化合物pre-LBK02之抗發炎作用及機轉之探討

Abstract

發炎反應(Inflammation)為組織受傷或受生物侵入時，體內所產生的自然防禦機制，一般具有紅、熱、腫、痛的症狀，會引起微血管擴張、血流加速、血管通透性增加並促使白血球轉移至發炎部位，進而吞噬病原體以及受傷組織。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或促進發炎反應活化的細胞激素(pre-inflammatory cytokine)來維持或增強發炎反應的進行以助於個體清除外來的有害刺激；然而，過度的發炎反應會造成個體的傷害，並觸發多種和發炎反應相關的疾病，例如：關節炎、多發性硬化症、氣喘、動脈硬化、自體免疫疾病、癌症、阿茲海默症以及急性敗血症。因此尋找具有抗發炎活性的藥物用以抑制過度的發炎反應成為現今藥物開發的重要方向。 在初步的藥物篩選過程中，我們發現nstbpb5185的衍生物， pre-LBK0具有抗發炎活性。且在活性測試的劑量下，不會影響細胞的存活率。化合物pre-LBK02在30 microM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， TNF-alpha和IL-6並且同時降低NO的產生。此外我們也使用了敗血症動物模式來探究pre-LBK02對於急性發炎的動物是否有抗發炎活性，我們發現pre-LBK02可以有效地降低發炎引發之病理反應，如降低敗血症小鼠血清中的發炎細胞激素，改善敗血症小鼠所受到的器官損傷。 在作用機制的探討方面，我們發現給予pre-LBK02後，對於抑制LPS所產生的下游訊息傳遞鏈主要以抑制MAPK、NF-kappaB、JAKs/STATs的活化為主。在MAPK活化路徑中，我們檢視了三種激酶ERK1/2、JNK、p38的磷酸化現象，發現化合物pre-LBK02對於三種激酶都有抑制作用；另外也檢視了NF-kappaB活化路徑的重要因子：I-kappa-B-alpha的磷酸化以及降解情形，發現化合物pre-LBK02能抑制I-kappa-B-alpha磷酸化和降解現象；pre-LBK02也可從轉錄層級去抑制NF-kappaB活化路徑下游產生的酵素像是iNOS、COX-2的表現。此外，我們也發現，pre-LBK02也能減少某些跟發炎反應相關的JAKs/STATs的活化(JAK2、STAT5、STAT3)。綜合上述結果，我們認為pre-LBK02是藉由抑制MAPK、NF-kappaB、JAKs/STATs的活化來達到抑制發炎反應的效果。

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 bacteria invasion, mammalian monocytes/ macrophages release a variety of inflammatory mediators such as pro-inflammatory and cytotoxic cytokines, nitric oxide and reactive oxygen 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, Alzheumer’s disease and cancer. Hence, targeting on uncontrolled inflammation seems feasible to control numerous inflammation-associated diseases. Under the drug screening process of nstpbp5185 derivatives, we discovered that compound pre-LBK02, a synthetic benzimidazole molecule, possessed anti-inflammatory effects in decreasing the release of pro-inflammatory cytokines including TNF-alpha and IL-6, as well as decreased the production of NO, and we also excluded its cytotoxicity by cell viability and LDH assay. Furthermore, we found that pre-LBK02 attenuates some pathological syndromes of LPS-induced endotoxemia in mice, such as decreasing the cytokine release, protecting the mice from tissue injury in septic conditions. These results suggest the beneficial effects of pre-LBK02 in septic animal models. Regarding protein expression of LPS-stimulated RAW264.7 cells, we observed that the molecular mechanism of pre-LBK02-mediated anti-inflammation is associated with decreasing phosphorylation of MAP kinases such as ERK1/2, JNK and p38. pre-LBK02 also inhibited phosphorylation of Ikappa B alpha and reversed Ikappa B alpha degradation and attenuated the expression of NF-kappaB-related downstream inducible enzymes such as iNOS and COX-2. In addition, we also identified that pre-LBK02 might be a potential anti-inflammatory agent by downregulating phosphorylation of some JAKs/STATs associated with inflammation such as JAK2, STAT5, STAT3. Taken together, these results indicate that pre-LBK02 inhibits LPS-induced inflammation by inhibiting phosphorylation of MAP kinases and Ikappa B alpha and downregulating phosphorylation of some JAKs/STATs.