蛋白激酶A调节亚基PKARIIα介导的cAMP-PKA非经典信号途径对肠道炎症的作用及机制研究

The classical cAMP-PKA pathway mediated by catalytic subunit plays an important role in IBD and many other inflammatory diseases. However, there are many arguments about the role of the classical cAMP-PKA pathway in inflammation. A recent published article demonstrates that PKA regulatory subunit PKARIIα, instead of PKA catalytic subunit, can regulate the inflammation after myocardial infarction. However, it is not clear whether PKARIIα participates in the development and progression of inflammatory bowel disease. Our previous research has found that the phosphorylation level of PKARIIα was signaficantly down regulated in the early phase of colitis. Moreover, PRKARIIA-/- mice were resistant to colitis induced by DSS administration compared to WT littermates. We speculate that PKARIIα may regulate the onset of DSS-induced colitis. Given the result we have gotten, we plan to further explore the impact of PKARIIα on colitis and related signaling pathway. Our research will be helpful in understanding the non-classical pathway of cAMP-PKA mediated by PKARIIα, and may provide new idea and theory foundation for medical treatment targeting cAMP-PKA pathway.

业已发现，cAMP-PKA通过催化亚基作为经典信号通路参与肠道炎症过程，但结果多不一致。新近有报道，cAMP-PKA可不依赖催化亚基，直接通过调节亚基(PKARIIα)，调控心肌缺血中的炎症过程，但PKARIIα是否在炎症性肠病中发挥作用及作用如何仍不清楚。基于我们前期工作发现，DSS诱导小鼠肠炎发生早期p-PKARIIα显著下降, 且PRKARIIA基因敲除后DSS诱导的肠炎显著减轻，我们推测cAMP-PKA可能通过调节亚基PKARIIα作为非经典信号通路参与肠炎过程。本研究拟进一步采用全身及细胞特异性PRKARIIA基因敲除小鼠肠炎模型、流式细胞分析、免疫共沉淀、Microarray等技术，探索PRKARIIA基因敲除后对肠炎的作用，分析其相关信号通路，阐明调节亚基PKARIIα介导的cAMP-PKA非经典信号通路对肠炎的调控作用及机制，为炎症性肠病的靶向治疗提供新的途径和理论依据。

目前炎症性肠病的发病机制仍然不完全清楚，包括蛋白激酶A在内的多种信号通路的异常参与炎症性肠病的发生发展。为了明确PKA活化后调节亚基PKAR2α是否参调控肠道炎症，我们应用基因敲除小鼠首次探索PKAR2α在结肠炎症中的作用。首先通过DSS诱导急性结肠炎症模型观察Prkar2a-/-小鼠和WT小鼠之间肠道炎症的差异以及Prkar2af/fVillinCre小鼠和Prkar2af/f小鼠肠道炎症的差异，其次通过RNA-seq等分析Prkar2a基因敲除后对炎症相关信号通路的影响，最后应用16S rRNA测序分析Prkar2a-/-小鼠和WT小鼠之间肠道菌群的差异，然后通过抗生素饮水和交叉喂养实验明确肠道菌群在Prkar2a-/-小鼠结肠炎症中的作用。实验发现DSS饮水诱导急性结肠炎症后，与WT小鼠相比Prkar2a-/-小鼠的结肠炎症显著减轻；此外在结肠上皮细胞中特异性敲除PKAR2α后发现Prkar2af/fVillinCre小鼠DSS诱导的结肠炎症与Prkar2af/f小鼠相比亦显著减轻；通过RNA-seq等发现Prkar2a基因敲除后IFN信号通路相关分子表达显著上升；16S rRNA测序分析发现Prkar2a-/-小鼠和WT小鼠之间的肠道菌群存在显著差异，且抗生素饮水和交叉喂养实验证实肠道菌群在Prkar2a-/-小鼠显著减轻的结肠炎症中发挥重要作用。我们研究发现：（1）Prkar2a基因敲除后小鼠结肠炎症显著减轻；（2）Prkar2a基因敲除后的肠炎保护作用可能来自于结肠上皮细胞；（3）Prkar2a基因敲除后IFN信号通路活化；（4）肠道菌群在Prkar2a基因敲除后的肠炎保护中发挥重要作用。