抑制活性氧可改善AGEs诱导的3T3-L1细胞脂联素水平下调

Reactive oxygen species inhibition improves the down-regulation of adiponectin in 3T3-L1 adipocytes induced by AGEs

目的 研究体外模拟细胞内活性氧（reactive oxygen species,ROS）抑制对晚期糖基化终末产物（advanced glycation end products,AGEs）引起的小鼠3T3-L1脂肪细胞脂联素（adiponectin,ADPN）表达水平的影响.方法 制备AGEs,体外培养3T3-L1前脂肪细胞,并将其诱导分化为成熟脂肪细胞,油红O染色鉴定脂肪细胞分化程度和脂质积聚情况.予以AGEs刺激脂肪细胞不同的时间,以活性氧（ROS）捕获剂双氢-乙酰乙酸二氯荧光黄（DCFH-DA）孵育细胞,通过流式细胞仪检测细胞内二氯荧光黄（DCF）的荧光强度而测得细胞内ROS水平,采用实时荧光定量PCR与酶联免疫吸附法检测脂联素的mRNA和蛋白水平;予以抗氧化剂N-乙酰-半胱氨酸（N-Acetylcysteine,NAC）预处理后,再次检测脂联素的mRNA和蛋白水平.结果 AGEs处理3T3-L1脂肪细胞后,细胞内ROS水平明显升高,脂联素mRNA及蛋白水平均显著下调;将ROS抑制后,脂联素的mRNA和蛋白水平均有不同程度回升.结论 AGEs诱导3T3-L1脂肪细胞ROS生成增高,从而抑制了脂联素的表达,参与了胰岛素抵抗的病理生理过程.

Objective To investigate the effects of reactive oxygen species （ROS） inhibition on the down-regulation of adiponectin （ADPN） in mouse 3T3-L1 adipocytes by advanced glycation end-products （AGEs）.Methods AGEs were prepared for incubating with cell.3T3-L1 preadipocytes were cultured in vitro and differentiated into mature adipocytes.Cell differentiation and lipid accumulation were determined by oil red O staining.After being intervened with AGEs,2＇,7＇-dichlorofluorescein diacetate （DCFH-DA） was used as a reactive oxygen species （ROS） capture agent and the fluorescent intensity of 2＇,7 ＇-dichlorofluorescein （DCF） was detected by flow cytometry.Adiponectin expression under AGEs in 3T3-L1 adipocytes pretreated with N-acetyl-L-cysteine（NAC） or not was detected by real-time fluorescent PCR and ELISA.Results The level of ROS in 3T3-L1 adipocytes treated with AGEs was increased.mRNA and protein of ADPN were down-regulated.After inhibition with ROS,mRNA and protein expressions of ADPN injured by AGEs were ameliorated.Conclusion Exposure of 3T3-L1 adipocytes to AGEs induces oxidative stress in vitro,which decreases the expression of ADPN,and causes functional impairment of adipose cells and insulin resistance.