探討Eupafolin抑制脂多醣體誘導RAW 264.7巨噬細胞發炎反應之效用與作用機制

Abstract

Eupafolin是一種類黃酮的物質，經由鴨舌癀Phyla nodiflora. (Verbenaceae) 此中草藥萃取而來。類黃酮通常是用來治療發炎、癌症及一些經細菌感染等的疾病。然而，Eupafolin的抗發炎效果及其作用機轉都尚未十分清楚，因此本篇的主要研究目的是利用老鼠的巨噬細胞 (RAW 264.7) 及小鼠腳掌與肺臟經脂多醣體 (LPS) 刺激發炎產生後，進一步觀察及釐清 Eupafolin 的抗發炎效果及作用機制。首先在in vitro方面，使用RAW 264.7細胞來進行體外實驗；以LPS作為刺激劑，作用於RAW 264.7細胞，來誘發發炎反應的產生，初步結果發現Eupafolin的預先作用能夠有效的降低LPS所誘發的inducible nitric oxide synthase (iNOS) 及cyclooxygenase (COX-2) 表現與NO的產生。藉由西方墨點法及免疫細胞化學染色法的分析，發現Eupafolin 是藉由抑制 LPS 所誘發的MAPKs中的c-Jun NH2-terminal kinase (JNK) 磷酸化的表現，與nuclear factor-kappa B (NF-κB) 及activator protein-1 (AP-1) 這兩種轉錄因子的活化來達到抗發炎的作用。除此之外，在in vivo 實驗方面，經由組織染色的實驗結果也發現，相較於 LPS 單獨注射的刺激組，Eupafolin的預先投予，能有效的降低LPS誘發小鼠後腳掌所產生的發炎症狀-嗜中性球的浸潤現象和腳掌水腫，在肺臟誘發發炎方面，Eupafolin能有效減緩LPS誘發小鼠肺臟所產生的組織發炎、充血和巨噬細胞、嗜中性白血球、淋巴球等發炎細胞聚集的現象。由以上實驗結果推論，Eupafolin可能主要是藉由調降JNK、NF-κB和AP-1等訊息傳遞路徑，抑制發炎因子COX-2、iNOS、NO的表現。綜合上述，此次實驗結果發現Eupafolin 提供了另一種新潁且有效治療發炎相關疾病的藥物選擇。

Eupafolin, a flavonoid isolated from Phyla nodiflora.(Verbenaceae), has been used traditionally to treat disease such as inflammation, cancer and infections by fungi, bacteria, and viruses. However, its mechanism remains unknown. The aim of this study was to evaluate the anti-inflammatory effects and underlying the regulatory mechanisms of the eupafolin on Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells in vitro and mouse paw, lung in vivo. First at all, in in vitro study, eupafolin significantly decreased LPS-induced NO production and expressions of iNOS and COX-2 in RAW 264.7 cells. Furthermore, eupafolin suppressed NF-κB p65 translocation and activator protein-1 (AP-1) activation as well as NH (2)-terminal kinase (JNK) phosphorylation. In in vivo assay, eupafolin reduced the neutrophil infiltration, edema and inflammatory mediators expression in LPS-induced mouse paw and lung. The present results showed that eupafolin exerts its anti-inflammatory effects through the decrease of inflammatory mediators, expression by suppressing pJNK, NF-κB and AP-1 signaling pathways. Taken together, our results indicate that eupafolin may provide a useful therapeutic approach for inflammation-associated diseases.