在这项研究中,我们证实凝血酶显著增加人气管平滑肌细胞(HTSMC)中COX-2和PGE2的产生,导致气道和肺部炎症。这些分子是众所周知的各种炎性疾病的贡献者。这里,我们使用特异性抑制剂和小干扰RNA(siRNA)详细研究了所涉及的信号通路.我们的结果表明,抑制剂靶向蛋白质,如蛋白激酶C(PKC)δ,富含脯氨酸的酪氨酸激酶2(Pyk2),c-Src,表皮生长因子受体(EGFR),磷脂酰肌醇3-激酶(PI3K),或激活蛋白-1(AP-1)有效降低凝血酶诱导的COX-2和PGE2的产生。此外,用siRNA转染针对PKCδ,Pyk2,c-Src,EGFR,蛋白激酶B(Akt),或c-Jun缓解了这些反应。此外,我们的观察显示,凝血酶刺激信号级联的关键成分的磷酸化,包括PKCδ,Pyk2,c-Src,EGFR,Akt,还有c-Jun.凝血酶通过AP-1激活激活COX-2启动子活性,被COX-2启动子内的点突变AP-1位点破坏的过程。最后,白藜芦醇(研究最多的天然多酚之一)被发现通过阻断Pyk2,c-Src的活化,有效抑制凝血酶诱导的COX-2表达和PGE2在HTSMCs中的释放,EGFR,Akt,还有c-Jun.总之,我们的研究结果表明,凝血酶诱导的COX-2和PGE2生成涉及PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt依赖性AP-1激活途径.该研究还表明白藜芦醇作为控制气道炎症的干预措施的潜在用途。
In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.