白藜芦醇减轻大鼠胰岛β细胞瘤细胞低氧损伤

RESVERATROL ALLEVIATES HYPOXIC INJURY OF RAT ISLET B CELL TUMOR CELLS

目的研究白藜芦醇减轻大鼠胰岛β细胞瘤细胞(INS-1)低氧损伤的作用。方法将INS-1细胞分为三组,即常氧对照组(control)、低氧组(hypoxia)、低氧及白藜芦醇组(hypoxia+RSV);采用正常培养方法培养常氧对照组,用CCK8检测不同低氧时间(6、12、24、48 h)的细胞增殖情况,选择合适的时间条件构建低氧模型并用于下一步研究;再以不同浓度的白藜芦醇(8、10、12、15μmol/L)提前干预12 h,并进行前期设定的低氧条件处理,用CCK8检测合适的浓度条件,构建白藜芦醇干预模型并用于下一步研究。以组织活性氧试剂盒及线粒体超氧化物试剂盒检测氧化应激水平;线粒体膜电位及ATP水平检测线粒体功能;流式细胞仪分析检测凋亡;胰岛素定量检测试剂盒检测胰岛素分泌量;实时定量反转录检测Sirt1、Pdx1、Glut2的表达水平;蛋白免疫印迹检测相应蛋白水平。结果与常氧对照组相比,低氧组细胞增殖降低(P<0.05),细胞凋亡率增加(P<0.01),线粒体功能降低(P<0.05),胰岛素分泌减少(P<0.05),Sirt1、Pdx1、Glut2基因及其相应蛋白表达水平降低(P<0.05)。而白藜芦醇干预可以明显减轻低氧应激损伤(P<0.05),促进增殖(P<0.05),逆转凋亡(P<0.01),改善线粒体功能(P<0.05),促进胰岛素分泌(P<0.05),促进Sirt1、Pdx1、Glut2基因及其相应蛋白表达水平(P<0.05)。结论白藜芦醇可以保护低氧造成的INS-1细胞氧化应激损伤,改善线粒体功能,抗凋亡,并促进胰岛素功能性基因表达,促进胰岛素分泌。

Objective To study the effects of resveratrol on hypoxia injury in rat islet beta tumor cells(INS-1).Methods The INS-1 cells were divided into three groups:control group,hypoxia group,hypoxia and resveratrol group(hypoxia+RSV).The cell proliferation at different hypoxia times(6,12,24,48 h)was detected by CCK8 kit.Resveratrol at the concentrations of 8,10,12,15μmol/L were used for intervention for 12 hours.Oxidative stress levels were detected by reactive oxygen species kit and mitochondrial superoxide kit.Mitochondrial membrane potential and ATP levels were detected for mitochondrial function,and apoptosis was detected by flow cytometry.Insulin quantitative assay kit was used to detect insulin secretion,and quantitative real-time reverse transcription was used to detect the mRNA levels of Sirt1,Pdx1 and Glut2genes.The corresponding protein levels were detected by Western blot.Results Compared with the normal oxygen control group,cell proliferation was decreased(P<0.05),apoptosis rate increased(P<0.01),mitochondrial function decreased(P<0.05),and insulin secretion decreased(P<0.05)in the hypoxia group.The expression levels of Sirt1,Pdx1,Glut2 genes and their corresponding proteins were also decreased(P<0.05).The intervention by resveratrol could significantly reduce hypoxia stress induced damage(P<0.05),promote proliferation(P<0.05),decrease apoptosis(P<0.01),improve mitochondrial function(P<0.05),and promote insulin secretion(P<0.05)in INS-1 cells exposed to hypoxia.The expression levels of Sirt1,Pdx1,Glut2 genes and their corresponding proteins were also improved in response to resveratrol intervention(P<0.05).Conclusion Resveratrol can protect INS-1 cells against oxidative stress caused by hypoxia,improve mitochondrial function,inhibit apoptosis,promote insulin function gene expression and increase insulin secretion.