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目的:研究西红花酸对晚期糖基化终产物(AGE)诱导牛内皮细胞(BEC)中晚期糖基化终产物受体(RAGE)mRNA表达的抑制作用,并探讨其可能机制。方法:不同浓度的西红花酸(1、0.1、0.01μmol/L)预孵BEC细胞12h后,用AGE(100mg/L)刺激细胞12h,RT-PCR法测定RAGEmRNA的表达水平;ELISA法测定细胞间黏附分子-1(ICAM-1)的表达;试剂盒分别检测胞外超氧阴离子和硫代巴比妥酸反应产物(TBARS)浓度;同时,还用2,7-二氯荧光素(DCFH)测定了胞内H2O2的浓度,并用罗丹明123(Rh123)荧光法及MTT法分别检测细胞线粒体膜电位(MMP)水平和其琥珀酸脱氢酶(MSDH)的活性。结果:与AGE模型组相比,西红花酸能显著抑制RAGE mRNA的表达(P<0.05),降低胞外超氧阴离子和TBARS(P<0.01或P<0.05)及胞内H2O2水平;结果还显示,西红花酸能提高细胞MMP水平和MSDH活性。对ICAM-1蛋白表达也有抑制作用,且呈时间和剂量依赖性。结论:西红花酸可能通过清除AGE与RAGE结合产生的活性氧(ROS)来抑制RAGE mRNA的高表达。提示西红花酸对糖尿病血管病变有潜在的治疗价值。
Objective: To investigate the inhibitory effect of crocetin on the expression of advanced glycation endproduct receptor (RAGE) mRNA in bovine endothelial cells (BEC) induced by advanced glycation end products (AGE), and to explore its possible mechanism. METHODS: After preincubation of BEC cells with different concentrations of crocetin (1, 0.1, and 0.01 μmol/L) for 12 h, cells were stimulated with AGE (100 mg/L) for 12 h. The expression level of RAGE mRNA was measured by RT-PCR and determined by ELISA. Expression of intercellular adhesion molecule-1 (ICAM-1); kits were used to detect extracellular superoxide anion and thiobarbituric acid reaction product (TBARS) concentrations; meanwhile, 2,7-dichlorofluorescein was also used ( The concentration of intracellular H2O2 was measured by DCFH, and the mitochondrial membrane potential (MMP) level and the activity of succinate dehydrogenase (MSDH) were determined by Rh123 fluorescence assay and MTT assay, respectively. RESULTS: Compared with the AGE model group, crocetin significantly inhibited the expression of RAGE mRNA (P<0.05), decreased extracellular superoxide anion and TBARS (P<0.01 or P<0.05) and intracellular H2O2 levels; It has also been shown that crocetin can increase cellular MMP levels and MSDH activity. It also inhibited the expression of ICAM-1 protein in a time and dose-dependent manner. Conclusion: Crocetin may inhibit the high expression of RAGE mRNA by removing reactive oxygen species (ROS) produced by the combination of AGE and RAGE. It suggests that crocetin has potential therapeutic value for diabetic vascular disease.