益气健脾化瘀祛痰方对脾虚痰浊动脉粥样硬化巴马小型猪心脏线粒体蛋白质组的影响

Effects of Yiqi Jianpi Huayu Qutan Formula on the heart mitochondrial protein of Bama mini pigs with spleen deficiency and phlegm turbidity atherosclerosis syndrome

目的:应用i TRAQ技术研究益气健脾化瘀祛痰方对脾虚痰浊动脉粥样硬化巴马小型猪心脏线粒体蛋白质组表达的影响。方法:9只巴马小型猪随机分为正常组、模型组和治疗组。模型组予高脂饲料喂饲,于第2周行冠脉球囊挤压伴拉伤,第6周进行跑步干预。治疗组予以益气健脾化瘀祛痰方混于饲料中饲喂。用药后分离提取心肌线粒体,ELISA法检测线粒体ATP含量,i TRAQ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,用Western blot方法验证部分差异蛋白。结果:与正常组比较,模型组心肌线粒体ATP含量明显降低(P<0.01);与模型组比较,治疗组心肌线粒体ATP含量显著上调(P<0.01)。i TRAQ技术分析:与模型组比较,治疗组共有56个蛋白上调,24个蛋白下调。治疗组心肌组织中phosphoenolpyruvate carboxykinase,GTP-binding protein Di-Ras2,GTP-binding protein SAR1b,ATP synthase(ATP5E),Succinyl-Co A,ADP-ribosylation factor 3(ARF3),NADH dehydrogenase差异蛋白表达量显著上调,这些蛋白参与的生物过程与能量代谢尤其是三羧酸循环的过程密切相关。Western blot验证ATP5E及ARF3结果与蛋白质组所得相同蛋白的结果表达一致。结论:益气健脾化瘀祛痰方可能通过影响三羧酸循环相关酶及蛋白的表达,从而促进ATP的合成,达到其保护心脏的作用。

Objective: To investigate the influence of Yiqi Jianpi Huayu Qutan Formula on the change of heart mitochondrial protein expression patterns in Bama mini pigs with spleen deficiency and phlegm turbidity atherosclerosis syndromebased on the i TRAQ technique. Methods: Nine Bama mini pigs were randomly divided into control group, model group and treatment group. Animals in the model group were fed with high fat diet and conducted with coronaryballoon extrusion with strain in the 2nd week. Running intervention was given in the 6th week. The treatment group was treated with Yiqi Jianpi Huayu Qutan Formula, mixed with feed. Myocardial mitochondria were isolated after administration. Using ELISA to detect mitochondrial ATP content. Samples were analyzed for mass spectrometry after i TRAQ labeling, and the differential expressed proteins were analyzed by software. The partial differential proteins were verified by Western blot. Results: Compared with the control group, mitochondrial ATP content in model group were significantly decreased(P<0.01); compared with the model group, mitochondrial ATP content in treatment group were significantly increased(P<0.05). ITRAQ analysis: compared with the model group, there were 56 proteins up-regulated and 24 proteins down regulated in the treatment group. In the treatment group, the differential protein expressions of phosphoenolpyruvate carboxykinase(PEPCK), GTP-binding protein Di-Ras2, GTPbinding protein SAR1 b, ATP synthase(ATP5E), Succinyl-Co A, ADP-ribosylation factor 3(ARF3), NADH dehydrogenase in the myocardium were up-regulated significantly. The biological processes involved in these proteins were closely related to the processes of energy metabolism, especially the three carboxylic acid cycle. The results of Western blot verification testing were consistent with the proteomics. Conclusion: Yiqi Jianpi Huayu Qutan Formula might promote the synthesis of ATP by affecting the expression of the related enzymes and proteins of the three carboxylic acid cycle, so as to protect the heart.