应用同位素标记相对和绝对定量技术分析水蛭对IgA肾病血瘀证大鼠差异蛋白的表达

Application of isobaric tags for relative and absolute quantitation in identifying differentially expressed proteins in rats with IgA nephropathy with blood stasis syndrome after leech treatment

目的:运用同位素标记相对和绝对定量技术(iTRAQ)分析水蛭对IgA肾病血瘀证大鼠差异蛋白的表达,并从血清蛋白质水平阐释水蛭治疗IgA肾病血瘀证的作用机制。方法:选用6周龄的Wistar雄性大鼠80只,运用脂多糖牛血清白蛋白+脂多糖+四氯化碳方法建立实验性IgA肾病模型,造模成功后,随机分为模型对照组、强的松治疗对照组和水蛭治疗组,每组各20只,另取20只为空白对照组,给药喂养至14周末后进行采血,血液样本经过SDS-PAGE电泳后进行iTRAQ标记,运用离线二维液相色谱分离与点靶以及质谱分析及数据库检索对各组样本进行相对定量分析,并利用生物信息学软件进行分析,以上实验再重复进行2次。结果:取4组均有表达且差异倍数≥1. 2或≤0. 83为筛选标准的蛋白纳入数据分析,经水蛭治疗组治疗后下调的差异蛋白共21个,这些差异蛋白主要参与低密度脂蛋白颗粒受体分解代谢、脂肪酸体内平衡、胆固醇稳态以及线粒体分裂等生物过程,其差异可能与补体和凝血级联通路、代谢信号通路、嘌呤代谢和嘧啶代谢途径等有关。结论:应用i TRAQ标记方法可筛选出水蛭治疗IgA肾病血瘀证大鼠的差异蛋白表达,并能揭示水蛭治疗IgA肾病血瘀证的物质基础及活血祛瘀的疗效机制。

Objective:To investigate the application of isobaric tags for relative and absolute quantitation(iTRAQ)in identifying differentially expressed proteins in rats with IgA nephropathy with blood stasis syndrome after leech treatment,and to clarify the mechanism of action of leech in the treatment of IgA nephropathy with blood stasis syndrome at the protein level.Methods:A total of 80 male Wistar rats aged 6 weeks were selected.A model of IgA nephropathy was established by bovine serum albumin+lipopolysaccharide+carbon tetrachloride.After modeling,the rats were randomly divided into model control group,prednisone treatment group,and leech treatment group,with 20 rats in each group.Another 20 rats were selected as blank control group.Blood samples were collected after 14 weeks of administration and was labeled by iTRAQ after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Two-dimensional offline chromatographic fractionation,mass spectrometry,and database retrieval were used for relative quantitative analysis,and a bioinformatics software was used for analysis.The experiment above was repeated twice.Results:The proteins with a fold change of≥1.2 or≤0.83 in all four groups were included in analysis.A total of 21 proteins were downregulated after leech treatment,which were mainly involved in the biological processes of catabolism of low-density lipoprotein particle receptor,homeostasis of fatty acids,cholesterol homeostasis,and mitochondrial fission.The differences in these proteins might be associated with the complement and coagulation cascade pathway,the metabolic signaling pathway,and the purine/pyrimidine metabolic pathways.Conclusion:The i TRAQ labeling method can be used to screen out differentially expressed proteins in rats with IgA nephropathy with blood stasis syndrome after leech treatment.It also helps to reveal the material basis of leech treatment for IgA nephropathy with blood stasis syndrome and the mechanism of blood-activating and blood stasis-removing effect of leech.