甘草黄酮醇降尿酸及抑制氧化应激作用及其机制研究

Study on Effect and Mechanism of Licoflavonol on Reducing Uric Acid and Inhibiting Oxidative Stress

目的探究甘草黄酮醇(licoflavonol,LCF)降尿酸及抑制氧化应激作用及其机制。方法建立腺苷诱导肾小管上皮细胞NRK-52E的高尿酸细胞模型,给予1.25,5,20μmol·L^(–1)LCF,利用HPLC检测培养液中尿酸、次黄嘌呤、黄嘌呤、腺嘌呤、肌苷、腺苷含量。使用15 mg·dL^(–1)尿酸诱导NRK-52E细胞氧化应激损伤,同时给予1.5,5,15μmol·L^(–1)LCF处理24 h,试剂盒测定细胞中超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)活力及还原型谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)及过氧化氢(H2O2)的含量。通过分子对接预测LCF与黄嘌呤氧化酶(xanthine oxidase,XO)的结合活性。进一步通过微量热泳动技术(microscale thermophoresis,MST)探究LCF和XO的结合能力。结果LCF可降低腺苷诱导的细胞尿酸水平,提高尿酸诱导后细胞SOD、CAT的活力及GSH含量,并降低H2O2、MDA含量;分子对接结果显示LCF与XO之间结合能为-9.1 kcal·mol^(-1),二者主要形成氢键,对应残基为Thr-262、Glu-263;MST结果表明两者间解离常数Kd=(0.81489±0.2373)mmol·L^(–1)。结论LCF可降低腺苷诱导的细胞尿酸水平并改善高尿酸所致肾小管上皮细胞氧化应激损伤,其降尿酸作用机制可能与XO有关。

OBJECTIVE To explore the effect and mechanism of licoflavonol(LCF)on reducing uric acid and inhibiting oxidative stress.METHODS The hyperuricemia cell model of renal tubular epithelial cells NRK-52E was induced by adenosine,1.25,5,and 20μmol·L^(–1)LCF were administered,then the contents of uric acid,hypoxanthine,xanthine,adenine,inosine and adenosine in the culture medium were detected by HPLC.NRK-52E cells were treated with 15 mg·dL^(–1)uric acid to induce oxidative stress injury,1.5,5,and 15μmol·L^(–1)LCF were administrated respectively for 24 h,the activity of superoxide dismutase(SOD),catalase(CAT)and the contents of glutathione(GSH),malondialdehyde(MDA)and H2O2in the cells were determined by kits.The binding activity of LCF to xanthine oxidase(XO)was predicted by molecular docking,and further investigated by microscale thermophoresis(MST).RESULTS LCF reduced the level of uric acid induced by adenosine,increased the activity of SOD and CAT and the content of GSH,decreased the content of H2O2and MDA after uric acid induction.The molecular docking results showed that the binding energy between LCF and XO was-9.1 kcal·mol^(-1),both were mainly formed hydrogen bonds,and the corresponding residues were Thr-262 and Glu-263.MST results showed that the dissociation constant Kd=(0.81489±0.2373)mmol·L^(–1).CONCLUSION LCF can reduce the level of cellular uric acid induced by adenosine and improve the oxidative stress damage of renal tubular epithelial cells induced by high uric acid.Its mechanism of lowering uric acid may be related to XO.