转录共激活子p300及表观修饰在高糖致人系膜细胞代谢记忆中的汇聚作用

Convergence role of transcriptional coactivator p300 and apparent modification on HMCs metabolic memory induced by high glucose

目的以人系膜细胞(HMCs)作为体外模拟代谢记忆的研究对象,观察转录共激活子p300及组蛋白乙酰化蛋白H3(Ac-H3)、Ac-H4的表达规律,并探讨p300在其中的潜在汇集点作用。方法将培养的HMCs按以下分组处理:①高糖诱导代谢记忆模型,分为正糖组(NG,5.5mmol/L D-葡萄糖×2d)、渗透压组(LG,NG+20mmol/L L-葡萄糖×2d),高糖组(HG,25mmol/L D-葡萄糖×2d)、记忆组(M1、M2、M3组,25mmol/L D-葡萄糖×2d+5.5mmol/L D-葡萄糖×3、6、9d)、持续正糖组(NC,5.5mmol/L D-葡萄糖×9d)。②糖其化终产物记忆模型,分为正糖组(NG,5.5mmol/L D-葡萄糖×2d);正糖+AGEs组(AGEs,5.5mmol/L D-葡萄糖+250μg/ml AGEs×2d);AGEs记忆组(AGEs-M,5.5mmol/L D-葡萄糖+250μg/ml AGEs×2d+5.5mmol/L D-葡萄糖×3d);BSA组(NG+BSA,给予同浓度BSA对照)。③H2O2模拟氧化应激记忆模型,分为正糖组(NG,5.5mmol/L D-葡萄糖×30min);正糖+H2O2组(H2O2,5.5mmol/L D-葡萄糖+100μmol/L H2O2×30min);H2O2记忆组(H2O2-M,5.5mmol/L D-葡萄糖+100μmol/L H2O2×30min+5.5mmol/L D-葡萄糖×3d);正糖对照组(NG3,5.5mmol/L D-葡萄糖×3d)。④蛋白激酶C(PKC)β2激活记忆模型,分为正糖组(NG,5.5mmol/L D-葡萄糖×2d);高糖组(HG,25mmol/L D-葡萄糖×2d);记忆组(M,25mmol/L D-葡萄糖×2d+5.5mmol/L D-葡萄糖×3d);空载体记忆组(HN,25mmol/L D-葡萄糖+Ad5-null×2d+5.5mmol/L D-葡萄糖×3d);PKCβ2激活记忆组(PO,25mmol/L D-葡萄糖+Ad5-PKCβ2×2d+5.5mmol/L D-葡萄糖×3d);PKCβ2抑制剂记忆组(PI,25mmol/L D-葡萄糖×2d+10μmol/L CGP53353+5.5mmol/L D-葡萄糖×3d)。采用二氢二氯荧光素(DCFDA)及酶标仪检测细胞内活性氧(ROS)的表达。Western blotting检测各组细胞p300、Ac-H3和Ac-H4及PKCβ2蛋白的表达水平。结果 HG组p300、Ac-H3和Ac-H4蛋白表达增加,分别较NG组增加了1.15倍、0.93倍和0.87倍(P<0.05),同时伴有PKCβ2蛋白及胞内ROS水平上调;M1、M2、M3组p300、Ac-H3、Ac-H4、PKCβ2蛋白水平及ROS表达水平与NG组比较仍显著升高,即使M3组亦较NG组分别增加75%、49%、47%、98%和48%(P<0.05)。AGEs组p300、Ac-H3和Ac-H4蛋白表达上调,较对照组分别升高了1.73倍、1.08倍和1.05倍(P<0.05),AGE-M组各蛋白较对照组分别增加了1.47倍、0.95倍和1.03倍(P<0.05)。H2O2组p300、Ac-H3和Ac-H4蛋白表达均升高,较对照组分别升高了1.03倍、0.85倍和0.79倍(P<0.05),而H2O2-M组各蛋白表达较对照组的差异无统计学意义。与M组比较,PO组p300、Ac-H3和Ac-H4蛋白表达进一步上调,分别为M组的1.25倍、1.06倍和1.10倍(P<0.05),选择性PKCβ2抑制剂CGP53353可以显著降低上述蛋白表达。结论 HMCs存在转录共激活子p300及表观修饰持续活化的记忆效应,p300可能系高糖致生化代谢及表观遗传记忆刺激的汇聚点。

Objective To investigate the protein expression of transcriptional coactivator p300, acetylated histone H3 （Ac-H3） and Ac-H4 in human renal mesangial cell （HMCs） as imitative “metabolic memory” in vitro, and explore the potential roleof convergence point of p300. Methods The HMCs were divided into the following groups： ① High glucose metabolic memory model： normal glucose group （NG, S.Smmol/L D-glucose ×2d）, high glucose group （HG, 25mmol/L D-glucose ×2d）, memory groups （M1, M2, M3, 25mmol/L D-glucose ×2days ＋ 5.5mmol/L D-glucose × 3d, 6d or 9d）, persisting normal glucose group （NG, 5.5mmol/L D-glucose ×9d）. ② Advanced glycation end products memory model： normal glucose group （NG, 5.5mmo1/ L D-glucose ×2d）, NG＋AGEs group （AGEs, 5.5mmol/L D-glucose＋250μg/ml AGEs ×2d）; AGEs memory group （AGEs-M, 5.Smmol/L D-glucose ＋ 250μg/ml AGEs ×2d ＋ S.Smmol/L D-glucose ×3d）; BSA control group （NG＋BSA, S.Smmol/L D-glucose ＋ 250μg/ml BSA×2d）. ③H2O2 was used to simulate oxidative stress memory model： normal glucose group （NG, 5.5mmol/L D-glucose ×2d）, NG＋H202 group （H202, 5.5mmol/L D-glucose ＋100μmol/L H2O2 ×30min）; H2O2 memory group [（5.5mmol/ L D-glucose ＋ 100μmol/L H2O2 × 30min） ＋ 5.5mmol/L D-glucose ×3d]; normal glucose control group （NG3, 5.5mmol/L D- glucose ×3d）. ④ Transfection with PKCβ2 memory model： normal glucose group （NG, 5.5mmol/L D-glucose×2d）; high glucose group （HG, 25mmol/L D-glucose×2d）; memory group （M, 25mmol/L D-glucose ×2d ＋ 5.5mmol/L D-glucose×3d）; AdS-null memory group （HN, 25mmol/L D-glucose ＋ AdS-null ×2d ＋ 5.5mmol/L D-glucose×3d）; PKCβ2 memory group （PO, 25mmol/L D-glucose ＋ AdS-PKCβ2×2d ＋ 5.5mmol/L D-glucose ×3d）; inhibitor of PKCβ2 memory group （PI, 25mmol/L D-glucose ×2d ＋ 10μmol/L CGP53353 ＋ 5.5mmol/L D-glucose×3d）. The expression ofintracellular reactive oxygen species （ROS） was detected by fluorescence microscope and fluorescence microplate reader. The expression levels of p300, Ac-H3, Ac-H4 and PKCβ2 proteins were determined by Western blotting. Results The expression levels of p300, Ac-H3 and Ac-H4 protein in HG group increased, being 2.15, 1.93 and 1.87 fold of those in group NG （P〈0.05）, accompanying with the up-regulation of PKCβ2 protein and ROS levels in HG group. The p300, Ac-H3, Ac-H4, PKCβ2 protein expression and ROS levels in M1, M2, M3 group were higher than those in NG group, and was 1.75, 1.49, 1.47, 1.98 and 1.48 fold higher in M3 group than in NG group. The protein expressions of p300, Ac-H3 and Ac-H4 in AGEs group were increased by 1.73, 1.08 and 1.08 folds, and in AGE-M group increased by 1.47, 0.95 and 1.03 folds of that in control group （P〈0.05）. The protein expression levels of p300, Ac-H3 and Ac-H4 in H2O2 group increased by 1.03, 0.85 and 0.79 folds of those in control group （P〈0.05）. However, no significantly difference in these indices was found between HEO2-M and control groups. The protein expression levels of p300, Ac-H3 and Ac-H4 in PO group increased more obviously by 1.25, 1.06 and 1.10 folds of those in M group （P〈0.05）. However, the elective PKCβ2 inhibitor CGPS33S3 could lower those indices significantly. Conclusion Persistent activation of transcriptional coactivator p300 and apparent modification may be normalized in HMCs. p300 may be the convergent point of glucose-induced metabolic “memory” stimulations.