BACKGROUND β2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear. METHODS We established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement. RESULTS We found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 μM, and ESI-09 promoted SOCE of ASMCs at 10 μM and 100 μM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365. CONCLUSIONS Our results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.

中文翻译：

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由cAMP（Epac）直接激活的交换蛋白可预防哮喘小鼠的气道炎症和气道重塑。

背景技术β2受体激动剂通过增加细胞内cAMP的产生来诱导气道平滑肌松弛。PKA是cAMP的传统下游信号通路。最近，由cAMP（Epac）直接激活的交换蛋白被鉴定为cAMP的另一个重要信号分子。Epac在哮喘气道炎症和气道重塑中的作用尚不清楚。方法我们建立了OVA致敏的，具有挑战性的急慢性哮喘小鼠模型，首先探讨了Epac的表达。然后，分别用Epac-选择性cAMP类似物8-pCPT-2'-O-Me-cAMP（8pCPT）和Epac抑制剂ESI治疗后，分别观察了急性哮喘小鼠模型的气道炎症和气道高反应性，以及慢性哮喘小鼠模型的气道重塑。 -09。下一个，通过CCK-8检测和Annexin-V染色检测了8pCPT和ESI-09对体外培养的小鼠气道平滑肌细胞（ASMCs）增殖和凋亡的影响。最后，通过共聚焦Ca 2+荧光测量研究了8pCPT和ESI-09对ASMCs的储库操作Ca 2+进入（SOCE）的影响。结果我们发现在急慢性哮喘小鼠模型的肺组织中，Epac的两个同工型Epac1和Epac2的mRNA和蛋白质表达均低于对照小鼠。在急性哮喘小鼠模型中，气道炎性细胞浸润，Th2细胞因子分泌和气道高反应性被8pCPT显着减弱，而ESI-09加剧。在慢性哮喘小鼠模型中 8pCPT减少了气道炎性细胞浸润和气道重塑指数，例如胶原蛋白沉积和气道平滑肌细胞增殖，而ESI-09则增加了气道炎症和气道重塑。体外培养的小鼠ASMCs，8pCPT剂量依赖性抑制，而ESI-09促进ASMCs增殖。有趣的是，8pCPT促进了ASMC的凋亡，而ESI-09对ASMC的凋亡没有影响。最后，通过共焦Ca2 +荧光检查发现8pCPT可以抑制100μM的ASMC中的SOCE，而ESI-09可以促进10μM和100μM的ASMC中的SOCE。此外，储库操作的Ca2 +通道阻滞剂SKF-96365抑制了ESI-09对ASMCs增殖的促进作用。结论我们的结果表明，Epac对哮喘气道炎症和气道重塑具有保护作用，