基于TMT标记定量蛋白质组学技术研究老龄肾阳虚证患者血浆差异蛋白的变化及意义

Changes and Significance of Plasma Differential Proteins in Aged Patients with Kidney-yang Deficiency Syndrome Were Studied Based on TMT-Labeled Quantitative Protein Omics

目的:基于TMT标记定量蛋白质组学技术挖掘老龄肾阳虚患者的血浆差异性表达蛋白的变化及意义。方法:收集8例符合中医辨证的老龄肾阳虚证患者及8例同年龄段健康者的血浆样品,分别在两组中随机挑选3例血浆样品,通过TMT蛋白质组学技术进行蛋白质定量分析并筛选出差异表达蛋白,对差异表达蛋白进行GO富集和KEGG富集分析,通过蛋白互作网络分析筛选出差异表达蛋白中的重要蛋白,然后将其中与阿尔茨海默病(AD)具有相互作用的蛋白在另外5例老龄肾阳虚患者和5例同年龄段健康人中进行ELISA验证。结果:老龄肾阳虚患者组与同年龄段健康人组对照共得到83个差异表达蛋白,其中73个上调,10个下调。差异表达蛋白涉及糖酵解、蛋白折叠等生物过程,胞浆、胞外空间等细胞成分,钙黏蛋白结合、蛋白结合等分子功能;涉及丙酮酸盐代谢、AD、雌激素信号通路、胰高血糖素信号通路等。与AD发生发展可能相关的蛋白验证表明,老年肾阳虚患者血浆中钙蛋白酶-1(Calpain1)、α-突触核蛋白(α-synuclein)表达显著高于对照组(P<0.05)。结论:老龄肾阳虚患者与同年龄段健康者血浆之间存在差异性蛋白表达,其中Calpain1和α-synuclein与AD的发生发展有关,为探索老年肾阳虚易患AD提供分子机制,在中医治未病中发挥重要作用。

Objective:To excavate the changes and significance of plasma differentially expressed proteins in aged patients with kidney-yang deficiency syndrome based on TMT-labeled quantitative protein omics.Methods:The plasma samples of eight aged patients with kidney-yang deficiency syndrome in line with TCM syndrome differentiation and eight healthy people of the same age were recruited,and three plasma samples were randomly selected from each group.Protein quantitative analysis was performed using TMT protein omics technology and the differentially expressed proteins were screened out,followed by GO enrichment and KEGG enrichment analysis for the differentially expressed proteins.The important proteins of the differentially expressed proteins were screened out by protein interaction network analysis,and the proteins that interacted with Alzheimer s disease(AD)were tested with ELISA in another five aged patients with kidney-yang deficiency and five healthy people of the same age.Results:A total of 83 differentially expressed proteins were obtained in the aged kidney-yang deficiency patient group and the control group of healthy people of the same age.73 of the 83 differentially expressed proteins were up-regulated and 10 were down-regulated.Differentially expressed proteins involve biological processes,such as glycolysis and protein folding,cellular components,such as cytoplasm and extracellular space,and molecular functions,such as cadherin binding and protein binding.These proteins also involves pyruvate metabolism,AD,estrogen signaling pathway,glucagon signaling pathway,etc.Protein validation related to the occurrence and development of AD showed that calpain 1 andα-synuclein expressions in the plasma of the aged patients with kidney-yang deficiency were significantly higher than those in the control group(P<0.05).Conclusion:There were differentially expressed proteins in plasma between the aged patients with kidney-yang deficiency and healthy people of the same age group,and among these differentially expressed proteins,calpain 1 andα-synuclein were related to the occurrence and development of AD.This finding provides a molecular mechanism for exploring the vulnerability of aged patients with kidney-yang deficiency to AD,and plays an important role in the prevention of disease by traditional Chinese medicine.