Wip1通过STING-TBK1信号通路介导的自噬流受阻引起急性胰腺炎损伤的作用机制研究

In acute pancreatitis (AP), the activation of STING pathway (DNA recognition) activated and the autophagic flux were blocked. The role and mechanism of Wip1, a phosphatase that regulates DNA damage, is still unclear. Previous studies have shown that cerulein-induced AP model has elevated Wip1 in pancreatic tissue. Wip1 knockdown could inhibited the release of inflammatory mediators IFNβ and TNFα. Meanwhile, it also inhibited the phosphorylation level of p-TBK1, and dereased the content of p62/STING. Thus, we hypothesized that Wip1 participated in the activation of STING signaling pathway through STING/TBK1 phosphorylation, and then affected the expression and activation of p62, which induced autophagic flux block, and leads to the pathogenesis of AP injury. In this study, Balb/c mice and primary pancreatic acinar cells were selected as the objects. Firstly, the Wip1 expression, STING signaling pathway activation and autophagic flux were detected during AP. Then, the effect of Wip1 or STING/TBK1 on AP was determined by using lentivirus technology. Finally, the gene knock down and rescue experiment was used to explore Wip1 regulate the STING/TNK1 signaling pathway and autophagic flux. This study aims to reveal the role and regulation mechanism of Wip1 in AP, and provide new ideas and evidence for the prevention and treatment of AP.

急性胰腺炎(AP)存在识别DNA的STING通路活化和自噬流受阻，而Wip1是调控细胞DNA损伤的磷酸酶，其在AP中的作用机制尚不清楚。前期研究显示雨蛙素诱导的AP模型胰腺组织Wip1升高，敲低Wip1能抑制雨蛙素所致的原代胰腺细胞IFNβ和TNFα的释放，并能降低TBK1磷酸化水平和STING/p62含量，增加自噬溶酶体数量。据此我们提出假说，AP时Wip1参与STING/TBK1通路的激活，影响p62的表达和活化，阻断自噬流，导致AP损伤的发生。本研究将选Balb/c小鼠和原代胰腺腺泡细胞为研究对象，首先检测AP时Wip1表达、STING通路活化和自噬流情况；再用慢病毒技术调节Wip1/STING，明确Wip1对STING通路活化和自噬流的影响；最后用基因敲除回补实验，验证Wip1通过STING-TBK1调控自噬流。本研究将揭示Wip1调控AP的分子机制，为AP的防治提供新的思路。

急性胰腺炎(AP)是由多种病因引起的胰腺内胰酶活化后胰腺组织自消化、水肿、出血甚至坏死的炎症反应。AP目前主要以多学科对症支持治疗为主，尚无特异性治疗方法，预后较差。因此，明确AP的发病机制，寻找新的有效的预防和治疗方法具有十分重要的意义。本项目利用雨蛙素构建AP大鼠和细胞模型，从而通过体内外实验阐明Wip1在AP中的作用和意义，探索Wip1通过影响STING信号通路和自噬流参与AP发病的作用及潜在分子机制。我们发现雨蛙素刺激后大鼠血清淀粉酶和脂肪酶含量升高明显，并可诱导AR42J细胞上清液中淀粉酶含量增加。雨蛙素还可以刺激大鼠胰腺组织中IFNβ、TNFα表达，且可促进AR42I细胞分泌IFNβ、TNFα，并随时间延长而逐步增加，说明雨蛙素确实可以用于构建AP的体内外实验模型。通过雨蛙素刺激大鼠胰腺组织形成AP后，组织中Wip1、STING、TBK1和IRF3蛋白表达显著上调。并刺激AR42I细胞不同时间点 Wip1、STING、TBK1和IRF3蛋白表达增加并呈时间依赖性。最后我们还发现敲低Wipl基因表达可以降低雨蛙素诱导的细胞AP模型中的细胞上清淀粉酶含量以及炎症因子IFNβ、TNFα水平，并下调雨蛙素诱导的细胞AP模型中STING、TBK1和IRF3蛋白的表达水平。通过本项目的开展，我们证实Wip1可能通过STING/TBK1/IRF3信号通路调控自噬并参与AP的发生发展。虽然Wip1 和STING-TBK1通路在AP中的研究仍然较少，但我们的研究已经体现出该通路在AP中的重要性，靶向Wip1和STING-TBK1通路将为AP治疗提供新的思路和策略。