Pcpc07f2抑制LPS活化噬菌細胞發炎反應之機轉探討

Abstract

發炎反應即個體遭遇到外界有害刺激，像是病原菌、有害化學物質、傷口感染、組織受損時，體內所產生的自然防禦機制。適當的發炎反應得以協助人體抵擋來自外界的傷害，清除病原菌，藉以恢復健康。然而過度甚而失去控制的強烈發炎反應，其產生的發炎相關激素與機制，往往會在體內不斷累積並放大反應，最終於器官組織造成損害，引發一系列的相關疾病。近來許多研究發現，發炎反應的確與關節炎、多發性硬化、氣喘、動脈硬化等慢性疾病有所相關，甚至某些癌症的發生也歸咎於持續而激烈的發炎反應。正因如此，研究開發有效的抗發炎藥物，減輕患者的發炎症狀，並試圖終止強烈的發炎反應，也成了現今許多專家學者與藥廠努力的方向與動力。 在實驗室初步篩藥過程中，發現一化學合成物質pcpc07f2具有抑制巨噬細胞釋放細胞激素的能力，引發我們進一步探究其抗發炎能力與機轉的興趣。首先確認了實驗中所用濃度並不會影響噬菌細胞株的生存率。接著在抑制噬菌細胞受到LPS 活化後釋放發炎性細胞激素之測試中，我們發現pcpc07f2在10μg/ml (約30μM)即能有效抑制由LPS 100ng/ml在原生性小鼠巨噬細胞、細胞株RAW264.7巨噬細胞、亦或細胞株THP-1單核細胞所引發之TNF-α與IL-6釋放，且此抑制作用呈現明顯的濃度相關性。除此之外，對於LPS 100ng/ml活化細胞株RAW264.7巨噬細胞後，其產生之移動現象(migration) 以及大量釋放nitric oxide的發炎反應，pcpc07f2也都能在10μg/ml (30μM)時達抑制作用，同樣此抑制作用呈現明顯的劑量相關性。最後，延伸pcpc07f2之抗發炎能力，將其運用至LPS誘發之敗血症動物模式中，結果發現pcpc07f2 (8μg/g)能有效降低敗血症小鼠血清中的發炎細胞激素含量(TNF-α與IL-6)，並明顯降低LPS誘發敗血症小鼠之死亡率。 而對於LPS刺激活化巨噬細胞後所產生的下游訊息傳遞鏈之中，我們檢視了MAPK 路徑中的三種重要激酶：p-38、ERK、JNK的磷酸化現象，以及NF-κB路徑中重要調控因子: IκB的降解情形 (degradation)。研究結果發現，在給予pcpc07f2之後，p-38、ERK、JNK這些MAPK重要激酶的磷酸化都有被抑制的現象，而抑制NF-κB活化的IκB，其降解情形也可被pcpc08f2所回復(reverse)，進而抑制NF-κB的活化。此外，我們也使用了MAPK和NF-κB inhibitor來研究兩種訊息傳遞路徑和發炎激素之間的關聯性，發現它們可以抑制LPS刺激活化之巨噬細胞釋放發炎性細胞激素與nitric oxide。 綜合以上所述，我們可以推測pcpc07f2可能經由抑制MAPK路徑中的三種重要激酶p-38、ERK、JNK的磷酸化，以及抑制NF-κB此轉錄因子的活化，來進行其抑制發炎的能力。當然，詳細的作用機轉還有待更多方面的實驗進一步來探討。

Macrophages play a vital role in inflammation and innate immune responses. Foreign pathogens recognized by macrophages trigger a serial of signaling cascades upon ligand-receptor stimulation, then resulting in releasing several pro-inflammatory and anti-inflammatory cytokines, and activating other immune cells. In this study, we used lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall for activating macrophages, as an experimental model. The pro-inflammatory cytokines, such as TNF-α and IL-6 increased significantly after LPS stimulation as measured by ELISA. We found pcpc07f2, a synthetic benzimidazole compound, concentration-dependently (10~90μM) inhibited LPS (100ng/ml)-induced TNF-α and IL-6 release in macrophages (RAW 264.7 cells), and the inhibition appeared more obvious in primary macrophages. However, pcpc07f2 did not affect the cell viability. Regarding the anti-inflammation effects of pcpc07f2 on LPS-induced septic animal model, we also found that pcpc07f2 significantly reduced pro-inflammatory cytokines release and also reduced LPS-induced mortality in a dose-dependent manner. Furthermore, pcpc07f2 also inhibited macrophage migration and NO production induced by LPS. We further examined if pcpc07f2 affects the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in LPS-induced RAW 264.7 cells by Western blotting analysis. Pcpc07f2 concentration-dependently inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2), JNK, and p38 phosphorylation, and suppressed LPS-induced NF-κB activation, suggesting that pcpc07f2 decreased TNF-α, IL-6 and NO production via the inactivation of ERK1/2, JNK, and p38 ,and NF-κB signal pathway. Taken together, we suggest that pcpc07f2 may be used as therapeutic approach for the treatment of inflammatory diseases.