基于iTRAQ技术分析脾虚痰浊动脉粥样硬化巴马猪小肠蛋白质组学变化

Proteomic Changes of Small Intestine of AS Bama Pig with Spleen Deficiency and Phlegm Turbid Based on iTRAQ Technique

目的应用iTRAQ技术研究脾虚痰浊动脉粥样硬化(atherosclerosis,AS)巴马猪小肠蛋白质组学变化,在蛋白质水平上探讨脾虚痰浊AS猪小肠功能改变机制。方法10只健康半岁雄性广西巴马小型猪,随机分为正常对照组和脾虚痰浊AS组,每组5只。正常对照组每日给予基础饲料喂饲。脾虚痰浊AS模型组采用中医经典复合因素造模法,即饮食不节加劳倦过度造脾虚模型,配合现代医学冠脉内皮损伤手术造动脉粥样硬化模型。分离提取小肠总蛋白,以iTRAQ试剂标记后进行质谱检测并以软件分析差异表达的蛋白质,分析与小肠消化吸收、细胞炎症反应、脂质代谢等有关系的代表通路。结果iTRAQ技术分析发现:蛋白定量结果脾虚痰浊AS组与正常对照组相比,得到99个上调蛋白和47个下调蛋白,共计146个差异蛋白,具代表性蛋白8个,主要有胰脂肪酶相关蛋白2前体;MHCⅠ类抗原1-3前体;1-酰基-sn-甘油-3-磷酸酰基转移酶前体;钠/葡萄糖协同转运体1;ATP结合盒亚家族G成员2;纤维蛋白原链前体等。这些蛋白存在于3条代表性通路中,分别为甘油脂类代谢通路、胆汁酸分泌通路、细胞黏附分子通路等,这些蛋白参与的生物过程可能与脂类代谢、胆汁分泌、细胞炎症反应等密切相关。结论脾虚痰浊AS巴马猪小肠蛋白质组学改变可能与甘油脂类代谢、胆汁酸分泌、细胞炎症反应等密切相关,脾虚高脂状态小肠消化吸收功能异常,细胞炎症反应可能与AS形成密切相关。

Objective To study the proteomic changes of the small intestine of atherosclerosis(AS)Bama pigs with spleen deficiency and phlegm turbid by iTRAQ technique and investigate the mechanism of intestinal function changes on the protein level.Method Ten healthy half-year-old male Guangxi Bama miniature pigs were randomly divided into normal control group and spleen deficiency and phlegm turbid AS group,five pigs in each group.The normal control group was given basic feeding daily.In the AS model group of spleen deficiency and phlegm turbid,the traditional Chinese medicine classic compound factor modeling method was adopted.The spleen deficiency model was set up with diet without restriction and excessive fatigue,and the atherosclerosis model was built with the operation of coronary endothelial injury in modern medicine.Total protein of the small intestine was isolated and extracted.The iTRAQ reagent was labeled for mass spectrometry detection,and the differentially expressed protein was analyzed by software.Representative pathways related to intestinal digestion and absorption,cellular inflammatory response and lipid Metabolism were analyzed.Result The iTRAQ technology analysis showed compared with the normal control group,the AS group with spleen deficiency and phlegm turbid obtained 99 up-regulated proteins and 47 down-regulated proteins,a total of 146 differentially expressed proteins and 8 representative proteins,mainly including pancreatic lipase-related protein 2 precursor,MHC class I antigen 1-3 precursor,1-acyl-sn-glycerol-3-phosphate acyltransferase beta precursor,sodium/glucose cotransporter,ATP-binding cassette sub-family G member 2,fibrinogen gamma chain precursor,etc.These proteins existed in three representative pathways,namely,the glyceroid Metabolic pathway,the bile acid secretion pathway,and the cell adhesion molecular pathway.The biological processes in which these proteins are involved in may be closely related to lipid Metabolism,bile secretion and cellular inflammation.Conclusion The proteomic changes in the small intestine of AS Bama pigs with spleen deficiency and phlegm turbid may be closely related to the Metabolism of glycerin,bile acid secretion and cellular inflammatory reaction.The intestinal digestion and absorption function is abnormal condition of spleen deficiency and high fat,and the cellular inflammatory reaction may be closely related to the formation of AS.