化合物9-2抑制LPS活化噬菌細胞發炎反應之機轉探討

Abstract

敗血症為一常見的全身性發炎重症，其根本源自於免疫系統針對感染或創傷的過度反應。巨噬細胞在發炎反應的啟動中扮演相當重要的角色，被活化的巨噬細胞將會激化許多下游反應，例如類Toll受體的活化，並藉此對抗外在環境的威脅。巨噬細胞的活化同時也會產生許多發炎性細胞激素以及抗發炎細胞激素，這些細胞激素能夠調控發炎狀態下的許多反應，諸如白血球的趨化以及血管內皮通透性增加，然而當免疫反應被過度激化時，可以偵測到血中的細胞激素持續維持在高峰，此類反應被認為是判斷發炎反應是否失去控制的標準之一。數十年來，許多研究報告已經證實多種慢性疾病與長期的發炎有關，例如阿茲海默症、類風濕性關節炎甚至是癌症，而雖然有許多報告針對慢性發炎所導致的疾病，嘗試提出完整或部分的解釋來與治療作連結，臨床上成功應用此一概念的醫療方法仍舊非常少見。目前臨床上對於新世代抗發炎藥物需才孔急，而我們利用細胞激素抑制效果的差異，初步地篩選一系列Benzimidazole類衍生化合物，發現其中的化合物9-2在老鼠巨噬細胞、人類單核細胞被內毒素刺激的情況下有效地抑制細胞激素產生。除此之外，其他發炎相關的調控因子如一氧化氮(NO)、環氧合脢(COX2)同樣也可以被化合物9-2有效地抑制。然而，一重要的調控因子:活性氧類(reactive oxygen species)在細胞中的積累僅有部分能被化合物9-2抑制。為了進一步確認化合物9-2是否具有實際應用的潛力，使用在老鼠腹腔注射致死劑量內毒素的活體實驗來檢視其效果，發現化合物9-2能夠有效提升老鼠24小時內的存活率。以上所述這些效果，其背後的機轉可能來自於促分裂原活化蛋白脢(MAPK)的磷酸化被抑制，此一可能性觀察自ERK1/2、JNK、p38三個MAPK亞型的磷酸化，在細胞實驗中被化合物9-2有效地抑制了。除此之外，另一條被化合物9-2有效抑制的訊息傳遞路徑為NF-κB，其活化需要將抑制性的結合因子IκB降解，而在實驗中能夠觀察到此降解反應同樣被化合物9-2所抑制。綜合以上，我們可得知化合物9-2具有抗發炎功效，能夠抑制部份發炎性調控因子及細胞激素的產生，改善致死性敗血症老鼠的存活率，而此效果極可能來自於化合物9-2對於MAPK、NF-κB路徑的負調控。

Sepsis is a common and severe medical condition characterized as a severe systemic inflammatory response. The core concept of sepsis is that the immune system overreact with the outer threat, whether infection or trauma. At the beginning of inflammation, the macrophage plays a pivotal role and would trigger several signaling such as Toll-like receptor(TLR) activation to counter against the threatening, resulting in release of diverse pro-inflammatory and anti-inflammatory cytokines, which activate further immune response including neutrophil recruitment and vascular permeability increase. For decades, more and more diseases have been found to be related to chronic inflammation, including Alzheimer’s disease, rheumatoid arthritis, atherosclerosis, and even the cancer. Although numerous studies have demonstrated the significance of regulating inflammation, only little clinical applications acquired real success. Regarding the urge of next generation anti-inflammation agent, we screened a few benzimidazole-based serial derivatives, and found the compound 9-2 demonstrating the concentration-dependent inhibition of cytokine production in LPS stimulated RAW 264.7 macrophage and THP-1 monocyte. Besides, other inflammatory mediators associated with LPS such as nitric oxide production and COX2 protein expression were also found to be suppressed effectively by compound 9-2, yet minor suppression was observed in reactive oxygen species(ROS) production. The in vivo LPS challenge was employed to examine whether compound 9-2 an applicable agent against severe systemic inflammation, and we found apparent improvement in 24-hour survival rate. All these effect might be achieved through the negative regulation of MPAKs, since the phosphorylation of ERK1/2, JNK and p38 were significantly inhibited under the exposure of LPS. Inhibition of IκB degradation was also observed and provided another explanation supporting the previous findings. These results indicate that compound 9-2 regulates LPS associated inflammation through MAPK and NF-κB.