CFTR在肠道缺血再灌注后肠道细菌移位中的作用及分子机制研究

Intestinal ischemia-reperfusion injury (IIRI) is a clinical phenomenon with high morbidity and mortality. Enteric bacterial translocation(EBT) is a keypoint in the pathogenesis of IIRI, but the underlying molecular mechanism remains unclear. Cystic fibrosis transmembrane signal transconductor (CFTR) is abundantly expressed in intestinal epithelial cells and is closely related to intestinal function. Our Previous studies have shown that hypoxia/reoxygenation causes significant reduction in CFTR of intestinal epithelial cells, and overexpression of CFTR can reduce the extent of bacterial translocation. We hypothesize that CFTR is involved in EBT via the following routes :①Downregulation of CFTR induced by H/R can medicate the increase of gut bacterial entry into intestinal epithelial cells, inducing EBT;②H/R induced downregulation of CFTR promote S-glutathionylation of MCU at Cys-97, Ca2+overload, ROS and mtDNA production, and then activate the NLRP3 inflammasome, which results in exaggerated downstream inflammation and destorys the mucosal barrier in EBT. In this study, Pseudomonas aeruginosa was used as the representative intestinal bacteria. In vitro, hypoxia-reoxygenationa was used to treat the cell. And Intestinal ischemia-reperfusion model was established in vivo to validate the role of CFTR in EBT. At the same time, overexpression of CFTR was used for negative demonstration. The results of the study can elucidate the role and mechanism of CFTR in EBT, providing a new theoretical basis and target contributing to diagnosis and treatment of IIRI.

肠缺血再灌注损伤（IIRI）是一个发病率及病死率都较高的临床现象。肠道菌群移位（EBT）是IIRI发生的关键环节，但其分子机制仍不清楚。囊性纤维化跨膜信号转导体（CFTR）在肠道上皮细胞大量表达，与肠道功能密切相关。前期研究显示缺氧复氧导致EBT发生以及CFTR表达下调，且过表达CFTR能够降低EBT程度。结合文献假设CFTR通过以下途径参与EBT:①CFTR降低能够介导细菌进入肠上皮细胞增多，诱发EBT；②CFTR降低导致MCU第97位半胱氨酸谷氨酰胺化，引起Ca2+超载，ROS及mtDNA产生，激活NLRP3小体，促发炎性反应及破坏黏膜屏障。本研究拟用绿脓杆菌为肠道代表菌,体外用缺氧复氧处理细胞，体内建立小鼠肠道缺血再灌注模型，分别验证CFTR在EBT中作用及其机制。同时过表达CFTR进行反面论证。研究结果将阐明CFTR在EBT作用和机制，为IIRI的诊治提供理论依据及治疗靶点。

背景：肠缺血再灌注损伤(IIRI)是一个发病率及病死率都较高的临床现象。肠道黏膜屏障损伤及肠道菌群移位是IIRI发生的关键环节。目前，关于肠黏膜上皮细胞屏障受损的信号级联反应仍是研究IIRI发生的盲区。本研究以肠黏膜上皮细胞膜中的重要通道蛋白为核心，探索其介导肠道黏膜屏障损伤及IIRI发生的分子核心机制，寻找干预其发生的新的关键靶点，对临床上预防和治疗 IIRI 具有重大意义。.主要研究内容及结果：利用体外H/R模型及体内I/R模型，通过分子生物学等手段明确了CFTR在炎症反应及细胞凋亡中的作用。阐明了H/R导致肠道上皮细胞 CFTR 降低，介导MCU过度激活和线粒体内钙超载，激发炎症反应及破坏紧密连接，导致细菌移位。同时，进一步阐明了H/R导致肠道上皮细胞 CFTR 降低，介导PI3K/NF-KB激活，诱导肠道上皮细胞凋亡，进而破坏紧密连接。而且，证明了PL120蛋白能够通过调节紧密连接的结合调整H/R诱导的肠道黏膜屏障损伤。.研究意义：本研究能够加深对肠上皮细胞中的 CFTR 功能的认识，并首次阐明了CFTR/MCU/NLRP3信号通路在其中作用，为预防 IIRI及黏膜屏障损伤的发生提供新的可能的干预靶点，从上游控制IIRI损伤的发生，对临床上预防和治疗 IIRI 具有战略意义。

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