高尿酸血症细胞模型的构建及其在降尿酸肽筛选中的应用

Establishment of the Cell Model of Hyperuricemia and Its Application in Screening Food-derived Anti-hyperuricemic Peptides

高尿酸血症是由于嘌呤代谢紊乱使尿酸生成增加和(或)排泄减少所致的代谢性疾病,是引起痛风的重要生化基础。本文建立了一种高尿酸血症肝细胞模型,并研究了乳清蛋白肽的降尿酸活性。具体步骤为:将肝细胞铺板,分为空白对照组和模型组,48 h后,模型组给予一定浓度的腺苷溶液,空白对照组给予基础培养基,36 h后,加入黄嘌呤氧化酶继续孵育12 h,收集细胞上清液,高效液相测定尿酸含量。结果显示模型组尿酸含量显著高于空白对照组,且腺苷的最佳诱导浓度、细胞铺板密度及诱导时间分别为2.5 mmol/L、10~5个/m L、36 h。采用最佳建模条件,将非布索坦和乳清蛋白肽用于该模型,结果显示相对于模型组,非布索坦组尿酸含量显著降低,并呈一定的浓度剂量效应,该结果进一步表明高尿酸血症模型构建成功。采用乳清蛋白酶解液孵育肝细胞后,当乳清蛋白肽达到一定浓度时,相对于模型组其尿酸生成量显著降低,表明乳清蛋白酶解物具有一定的降尿酸活性。

Hyperuricemia is a metabolic disease caused by purine metabolic disorders that lead to the overproduction and（or） reduced excretion of uric acid, and is an important biochemical basis for gout. In the present study, a cell model of hyperuricemia was established and the anti-hyperuricemic activity of whey protein-derived peptides was studied. Thehepatic（LO2） cells were seeded into a 24-well plate and divided into two groups–normal control and model groups. After 48 h of incubation, the model group was incubated with adenosine-containing medium, while the control group was incubated with fresh basal medium. After further incubation for 36 h, xanthine oxidase was added and the incubation was continued for another 12 h. Finally, the cell culture supernatants were collected and analyzed by high performance liquid chromatography（HPLC） assays. The results demonstrated that the uric acid level of model group was significantly higher than the normal control group, and the optimal adenosine concentration for incubation, cell plating density, and optimal incubation time were 2.5 mmol/L, 10~5cell/m L, and 36 h, respectively. Under the best conditions for model construction, febuxostat and whey protein hydrolysate were used to evaluate the anti-hyperuricemic activity of the model, and the results showed that the uric acid content of febuxostat group was markedly decreased in a dose-dependent manner, further suggesting that the hyperuricemia model was successfully established. After hepatic cells were incubated with whey protein hydrolysate, when whey protein-derived proteins reached a certain concentration, the level of uric acid decreased significantly compared with that of model group, indicating that whey protein hydrolysates exhibited anti-hyperuricemic activity.