白藜芦醇对波动性高糖培养的大鼠肾小球系膜细胞脂联素受体表达的影响

摘要

目的 探讨白藜芦醇( resveratrol)对波动性高糖培养的大鼠肾小球系膜细胞脂联素受体(AdipoR1和AdipoR2)表达的影响及机制.方法 采用脂质体介导的方法将叉头转录因子O1(FoxO1)短发夹样RNA( FoxO1 shRNA)质粒载体稳定转染大鼠肾小球系膜细胞;波动性高糖(5.6 mmol/L或30mmol/L,每隔8h轮换)培养转染后的大鼠肾小球系膜细胞,并用白黎芦醇(20μmol/L)处理.以葡萄糖浓度( 5.6 mmol/L)培养的大鼠肾小球系膜细胞作为正常对照组(NG),将波动性高糖培养的大鼠肾小球系膜细胞分为5组:波动性高糖组(IHG)、IHG+白黎芦醇组、IHG+FoxO1 shRNA转染组、IHG+白黎芦醇+FoxOlshRNA转染组、IHG+转染阴性对照组(shNC).培养72 h后,用荧光酶标仪检测各组大鼠肾小球系膜细胞内活性氧簇(ROS)水平；RT-PCR检测去乙酰化酶(Sirt1)、FoxO1、AdipoR1、AdipoR2 mRNA的表达,Western印迹检测FoxO1、磷酸化FoxO1( p-FoxO1)、AdipoR1和AdipoR2蛋白的表达.结果 (1)与NG组相比,IHG组Sirtl mRNA表达下降,p-FoxO1水平升高,转录活性降低,AdipoR1表达下降,ROS水平升高(均P＜0.05)；(2)与IHG组相比,IHG+FoxO1 shRNA转染组,FoxO1表达被抑制,AdipoR1表达更低,ROS水平更高(均P＜0.05)；(3)与IHG组相比,IHG+白黎芦醇组Sirt1 mRNA表达升高,p-FoxO1水平下降,转录活性升高,AdipoR1表达升高,ROS水平下降(均P＜0.05)；(4)与IHG+白黎芦醇组相比,IHG+白黎芦醇+FoxO1shRNA转染组,FoxO1表达被抑制,AdipoR1表达降低,ROS水平升高(均P＜0.05)；(5)各组间AdipoR2表达无统计学意义(P＞0.05).结论 (1)白藜芦醇能显著上调波动性高糖诱导的大鼠肾小球系膜细胞内AdipoR1的表达.(2)FoxO1在白藜芦醇调节AdipoR1表达中发挥重要作用.%Objective To study the effects and mechanisms of resveratrol (Resv) on the expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in rat mesangial cells (MCs) cultured in intermittent high glucose medium.Methods The MCs cultured in normal glucose ( 5.6 mmol/L) medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs ( FoxO1 shRNA ),and then cultured in intermittent high glucose medium (5.6mmol/L or 30 mmol/L,alternately every 8 hours ) and resveratrol ( Resv 20 μmol/L).The MCs cultured in normal glucose (5.6 mmol/L) medium served as normal glucose control group (NG),and the MCs exposed to iutermittent high glucose medium were divided into five groups:intermittent high glucose group (IHG),IHG+Resv group,IHG+FoxO1 shRNA group,IHG+ Resv + FoxO1 shRNA group,IHG + Negative control group ( shNC ),each group was cultured for 72 h.The level of reactive oxygen species (ROS) was assessed by Fluorescence microplate reader.The mRNA levels of Sirt1,Foxo1,AdipoR1,and AdipoR2 were assessed by RT-RCR.The protein levels of FoxO1,phosphorylation FoxO1 ( p-FoxO1 ),AdipoR1,and AdipoR2 were assessed by Western blotting.Results ( 1 )Compared with NG,intermittent high glucose significantly decreased the mRNA expression of Sirt1,but markedly increased the level of p-FoxO1.Furthermore,the mRNA and protein expression levels of AdipoR1 were obviously decreased while the level of ROS was enhanced in IHG ( all P＜0.05 ).(2) Compared with IHG,the mRNA and protein expression levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was enhanced in group IHG+FoxO1 shRNA ( all P＜0.05 ).( 3 ) Compared with IHG,after treating with Resv,the mRNA expression level of Sirtl was increased,whereas the level of p-FoxO1 was decreased.Moreover,the mRNA and protein levels of AdipoR1 was increased while the level of ROS was lowered in group IHG+Resv (all P＜0.05 ).(4) Compared with group IHG+Resv,the mRNA and protein levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was increased in group IHG+Resv+FoxO1 shRNA (all P＜O.05).(5) In addition,the mRNA and protein expression level of AdipoR2 showed no significant difference among these groups ( P＞0.05 ).Conclusion ( 1 ) Resveratrol significantly increases the expression of AdipoR1 in MCs induced by intermittent high glucose.( 2 ) FoxO1 plays an important role in regulating the expression of AdipoR1 by resveratrol.