大豆異黃酮genistein及daidzein對於potassium oxonate和uric acid誘發高血尿酸小鼠發炎作用及胰島素阻抗之影響

Effects of soy isoflavones genistein and daidzein on inflammation and insulin resistance in hyperuricemia mice induced by potassium oxonate and uric acid.

張雅娟(Ya-Chuan Chang)

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/CSMU201900201

摘要

已知血尿酸值提升是造成胰島素阻抗及相關共病症之獨立因子，而近期研究指出，飲食大豆可能改善血尿酸值升高及降低痛風發生風險，且有助於血糖調控，但飲食大豆中何種有效成分有助於改善血尿酸值提升相關之胰島素阻抗及其與發炎反應之關聯尚待釐清。本研究於十週齡雄性C57BL/6小鼠，以含2% potassium oxonate及3% uric acid之soy-free飼料餵食十天提升小鼠血尿酸值，並將其隨機分為7組，自第一天起分別每日灌食載劑丙二醇 (0.05 ml/隻，HUA組)、genistein高或低劑量 (160、10 mg/kg，分別為GH、GL組)、daidzein高或低劑量 (160、10 mg/kg，分別為DH、DL組)、allopurinol (5 mg/kg，allopurinol組)，並與餵食soy-free飼料且灌食丙二醇之小鼠 (ND組) 對照。於第九次灌食後飼育於代謝籠並收集二十四小時尿液備用於分析creatinine及尿酸含量，於灌食第十天當晚禁食十二小時後，各誘發組以腹腔注射 potassium oxonate (300 mg/kg) 及胃灌 hypoxanthine (500 mg/kg) 以進一步提升小鼠血尿酸濃度，ND組則處以載劑。小鼠於注射及灌食後第三十分鐘執行腹腔注射葡萄糖耐受試驗 (IPGTT) 或腹腔注射胰島素耐受試驗 (IPITT)，最後在誘發後第2.5小時以二氧化碳犧牲並收集血清及組織樣品進行分析。結果發現，此模式可顯著提升血尿酸濃度、24小時尿液尿酸總排出量，且介入genistein及daidzein均顯著降低血中尿酸濃度。此模式造成小鼠胰島素阻抗，其反映於顯著增加之IPGTT及IPITT期間第0分鐘至第30分鐘血糖值曲線下面積，及顯著增加IPGTT後之血清insulin濃度，介入genistein及daidzein可在不影響空腹血糖的情況下顯著降低IPGTT後insulin濃度。此外，genistein及daidzein逆轉高血尿酸所造成之副睪脂肪中TLR4、TLR2蛋白質表現量及IL-1β、TNF-α、MCP-1含量增加，以及逆轉副睪脂肪中adiponectin含量減少的情況，並伴隨減少副睪脂肪及腸系膜脂肪組織中巨噬細胞 (F4/80+) 浸潤程度。在免疫組織發炎反應方面，與控制組相較，此模式顯著增加脾臟TLR4、MyD88蛋白質表現量，且提升脾臟與淋巴結中IL-1β、TNF-α、MCP-1含量及caspase-1活性，介入genistein及daidzein均可改善以上發炎指數。綜合以上結果可知，誘發小鼠血尿酸值提升期間介入genistein及daidzein具有改善血尿酸濃度及減緩尿酸所造成之免疫組織及脂肪組織發炎反應活化之效果，並有助於回復脂肪組織分泌adiponectin並改善尿酸相關之胰島素阻抗現象。

關鍵字

氧嗪酸鉀 ； 高尿酸血症 ； 大豆異黃酮 ； 金雀異黃酮 ； 大豆異黃酮苷素 ； 胰島素阻抗

並列摘要

Elevated blood uric acid level is known an independent cause of insulin resistance and related comorbidities. Recent studies have pointed out that higher dietary soybean intakeis associated with lower serum uric acid and with reduce the risk of gout. Beneficial effect on blood glucose by dietary soybean has also been reported. However, whether or not soybean is able to improve hyperuricemia-related insulin resistance through its anti-inflammatory effects and or with what active compositions remain unknown. Here, male C57BL/6 mice were randomly assigned to seven groups, one of which was given normal control diet, and the others were given hyperuricemia-inducing (HUA) diet containing 2% potassium oxonate and 3% uric acid for 10 days. During the induction period with HUA diet, each group of mice that received HUA diet were gavaged with vehicle (propylene glycol, 0.05 ml), GH (genistein 160 mg/kg), GL (genistein 10 mg/kg), DH (daidzein 160 mg/kg), DL (daidzein 10 mg/kg), or allopurinol (5 mg/kg). Mice fed normal diet were gavaged vehicle (ND group). All HUA diet fed mice were then fasted overnight for 12 hours followed by injection (ip) with potassium oxonate (300 mg/kg) and gavaged hypoxanthine (500 mg/kg) to boost the level of fasting serum uric acid, the ND group was given vehicle. Intra-peritoneal glucose tolerance test (IPGTT) or intra-peritoneal insulin tolerance test (IPITT) were conducted at 30 minutes after the boosting followed by sacrifice of the mice and the collection of blood and tissue samples at 2.5 hours after the boosting. Compared with control diet, HUA diet significantly elevated the levels of 24h urinary uric acid and serum uric acid and the latter was significantly reversed by the intervention of genistein and daidzein. Insulin resistance in mice receiving HUA diet was found reflected by significantly increased under curve area of blood glucose value during the period of 0 to 30 minutes of IPGTT and IPITT, and by significantly increased serum insulin concentration at 120 min of IPGTT. Genistein and daidzein significantly reduced post-IPGTT concentration of insulin without affecting blood glucose level at the same time point. In addition, the treatment of genistein and daidzein reversed HUA-induced elevation of the levels of TLR4, TLR2, IL-1β, TNF-α, and MCP-1, and HUA-induced reduction of adiponectin content in adipose tissue. These findings were consistent with the reversal effect of genistein and daidzein on HUA-induced macrophage infiltration reflected by lowered F4/80+ expression level in the tissue. The present study also found that HUA diet increased the expression of TLR4、MyD88 of spleen and increased IL-1β, TNF-α, MCP-1 content and caspase-1 activity of spleen and lymph nodes, levels of these indices of inflammation were reduced by genistein and daidzein. In conclusion, the results of this study suggest that genistein and daidzein can ameliorate serum uric acid level in mice fed with HUA diet in accompany with reversed inflammatory condition of immune and adipose tissues. These effects in addition to restored adiponectin secretion by adipose tissue may explain the beneficial effect of genistein and daidzein on uric acid-related insulin resistance found in the presen study.

並列關鍵字

potassium oxonate ； hyperuricemia ； soy isoflavone ； genistein ； daidzein ； insulin reisistance

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