伤后二次打击造成的MODS，最终有助于改善临床预后。