高糖、高脂对2型糖尿病单个核细胞内信号蛋白RasGRP4表达的影响

Effects of high glucose and high lipid on the expression of signaling protein RasGRP4 in mononuclear cells of type 2 diabetes mellitus

目的探讨高糖、高脂对2型糖尿病(T2DM)患者外周血单个核细胞(PBMCs)中Ras鸟苷酸释放蛋白4(RasGRP4)表达水平的影响。方法纳入2016年8月至2017年2月于天津医科大学朱宪彝纪念医院住院的T2DM患者120例(T2DM组)和年龄、性别相匹配的健康志愿者50例(NC组),采用实时荧光定量聚合酶链式反应(RT-qPCR)检测受试者PBMCs中RasGRP4及其变异体mRNA的表达情况。采用双变量相关分析和多元线性回归分析糖尿病病程及主要临床指标与RasGRP4及其变异体mRNA表达水平之间的关系。采用不同浓度葡萄糖(低糖:5.5 mmol/L,高糖:12.5 mmol/L和25 mmol/L)、不同浓度的棕榈酸(低脂:0.2 mmol/L,高脂:0.5 mmol/L)及高糖高脂(25 mmol/L葡萄糖+0.5 mmol/L棕榈酸)分别处理人单核细胞系(THP-1)细胞6、12和24 h,采用RT-qPCR技术检测RasGRP4及其变异体mRNA的水平,采用蛋白质免疫印迹技术(Western blot)检测RasGRP4蛋白表达情况。结果与NC组相比,T2DM组PBMCs中RasGRP4 mRNA及其变异体a、b、c、d、f和g的mRNA水平均升高(t=4.308、5.432、5.654、5.931、4.018、3.958、5.073,均P<0.05),而变异体e的mRNA水平下降(t=-3.821,P<0.05)。双变量相关分析显示T2DM组RasGRP4 mRNA水平与病程、血清甘油三酯(TG)、超敏C反应蛋白(hs-CRP)、糖化血红蛋白A1c(HbA1c)呈正相关(r=0.973、0.237、0.472、0.245,均P<0.05);多元线性回归分析显示T2DM病程和HbA1c是主要影响因素。高糖、高脂可促进THP-1细胞表达RasGRP4,且高糖、高脂同时干预对RasGRP4表达的影响具有明显的协同促进作用。结论T2DM患者PBMCs内信号蛋白RasGRP4高表达且出现变异体,慢性高血糖、高血脂可能是激活RasGRP4及其变异体产生的重要因素。

Objective To explore the effects of high glucose and high lipid on the expression of Ras guanine nucleotide-releasing protein-4(RasGRP4)in peripheral blood mononuclear cells(PBMCs)of patients with type 2 diabetes mellitus(T2DM).Methods From August 2016 to February 2017,a total of 120 T2DM patients(T2DM group)who were hospitalized in the Metabolic Disease Hospital of Tianjin Medical University and 50 healthy volunteers(NC group)were included.The mRNA expression of RasGRP4 and its variants of PBMCs were determined by quantitative real-time PCR(RT-qPCR).Bivariate correlation analysis and multivariate linear regression analysis were performed to investigate the relationship between the course of disease and primary clinical indicators and the mRNA expression levels of RasGRP4 and its variants.THP-1 cells were treated with different concentrations of glucose(low glucose:5.5 mmol/L,high glucose:12.5 mmol/L and 25 mmol/L),different concentrations of palmitic acid(low lipid:0.2 mmol/L,high lipid:0.5 mmol/L)and high glucose with high lipid(25 mmol/L glucose+0.5 mmol/L palmitic acid)for 6 h,12 h and 24 h,respectively.RT-qPCR was used to detect the mRNA levels of RasGRP4 and its variants.Western Blot was used to detect the protein expression level of RasGRP4.Results Compared with NC group,the mRNA levels of RasGRP4 and its variants a,b,c,d,f and g in PBMC of T2DM group were significantly increased(t=4.308,5.432,5.654,5.931,4.018,3.958,5.073,all P<0.05),while the mRNA level of variant e was decreased(t=-3.821,P<0.05).Bivariate correlation analysis showed that RasGRP4 mRNA level was positively correlated with disease course,serum triglyceride(TG),hypersensitivity C protein(hs-CRP)and HbA1c in T2DM group(r=0.973,0.237,0.472,0.245,all P<0.05).Multiple linear regression analysis suggested that the course of T2DM and HbA1c were the main factors influencing the expression of RasGRP4.The expression of RasGRP4 in THP-1 cells was promoted under high glucose or high lipid condition,and the effect of high glucose with high lipid on the expression of RasGRP4 had a synergistic effect.Conclusion Signal protein RasGRP4 and its variants are highly expressed in PBMCs of T2DM patients.Chronic hyperglycemia and hyperlipidemia may be critical factors that activate RasGRP4 and induce the production of its variants.