| Abstract: | 前言：IL-1beta在退化性關節炎患的發病過程，扮演發炎重要的角色，IL-1beta在軟骨組織能誘發趨化因子 (IL-8, MCP-1, IP-10)，分別能吸引並活化嗜中性白血球、單核球細胞及毒殺性T淋巴細胞，涵蓋先天及後天免疫，影響病程的進展，這些基因可能由不同的訊號傳遞路徑所調控，另一方面IL-1beta能提高細胞鈣離子的活化，鈣離子的濃度能影響訊息傳遞路徑及基因表現，也可能影響細胞的存活及退化性關節炎疾病的發展。而葡萄糖胺(glucosamine)能夠減緩退化性關節炎患者疼痛感及發炎反應，但其抗發炎機制還未完全明瞭。
研究目的：本篇研究在探討葡萄糖胺的抗發炎機制，是否對於IL-1beta誘發細胞質鈣離子活化、以及相關訊息傳遞路徑(包括CaMK-II、MAPK磷酸化)有抑制作用，並探討glycogen synthase kinase 3beta (GSK-3beta) 在其中扮演的角色。
實驗方法：在SW-1353細胞中加入葡萄糖胺(glucosamine)和細胞激素IL-1beta，利用Fura-2AM (Fura-2 acetoxymethyl ester) 測定鈣離子濃度的變化；以Western blot來分析ERK、p38以及CaMK-II磷酸化程度。
結果：葡萄糖胺會抑制IL-1beta所誘發鈣離子的驅動，並導致細胞中鈣離子濃度下降以及ERK、CaMK-II、p38的磷酸化下降。在GSK-3beta的抑制劑LiCl存在下，能減輕葡萄糖胺對於IL-1beta所引發的發炎反應(包括鈣離子活化，及MAPK磷酸化)的抑制作用。在葡萄糖胺抑制IL-1beta誘發發炎反應中，Ionomycin能補充細胞質鈣離子濃度，也能恢復IL-1beta所誘發的ERK、CaMK-II的磷酸化程度，相反的卻造成p38磷酸化更為減弱。
討論：臨床上葡萄糖胺的抗發炎作用除了和其抑制IL-1beta所誘發NF?B的活化有關之外，本篇的研究發現可能也和其抑制鈣離子驅動及MAPK活化有關，結果顯示葡萄糖胺可能引發全面性的抑制作用，而非專一性的抑制單一路徑。而這些抑制作用可能是藉由GSK-3beta所調控。GSK-3beta活化可能抑制鈣離子驅動，進而影響ERK及CaMK-II的活化；另一方面透過其他機制，抑制p38的活化。
Background: Pro-inflammatory cytokine IL-1beta plays a central role in the pathogenesis of osteoarthritis (OA). Chemokines IL-8, MCP-1, IP-10 induced by this cytokine are repectively capable of attracting and activating neutrophils, monocytes and T cells which are involved in innate or adaptive immunity, and thus affect the development of the disease. Glucosamine has been used as supplement to treat OA patients. In vitro this drug has suppressive effect on IL-1beta induced NF?B activation. However the anti-inflammatory effect of this drug is not completely clear.
Aims of study: To clarify the anti-inflammatory effect of glucosamine, we investigated the effects of this drug on IL-1beta -induced activation of intracellular [Ca2+], and molecules might be involved in the inflammatory responses, including CaMK-II, MAPK. We further investigated the involvement of glycogen synthase kinase 3beta (GSK-3beta) in the anti-inflammation of this drug.
Methods: SW-1353 cells were treated with IL-1beta alone or in the presence of glucosamine. Fura-2AM staining was used for cytosolic [Ca2+] measurement. Phosphorylation of ERK, p38 and CaMK-II was analyzed by Westen blot.
Results: Glucosamine decreased cytosolic [Ca2+] and suppressed IL-1 beta -induced Phosphorylation of ERK, p38 and CaMK-II. In the presence of LiCl, an inhibitor of GSK-3 beta, both suppressive effects on cytosolic [Ca2+] and kinases by glucosamine were relieved. Addition of ionomycin, which raised cytosolic [Ca2+], relieved the suppressive effect of glucosamine on IL-1beta -induced phosphrylation of ERK and CaMK-II, but enhanced the suppressive effect on IL-1beta -induced Phosphorylation of p38.
Conclusions: The anti-infammatory effects of glucosamine in vivo may be related to its suppressive effects on NF?B activation, and in addition, on cytosolic [Ca2+] and suppressed IL-1beta -induced Phosphorylation of ERK, p38 and CaMK-II. Glucosamine may elicit general suppression on signal transduction pathways, but not a specific single pathway. These suppressive effects may be mediated by GSK-3 beta. Activation of this kinase may result in decreased cytosolic [Ca2+], which in turn suppresses IL-1beta -induced Phosphorylation of ERK and CaMK-II. And via unknown mechanism it suppresses IL-1beta -induced Phosphorylation of p38. |
