镇肝熄风汤对脑出血肝阳化风证患者外周血单个核细胞蛋白质组学的影响

Effect of Zhengan Xifeng decoction on Proteomics of peripheral blood mononuclear cell in intracerebral hemorrhage patients with liver yang forming wind syndrome

目的:观察镇肝熄风汤治疗脑出血肝阳化风证(方证相应)前后外周血单个核细胞蛋白质双向凝胶电泳图谱的动态变化,探讨脑出血肝阳化风证的蛋白质表达规律,寻找镇肝熄风汤所作用的蛋白及疗效机制。方法:利用固相梯度双向凝胶电泳技术建立脑出血(10例)肝阳化风证镇肝熄风汤治疗10日前后及健康对照组(10例)外周血单个核细胞蛋白双向凝胶电泳图谱,经PDQuest8.0软件分析,选取差异表达的蛋白质点,应用MALDI-TOF-MS质谱仪获得肽质量指纹图谱,用Mascot软件搜索数据库鉴定部分差异蛋白质。结果:筛选出22个差异表达蛋白质点,其中脑出血肝阳化风证与健康对照组比较有20个差异表达蛋白质点,8个表达下调,7个表达上调,5个缺失;脑出血肝阳化风证经镇肝熄风汤治疗后与健康对照组比较4个表达下调,5个表达上调,2个缺失,9个表达与健康人无明显差异;在挖取的20个蛋白质点中有8个得到鉴定,其中脑出血肝阳化风证与健康对照组比较6个表达下调,1个表达上调,1个缺失;脑出血肝阳化风证经镇肝熄风汤治疗后与健康对照组比较2个表达下调,1个蛋白缺失,5个表达与健康人无明显差异。结论:初步建立了脑出血肝阳化风证外周血单个核细胞蛋白双向凝胶电泳图谱,初步鉴定了部分与脑出血肝阳化风证相关的蛋白及镇肝熄风汤可能的多靶点蛋白调节机制,这些蛋白功能涉及细胞代谢、信号转导、电子传递、细胞骨架等。

Objective： With proteomic approach, to find the differentially expressed proteins spots in the peripheral blood mononuclear cells （PBMCs） of healthy people and intracerebral hemorrhage patients with liver yang forming wind syndrome （LYFWS） treated by Zhenganxifeng decoction（ZGXFD） to investigate the possible proteins of LYFWS and the cure mechanism of ZGXFD. Methods： 10 intracerebral hemorrhage patients with LYFWS were treated by ZGXFD for 7-10 days. 10 healthy people were selected taken as healthy controls.10ml venous blood was drawed from every subjects and PBMCs were isolated from blood by using lymphocytes separation medium. The total proteins was extracted from PBMCs. The total proteins from either intracerebral hemorrhage patients or healthy controls were prepared by means of immobilized pH gradient based on two-dimensional gel electrophoresis. After Coomassie brilliant blue G250 staining, gel-image analysis was performed by PDQuest. The differentially expressed proteins spots were identified by matrix-assisted laser desorption ionization-time-of- flight mass spectrometry （MALD I-TOF-MS）. Results： Gel-image analysis revealed that there were 22 proteins spots expressed differentially.20 proteins spots were expressed differentially in the intracerebral hemorrhage patients with LYFWS as compared with healthy control, of which 8 proteins were down-regulated, 7 proteins were up-regulated and 5 proteins were lost. After treated by ZGXFD, 4 proteins were down-regulated, 5 proteins were up-regulated and 2 proteins were lost, 9 proteins weren＇t expressed differentially as compared with healthy control. 8 of 20 differentially expressed proteins spots in the intracerebral hemorrhage patients with LYFWS identified by MALDI-TOF-MS as compared with healthy control. These proteins are related to cell metabolism, signal transduction and so forth, of which 6 proteins were down-regulated, 1 proteins wel：e up-regulated and I proteins were lost. After treated by ZGXFD,2 proteins were down-regulated, 1 proteins were lost, 5 proteins weren＇ t expressed differentially as compared with healthy control. Conclusion： 2-DE profiles of intracerebral hemorrhage patients with LYFWS in PBMCs had been established. The proteins of intracerebral hemorrhage patients with LYFWS had been identified, which were related to cell metabolism, signal transduction and so forth.This research presented that ZGXFD could adjust multiple differentially expressed proteins of intracerebral hemorrhage patients with LYFWS.It was also indicated that the cure mechanism of ZGXFD was to regulate multitarget proteins.