三聚氰胺致NRK-52e细胞氧化应激与p38MAPK通路介导细胞凋亡的研究

【作者】 郭成志； 袁莉芸； 李荣芳； 邬静； 文利新； 袁慧；

【机构】 湖南农业大学动物医学院；

【摘要】 通过研究三聚氰胺导致大鼠。肾小管上皮细胞氧化损伤的程度和细胞凋亡及Trolox对肾小管上皮细胞的保护作用探讨其细胞的凋亡通路;将三聚氰胺直接溶解于DMEM培养基中,分别稀释至5mg/ml、4mg/ml、3mg/ml、2mg/ml、1mg//ml、0mg/ml,通过MTT实验确定三聚氰胺对该细胞的半数抑制浓度IC₅₀。根据三聚氰胺对该细胞的IC₅₀选择三个剂量4mg/ml、2mg/ml、1mg/ml作为实验组,并每组都设立向对应的Trolox保护组,其中Trolox的终浓度为5mM。用三聚氰胺和Trolox于培养基中混匀作用于细胞8h后,收集细胞培养液并将细胞裂解收集细胞裂解液。进行相关指标的检测和研究;结果表明,三聚氰胺可以降低抗氧化酶的活性,加速细胞的脂质过氧化程度,破坏了细胞膜的完整性。同时,三聚氰胺还可以促进细胞产生过多的活性氧,这也可能是引起细胞损伤的重要因素之一,同时在加入保护剂Trolox后,三聚氰胺造成的氧化损伤程度有所降低,并且trolox可以通过提高抗氧化酶的活性、清除细胞内氧自由基来保护细胞,使细胞凋亡发生的几率降低;此外,三聚氰胺致NRK-52e细胞凋亡可能与p38MAPK通路有关,阻断p38MAPK可以阻断三聚氰胺所致的细胞凋亡现象。更多还原

【Abstract】 To study the oxidative damage of renal tubular epithelial cells（NRK-52e cell line） induced by melamine and the cytoprotective effect of Trolox,a water-soluble antioxidant,to NRK-52e cells and the role of p38MAPK pathway on apoptosis induced by melamine.Melamine was directly dissolved in DMEM medium and was diluted to 40mM,30mM,24mM,16mM,8mM,0mM.Half inhibition concentration(IC₅₀) of melamine was test by MTT assay.According to the IC₅₀,three doses were selected as experimental groups,as follows 24mM,16mM,8mM. At the same time,each group was set up to correspond to the protection group of Trolox,which the final concentration was 1 mM.In the experiment,trolox and melamine in protection groups were mixed to add to the medium.After 8 hours,the cell culture medium was collected for LDH activity ? total SOD activity（NBT assay）, GSH-Px activity,UV colorimetric assay and MDA content was determinated.The results showed that melamine decrease the activity of antioxidant enzymes to accelerate cell lipid peroxidation,destroy the cell membrane integrity.Meanwhile,the melamine can also promote the cells to produce excessive reactive oxygen species,which may also cause cell damage and induce cell apoptosis.In addition,the damage melamine-induced decrease by adding membrane protective angent trolox.It means that trolox can enhance the activity of antioxidant enzymes to eliminate oxygen free radicals to protect cells from cell apoptosis.In additional,this existed a relationship between the melamine-induced apoptosis of NRK-52e cells and the p38MAPK pathway.And the inhibition of p38MAPK pathway could stop the apoptosis induced by melamine.更多还原