介入大豆異黃酮daidzein對於尿酸鈉結晶誘發小鼠腹腔及腳踝關節急性痛風性發炎反應之影響

The effect of the soybean isoflavone daidzein on monosodium urate crystal-induced acute gouty inflammatory response in peritoneum and ankle joint of mice.

曹悅哲(Yueh-Che Tsao)

指導教授 ： 劉承慈

中山醫學大學/健康管理學院/營養學系/碩士(2019年)

https://doi.org/10.6834/CSMU201900183

摘要

痛風（gout）是一種常見的發炎性關節疾病，且在台灣和全球發生率有增加的趨勢。目前臨床上用於預防及治療痛風的藥物皆含有許多副作用，因此專家學者仍致力於找出副作用較低或是無副作用的防治方法。近來有研究發現大豆攝取量較高的華人有較低的痛風發生風險，本研究推論此可能與大豆異黃酮抗發炎機能有關。本研究目的為以小鼠腹腔或關節注射尿酸鈉結晶（monosodium urate, MSU）誘發痛風性發炎反應，觀察大豆異黃酮daidzein對於MSU結晶誘發之痛風性發炎反應的影響。本研究第一個模式將十週齡C57BL/6小鼠隨機分為五組，分別灌食載劑丙二醇（propylene glycol, 0.05 ml/mice）、不同劑量daidzein（10、40或160 mg/kg BW；分別為DL組、DM組、DH組）及作為對照組之colchicine（1.5 mg/kg BW；Colchicine組），連續十日。最後一次灌食後，於灌食載劑組小鼠之腹腔注射（i.p.）PBS（0.5 ml/mice；Con組）或MSU（3 mg/0.5 ml PBS/mice；MSU組），其餘組別小鼠均注射（i.p.）上述同等劑量之MSU結晶，腹腔注射之小鼠於禁食12小時後犧牲。第二個模式與第一個模式灌食之載劑及daidzein劑量皆相同，唯一不同的是於連續灌食十日後，於灌食載劑組小鼠之關節注射PBS（10 μl /mice；Con組）或MSU（0.5 mg/10 μl PBS/mice；MSU組），其餘組別小鼠均注射上述同等劑量之MSU結晶，並於誘發後3.5小時犧牲。犧牲時心臟採血收集血液，腹腔注射之小鼠自腹腔注射3 ml之PBS（含25 unit/ml heparin）收集腹腔灌洗液進行分析，而關節注射之小鼠收集腳踝關節部分進行分析。結果顯示，介入daidzein各劑量均不影響小鼠生長代謝參數。而於誘發前介入daidzein呈劑量依賴性降低浸潤腹腔之嗜中性球數量及浸潤至關節的MPO表現量。在腹膜炎模式中，daidzein可以降低腹腔灌洗液中IL（interleukin）-6、G-CSF （granulocyte colony-stimulating factor）、TGF（transforming growth factor）-β和血液中IL-6、G-CSF的濃度，並於中、高劑量達統計學意義，但不影響腹腔灌洗液中IL-1β、MCP（monocyte chemoattractant protein）-1和MIP（macrophage inflammatory protein）-1α的濃度。而在關節炎模式中，daidzein可以降低血液中IL-6和G-CSF濃度，並於中、高劑量達統計學意義。此外，介入daidzein也在MSU結晶誘發浸潤至腹腔的細胞抑制嗜中性球胞外網狀陷阱（NETs）之形成，且上述daidzein之作用趨近於臨床治療痛風藥物colchicine之效果。綜合以上結果，於MSU結晶誘發前介入daidzein可以降低發炎病灶中趨化性細胞激素IL-6、G-CSF、TGF-β濃度而減少嗜中性球的分化及浸潤至發炎病灶的數量，並降低周邊血中促發炎細胞因子IL-6和G-CSF濃度以及抑制已趨化至痛風性發炎部位的嗜中性球之活性和NETs的形成來改善痛風性發炎反應，據此可建議daidzein未來在預防痛風性發炎反應之應用。

關鍵字

大豆異黃酮 ； daidzein ； 尿酸鈉結晶 ； 痛風性發炎 ； 嗜中性球 ； NETs

並列摘要

Gout is a common inflammatory joint disease with an increasing incidence in Taiwan and globally. Current medications for gout attack may sometime have unwanted side effects. Consequently, researches are still working to find novel remedy with low or even no side effects. Recently, studies have found that Chinese with higher intake of soybeans have a lower risk of gout. We suggested that this may be related to the anti-inflammatory function of isoflavones. The purpose of this study was to investigate the effect of isoflavone daidzein on the gouty inflammatory response induced by monosodium urate (MSU) crystals in mice intra-articularly or -peritoneally. Ten-week-old C57BL/6 mice were divided into five groups, gavaged once a day with vehicle (propylene glycol, 0.05 ml/mice), different doses of daidzein (10, 40, and 160 mg/kg BW; DL group, DM group, and DH group, respectively) and colchicine (1.5 mg/kg BW; Colchicine group) for 10 days. For MSU-induced peritonitis model, after the last gavage, mice were injected intraperitoneally PBS (0.5 ml/mice; Con group) or MSU (3 mg/0.5 ml PBS/mice; MSU group), and the other groups of mice were given the same dose of MSU crystals as the MSU group. All of the mice were sacrificed after 12 hours of fasting followed by the collection of blood samples and the preparation of peritoneal lavage fluid with 3 ml of PBS (containing 25 unit/ml heparin) for analysis. For MSU-induced arthritis model, after the last gavage, mice were injected intraarticularly at ankle joint with PBS (10 μl /mice; Con group) or MSU (0.5 mg/10 μl PBS/mice; MSU group), the other groups of mice were injected with the same dose of MSU crystals as the MSU group, and sacrificed 3.5 hours after induction. Subsequently, blood and ankle joint samples were collected for analysis. The results showed that the intervention of daidzein did not affect the growth and metabolism of mice. The prophylactic treatment with daidzein dose-dependently significantly reduced the number of neutrophils infiltrating into the abdominal cavity and the MPO expression in the joint. In the peritonitis model, daidzein reduced IL (interleukin)-6, G-CSF (granulocyte colony-stimulating factor), TGF (transforming growth factor)-β levels in the peritoneal lavage fluid, and IL-6 and G-CSF levels in the plasma with statistical significantly at medium and high doses. In arthritis model, daidzein reduced blood concentrations of IL-6 and G-CSF with statistical significantly at medium and high doses. In addition, daidzein also inhibits MSU crystal-induced neutrophils to form extracellular traps (NETs). Furthermore, the above mentioned effects of daidzein approache those of colchicine. In summary, the present study showed prophylactic potency of daidzein on gouty inflammation evidenced by reduced levels of several inflammatory biomarkers in blood and/or inflammatory lesions as well as reduced infiltration level and NETs formation of neutrophils in the inflammatory lesions. Thus, the findings of the present study suggest future use of daidzein for prevention of gouty inflammatory response.

並列關鍵字

isoflavones ； daidzein ； MSU crystal ； gout ； neutrophils ； NETs

參考文獻

Abderrazak, A., Syrovets, T., Couchie, D., El Hadri, K., Friguet, B., Simmet, T., and Rouis, M.（2015）. NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biol 4, 296-307.

Alexander, J.S., Zhu, Y., Elrod, J.W., Alexander, B., Coe, L., Kalogeris, T.J., and Fuseler, J.（2001）. Reciprocal regulation of endothelial substrate adhesion and barrier function. Microcirculation（New York, NY : 1994）8, 389-401.

Amaral, F.A., Costa, V.V., Tavares, L.D., Sachs, D., Coelho, F.M., Fagundes, C.T., Soriani, F.M., Silveira, T.N., Cunha, L.D., Zamboni, D.S., et al.（2012）. NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B（4）in a murine model of gout. Arthritis Rheum 64, 474-484.

Arnaout, M.A.（2016）. Biology and structure of leukocyte β（2）integrins and their role in inflammation. F1000Research 5.

Atkinson, C., Frankenfeld, C.L., and Lampe, J.W.（2005）. Gut bacterial metabolism of the soy isoflavone daidzein: exploring the relevance to human health. Experimental Biology and Medicine（Maywood, NJ）230, 155-170.