人角质形成细胞用黎豆叶提取物处理后的蛋白质组学分析及翻译后修饰。

Proteomic profiling and post-translational modifications in human keratinocytes treated with Mucuna pruriens leaf extract.

机构信息

Department of Medical Biotechnologies, University of Siena, Siena, Italy; Child Neuropsychiatry Unit, University Hospital AOUS, Siena, Italy.

Department of Biotechnology, Chemistry and Pharmaceutical Sciences, University of Siena, Siena, Italy.

出版信息

J Ethnopharmacol. 2014 Feb 3;151(2):873-81. doi: 10.1016/j.jep.2013.11.053. Epub 2013 Dec 5.

DOI:10.1016/j.jep.2013.11.053

PMID:24315849

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE

Mucuna pruriens (Mp) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure.

MATERIAL AND METHODS

The effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS).

RESULTS

Eighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9.

CONCLUSIONS

Our findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs).

摘要

民族药理学相关性

Mucuna pruriens（Mp）是豆科植物，具有多种药用特性，其中包括治疗活性氧（ROS）在发病机制中起重要作用的疾病的潜力。目的是研究 Mp 叶甲醇提取物（MPME）对人角质形成细胞蛋白表达的影响及其在氧化应激（OS）暴露后预防蛋白氧化的作用。

材料与方法

通过用不同浓度的 MPME、葡萄糖氧化酶（GO，OS 的来源）以及随后用 GO 处理的 MPME 处理细胞，评估 MPME 对 HaCaT 细胞蛋白表达的影响。用二维凝胶电泳（2-DE）分析处理 HaCaT 细胞的蛋白图谱，并与未经处理的 HaCaT 细胞进行比较。然后用免疫印迹法评估 MPME 防止 4-羟基壬烯醛蛋白加合物（4-HNE PAs）形成（OS 的标志物）的作用。

结果

通过质谱（LC-ESI-CID-MS/MS）鉴定，MPME 明显调节了 HaCaT 中的 18 种蛋白质。总体而言，MPME 拮抗 GO 的作用，减少了 GO 诱导的几种应激反应相关蛋白（T 复合物蛋白 1、蛋白二硫键异构酶 A3、蛋白 DJ-1 和应激诱导磷酸蛋白 1）、细胞能量代谢（无机焦磷酸酶、磷酸丙糖异构酶 1 型、2-磷酸丙酮酸水合酶 α-烯醇酶、果糖二磷酸醛缩酶 A 同工型 1）、细胞骨架组织（细胞角蛋白 18、9、2、丝切蛋白 1、膜联蛋白 A2 和 F-肌动蛋白加帽蛋白亚基β同工型 1）和细胞周期进展（真核翻译起始因子 5A-1 同工型 B）的过度表达。此外，MPME 降低了 4-HNE PAs 水平，特别是 2-磷酸丙酮酸水合酶 α-烯醇酶和细胞角蛋白 9。