探討酪胺酸激酶抑制劑對家塵螨誘發炎症之影響及機制

家塵螨引發的氣喘是常見的炎症之一，其中Dermatophagoides pteronyssinus是造成氣喘的主要過敏原，會誘發特異性抗原CD4+ Th2產生免疫反應。過去研究發現呼吸道上皮細胞中活化的鐸受體(Toll-like receptor, TLR)外，表皮生長因子接受器 (Epidermal growth factor receptor, EGFR) 也是調控過敏性呼吸道疾病的重要因子之一。本實驗將探討酪胺酸激酶抑制劑 (Tyrosine Kinase Inhibitors, TKIs) 是否抑制家塵螨誘發的免疫反應及其機制，並釐清家塵螨對不同型態細胞種類及其微環境的致敏關係。首先，我們在人類周邊血液單核球細胞(HPBMC)預處理TKIs (Tarceva、AZD-9291) 並用Dermatophagoides pteronyssinus重組蛋白-Der pII刺激，以酵素結合免疫吸附分析 (ELISA) 來測定相關激素之變化，結果發現 TKIs 可以有效減IL-6和IL-8之分泌。其次，結果發現在人類肺上皮細胞(BEAS-2B) 中，以 ELISA、RT-PCR和Real-time PCR，TKIs皆可有效抑制 Der pII 所誘發的IL-6和 IL-8之表現，以西方墨點法分析EGFR以及AMPK之活化，則觀察到Tarceva可抑制p-EGFR之活化以及其下游相關蛋白p-AKT，而AZD-9291雖無法抑制p-EGFR之活化，但仍可有效抑制其下游相關蛋白p-STAT3和p-AKT，而TKIs抑制上皮細胞發炎之機轉並非透過AMPK之活化；在核質分離法中發現AZD-9291主要是抑制NF-κB之進核作用。另外，結果發現在大鼠肺泡巨噬細胞 (NR8383) 中，以 Griess Reagent System 測定一氧化氮 (NO) 之產生，則 AZD-9291 可有效抑制 Der pII 所誘發的 NO 之產生，但無法抑制 iNOS 基因之表現。最後，結果發現在單核球和巨噬細胞中，以RT-PCR觀察巨噬細胞相關指標CD14和 CD36，結果發現AZD-9291可抑制Der pII所誘發CD14和CD36之基因表現，在西方墨點法中發現AZD-9291可促使p-AMPK之表現，但無法抑制 Der pII所誘發之capase-1。綜合以上實驗結果，AZD-9291可透過抑制p-AKT / p-STAT3訊號路徑、NF-κB之進核作用及AMPK活化來減緩 Der pII 所造成之IL-6、IL-8之分泌。因此AZD-9291可發展為減緩家塵蟎 Der pII所引起發炎反應的臨床治療藥物。

House dust mites (HDM；Dermatophagoides pteronyssinus.) is one of the common sources of airborne allergens worldwide. It activates specific CD4+ Th2 cells to orchestrate the HDM-induced allergic response. Previous study demonstrated that both Toll-like receptor (TLR) and epithelial growth factor receptor (EGFR) signaling regulated the key features of allergic airway disease in airway epithelial cells. The aim of study investigated whether tyrosine kinase inhibitors affect Derp II -induced inflammation in various cell types and their microenvironment. To determine related cytokines stimulated by recombinant protein Der pII in HPBMC, we demonstrated that pre-treatment of TKIs such as Tarceva and AZD-9291 effectively inhibits the production of IL-6 and IL-8 by ELISA. Second, Tarceva and AZD-9291 inhibited the Der pII-induced expression of IL-6 and IL-8 in human lung epithelial cells (BEAS-2B) by ELISA, RT-PCR and Real-time PCR. On Western blot, Tarceva decreased both phosphorylated EGFR and its downstream p-AKT, whereas AZD-9291 only decreased its downstream p-STAT3 and p-AKT, indication mechanism of TKIs inhibition wsa not associated with AMPK activation in human lung epithelial cells. Moreover, AZD-9291 inhibited NF-κB transloction to nucleus. Third, AZD-9291 effectively decreased Der p-induced nitric oxide production not via inhibition of iNOS gene in rat alveolar macrophages (NR8383). We determined the expression of macrophage related marker of CD14 and CD36 by RT-PCR in monocyte and macrophage. Our data indicated AZD-9291 was shown to inhibit Der pII-induced gene expression of CD14 and CD36. Furthermore, AZD-9291 activated phosphorylated AMPK, but can’t inhibit Der pII-induced caspase-1. Take together, pur data indicate AZD-9291 alleviates Der pII-stimulated secretion of IL-6 and IL-8 via inhibiting p-AKT /p-STAT3 signaling pathway, NF-κB transloction and AMPK activation. Therefore, AZD-9291 can be developed as a clinical potential drug against Der pII-induced inflammation.