急性痛風關節炎自我緩解分子機轉之研究

Abstract

摘要 痛風是因單鈉尿酸鹽(monosodium urate, MSU)結晶體沉澱於關節內或關節周邊的柔軟組織所引起的自體發炎性及自我緩解性的常見疾病。最近的研究報告指出由於飲食及生活習性的改變、醫療照護的進步、及普遍性的長壽等諸多因素的影響，痛風成為相當常見的疾病之一。急性痛風關節炎(acute gouty arthritis, acute GA)與其它關節炎疾病不同，會有炎症自我緩解的現象，然而其分子調控機轉至今仍不清楚。本研究即對此進行探討，並從細胞內、外的調控機制來進行解析。GA的標準診斷方法為抽取患者關節液，檢驗是否有被嗜中性白血球吞噬的MSU結晶。有鑑於此，細胞外的分析則以GA患者的關節液為檢體，藉由ELISA技術，量測關節液中的transforming growth factor β1 (TGF-β1)、interleukin 1 (IL-1) receptor antagonist (IL-1ra)、IL-10、soluble tumor necrosis factor (TNF) receptor I (sTNFR-I)、sTNFR-II 等抗發炎因子的濃度。結果顯示，急性痛風關節炎病患的關節液中抗發炎因子TGF-β1、IL-1ra、IL-10、sTNFR-I/II 等濃度顯著較OA(退化性關節炎)患者為高。 細胞內調控機轉的分析則是利用小鼠巨噬細胞株(RAW264.7)為研究材料，添加MSU結晶體刺激發炎反應後，利用reverse transcription (RT)-PCR 技術，偵測細胞激素信號抑制因子cytokine inducible SH2-containing protein (CIS)、及各種suppressors of cytokine signaling (SOCS) 1-7 mRNA表現量是否受MSU結晶體調控。結果顯示MSU結晶體可顯著增加CIS和SOCS3的表現量。 為了進一步確認CIS和SOCS3於GA之病態生理學角色，本研究以急性痛風關節炎患者的: (1)滑膜組織(synovial tissue) (2)關節液中分離出的白血球(synovial fluid mononuclear cells, SFMCs) 及 (3)受MSU結晶剌激過的人類初代巨噬細胞為材料。利用免疫組織化學分析 (immunohistochemistry)、定量PCR (quantitative PCR) 及西方點墨法 (immunoblotting)，檢測CIS以及SOCS3的表現情形。結果顯示上述3種檢體中皆可見CIS與SOCS3的表現量顯著增加。 為了更深入瞭解CIS在緩解MSU晶體誘發的急性炎症過程中扮演的真正分子機轉為何，本研究首先建立CIS穩定表現之RAW264.7細胞株，在CIS過度表現的細胞中，MSU晶體所誘發的IL-1β及TNF-α之分泌現象會被抑制。此外CIS的過度表現會促進signal transducer and activator of transcription 3 (STAT3) 和TGF-β1 基因啟動區 (gene promoter region)鍵結，產生更多的TGF-β1。相反地，在CIS knockdown的細胞中，則會增強IL-1β及TNF-α的表現量並削減TGF-β1之生成。 本研究首先發現細胞外抗發炎因子TGF-β1、IL-1ra、IL-10、sTNFR-I/II的濃度提昇及細胞內CIS、SOCS3之活化，有助於急性痛風關節炎之自我緩解。透過這個研究成果，不僅有助釐清急性痛風關節炎的自我緩解機轉，更開創了新的思考及研究方向，亦為痛風疾病預防暨藥物開發提供了新契機。

ABSTRACT

Acute gouty arthritis (GA) is an autoinflammatory disease that is caused by the deposition of monosodium urate (MSU) crystals in articular joints and periarticular tissues. The inflammatory response in acute GA is typically self-limiting. Recent studies have indicated that the rising prevalence of GA is primarily due to factors such as changes in dietary habits and lifestyle, improved medical care and increased longevity. To date, the molecular basis that underlies spontaneous resolution of acute gouty inflammation remains poorly understood. In this study, we tested the hypothesis that extracellular anti-inflammatory factors and intracellular negative regulators of cytokine signaling play a role in the spontaneous resolution of acute GA. The gold standard for the diagnosis of GA is to confirm the presence of MSU crystals in the synovial fluid, and the measurement of cytokines in the synovial fluid serves as a tool for studying the inflammatory process in vivo. Using ELISA, we assessed the levels of the primary anti-inflammatory cytokines in the synovial fluid of patients with acute GA in relation to patients with osteoarthritis (OA). The synovial fluid levels of transforming growth factor β1 (TGF-β1), interleukin 1 (IL-1) receptor antagonist (IL-1ra), IL-10 and soluble tumor necrosis factor (TNF) receptors I (sTNFR-I) and II (sTNFR-II) were all significantly elevated in patients with acute GA relative to the levels in patients with OA. Next, we determined the mRNA levels of intracellular cytokine-inducible SH2 protein (CIS) and suppressors of cytokine signaling (SOCS) 1-7 in MSU crystal-stimulated RAW264.7 mouse macrophages by reverse transcription (RT)-PCR. In this context, the levels of CIS and SOCS3 mRNA were increased in response to stimulation with MSU crystals. Using immunohistochemical analyses, quantitative PCR and immunoblotting, we further confirmed the expression levels of CIS and SOCS3 in both the synovial tissue and synovial fluid mononuclear cells (SFMCs) from patients with GA, and MSU crystal-stimulated peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages from healthy donors. Finally, we used CIS overexpression and small interfering RNA-mediated knockdown in RAW264.7 cells to investigate the role of CIS in resolving MSU crystal-induced acute inflammation. CIS overexpression in RAW264.7 cells attenuated the MSU crystal-induced IL-1β and TNF-α production but enhanced TGF-β1 production via increased binding of signal transducers and activators of transcription 3 (STAT3) to the TGF-β1 promoter. Conversely, CIS knockdown reversed the effect of CIS overexpression, resulting in enhanced IL-1β and TNF-α production but reduced TGFβ1 production in MSU crystal-stimulated RAW264.7 cells. In conclusion, we demonstrate for the first time that increased production of TGF-β1, IL-1ra, IL-10 and sTNFR-I/II and upregulation of intracellular CIS and SOCS3 expression are associated with the spontaneous resolution of acute GA. These results both advance our understanding of the molecular basis of gouty inflammation resolution and have the potential to provide insights into new therapeutic strategies that can be aimed at triggering the resolution of gouty inflammation, thus preventing the clinical progression of the disease.