| 摘要: | 在發炎疾病中，活化的人類單核球會製造、分泌許多的前發炎細胞激素(pro-inflammatory cytokines)及基質金屬蛋白酶(matrix metalloproteinases, MMPs)。本次研究欲探討從真菌Nectria balsamea #YMJ94052402萃取而來的天然物radicicol對人類單核球細胞釋放MMPs及前發炎細胞激素之影響與機轉。實驗結果顯示，radicicol 可以抑制腫瘤壞死因子???n?n( tumor necrosis factor-alpha, TNF-alpha)、白細胞介素1beta(interleukin-1beta, IL-1beta)或脂多醣體 (lipopolysaccharide, LPS)誘發人類單核球細胞製造及釋放MMP-9。利用西方墨點法，發現radicicol有濃度梯度效應的降低受LPS活化THP-1細胞的MMP-9蛋白質表現。Radicicol是一種熱休克蛋白90 (heat shock protein 90, HSP90) 抑制劑，發現其促進HSP70蛋白質的表現間接證明了抑制HSP90活性的能力。利用反轉錄-聚合酵素連鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR) ，發現radicicol可以減少活化的THP-1之MMP-9的mRNA表現。在受到LPS刺激的THP-1細胞中，radicicol有意義反轉inhibitor-kappaB-alpha(IkappaB-alpha)的降解作用，並且發現radicicol能夠促進Akt降解。除此之外，利用酵素連結免疫吸附分析法(Enzyme-Linked Immunosorbent Assay, ELISA)發現 radicicol依濃度梯度效應降低受LPS活化之THP-1細胞釋放TNF-alpha或IL-6。綜合結論，本研究顯示radicicol可藉由調控NF-kappaB或PI3K/Akt pathway抑制MMP-9的表現及前發炎細胞激素的製造，並提供未來發展抗發炎藥物之新的方向。
There is much evidence indicating that activated human monocytes / macrophages synthesize, and secrete several pro-inflammatory cytokines and matrix metalloproteinases (MMPs), and participate in the inflammatory diseases. In this study, we investigated the effects and mechanism of radicicol, as a natural product, extracted from Nectria balsamea #YMJ94052402, on human monocytic MMPs and pro-inflammatory cytokine expression. Radicicol exhibited concentration-dependent inhibition of MMP-9 enzymatic activation induced by tumor necrosis factor-alpha(TNF-alpha), interleukin-1beta(IL-1beta) or lipopolysaccharide (LPS) in human monocytic THP-1 cells. Western blot analysis showed that radicicol concentration-dependently suppressed monocytic MMP-9 protein expression. Radicicol is a heat shock protein 90 (HSP90) inhibitor. We found that radicicol enhanced HSP70 expression, and that indicated the inhibition ability of HSP90 in LPS-activated monocytic cells. Besides, reverse transcription- polymerase chain reaction (RT-PCR) showed that radicicol concentration-dependently suppressed monocytic MMP-9 mRNA level. Furthermore, Western blot analysis showed that radicicol could significantly reduce the degradation of inhibitor-kappaB-alpha (IkappaB-alpha) induced by LPS. In addition, we found that radicicol showed reduction of Akt expression. On the other hand, enzyme-linked immunosorbent assay (ELISA) showed that radicicol presented an inhibitory effect on TNF-alpha or IL-6 release in a concentration-dependent manner. In conclusion, we demonstrate that radicicol might involve interference with the NF-kappaB or PI3K/Akt pathway to attenuate MMP-9 expression and production of pro-inflammatory cytokines, and provide new opportunities for the development of new anti-inflammatory durgs. |
