化合物Ctkf6f2之抗發炎作用及機轉之探討

Abstract

發炎反應(Inflammation)為組織受傷或受微生物侵入時，體內所產生的自然防禦機制，一般具有紅、腫、熱、痛之典型特徵，引起微血管擴張、血流加速、血管通透性增加及白血球移行至發炎部位，進而吞噬病原體及受傷組織。在發炎反應的過程中，活化的免疫細胞還會分泌多種具有細胞毒殺作用(cytotoxic)或促發炎反應的細胞激素(proinflammatory cytokine)來促進並維持發炎反應的進行以助於個體清除外來的有害刺激；然而，在過度的發炎反應中，上述物質會大量產生而對個體產生傷害。近來研究指出，發炎反應和多種疾病狀態有關，例如：關節炎、阿茲海默症、多發性硬化症、氣喘、動脈硬化、自體免疫疾病、癌症甚至是急性的敗血症。因此尋找具有抗發炎活性的藥物來抑制過度的發炎反應便成為藥物開發的重要方向。 我們發現化合物5185的衍生物ctkf6f2具有抗發炎活性，並且在活性測試所使用的劑量下，不會影響細胞的存活率。實驗指出化合物ctkf6f2在10 μM時即可有效抑制受細菌內毒素(LPS)活化的鼠源巨噬細胞或人類的單核細胞所分泌的細胞激素，像是TNF-α及IL-6；和趨化素，像是MCP-1；也會抑制nitric oxide及自由基(ROS)的釋放。 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,因此在活體實驗中，我們分別使用了內毒素(LPS)引發的內毒素血症及盲腸結紮穿刺手術(CLP)誘導敗血症此兩種動物模式來研究化合物ctkf6f2在活體發炎中的抗發炎活性。我們發現在此兩種模擬敗血症的發炎狀況中，化合物ctkf6f2都具有抑制發炎的現象，例如：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)與趨化素含量(MCP-1)、降低LPS和CLP誘發敗血症小鼠之死亡率、改善敗血症小鼠所受到的器官損傷。 在機制的探討方面，LPS活化巨噬細胞後所產生的下游訊息傳遞鏈以ROS產生、MAPK路徑及NF-κB路徑為主。在ROS生成路徑中，我們發現給予Ctkf6f2的情況下可以抑制NADPH oxidase 次單位p47及p67從細胞質移動到細胞膜的情形，而透過抑制NADPH oxidase的活化降低ROS的表現量；而在MAPK 路徑中，我們檢視了三種重要的激酶包括p38、ERK1/2、JNK的磷酸化現象，發現化合物ctkf6f2對於這三種激酶的磷酸化都有抑制作用；另外也檢視了NF-κB活化路徑中重要的因子：IκBα的磷酸化和降解(degradation)情形，發現化合物ctkf6f2也可抑制IκBα磷酸化和降解現象;此外，ctkf6f2也可從轉錄層級(transcriptional level)去抑制活化NF-κB路徑後所誘發的下游酵素，例如：iNOS及COX-2的表現。因此我們推測化合物ctkf6f2藉由抑制ROS產生和MAPK及NF-κB訊息傳遞路徑來抑制發炎反應。

Inflammation, usually characterized by swelling, redness, pain, and heat, is a crucial function of the innate immune system for protecting the host against pathogens. Upon bacterial invasion, mammalian monocytes/ macrophages release a variety of inflammatory mediators such as pro-inflammatory and cytotoxic cytokines, chemokines and reactive oxygen/nitrogen species to defense harmful stimuli, but excessive inflammatory reaction leads to extensive tissue damage and manifestation of pathological states such as multiple sclerosis, asthma, arthritis, atherosclerosis, Alzheimer’s disease and cancer. Therefore, targeting on uncontrolled inflammation seems feasible to control numerous inflammation-associated diseases. We discovered compound ctkf6f2 , a synthetic benzimidazole molecule , derivative of compound 5185, possesses anti-inflammatory effects in decreasing the release of pro-inflammatory cytokines including TNF-α and IL-6, chemokines including MCP-1 ,as well as decreased the production of nitric oxide and reactive oxygen species (ROS), and we also excluded its cytotoxicity by cell viability and LDH release assay. Furthermore, we found that ctkf6f2 attenuates some pathological syndromes of LPS-induced endotoxemia and CLP-induced sepsis in mice, such as decreasing the cytokine production, protecting the mice from tissue injury in septic conditions . These results suggest the beneficial effects of ctkf6f2 in septic animal models. In protein expression of LPS-stimulated macrophages, we observed that the molecular mechanism of ctkf6f2-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules such as ERK1/2, JNK and p38. Ctkf6f2 also inhibited phosphorylation of IκBα and reversed IκBα degradation and attenuated the expression of NF-κB-related downstream inducible enzymes like iNOS and COX-2. In addition, ctkf6f2 has been identified as an anti-inflammatory agent by inhibiting NADPH oxidase activity which leads to the reduction of reactive oxygen species (ROS) by inhibiting the membrane translocation of NADPH oxidase subunit p47phox and p67phox. Taken together, these results indicate that ctkf6f2 inhibits LPS-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production and both the blockade of NF-κB and MAPK pathways.