基于同位素标记相对和绝对定量技术的联合用药抗肝纤维化作用的蛋白质组学研究

Proteomics research on antifibrotic mechanism of combination therapy based on isobaric tags for relative and absolute quantitation technique

目的以表没食子儿茶素没食子酸酯(EGCG)、牛磺酸和染料木素3种具有不同抗纤维化作用的药物组成联合用药,应用同位素标记相对和绝对定量(i TRAQ)技术研究联合用药对肝纤维化大鼠治疗作用的蛋白质基础及分子机制。方法将SD大鼠分为对照组、模型组和联合用药组,模型组和联合用药组大鼠分别ig 50%CCl4溶液,对照组给予花生油,连续14周。于第9周开始联合用药组进行ig给药(牛磺酸200 mg/kg+EGCG 30 mg/kg+染料木素20 mg/kg)治疗,连续6周。采集大鼠血清和肝组织样本,肝组织进行HE染色观察病理变化,模型组和联合用药组血清样本组内等量混合后去除高丰度蛋白,i TRAQ试剂标记后应用液相色谱分离,经质谱鉴定,进行相对定量及生物信息学分析,用ELISA法对有代表性的血清差异蛋白Txn1进行验证。结果质谱鉴定出置信度在95%以上的蛋白质共359个,其中差异表达的蛋白有78种,上调的蛋白有51种,下调的有27种。与模型组相比,差异蛋白Txn1在联合用药组血清中的量明显升高(P<0.05)。结论联合用药能通过对多个蛋白质、多途径的综合调节达到抗肝纤维化的效果,其中对抗氧化防御系统和血液凝固级联活化途径的调节可能是联合用药抗肝纤维化作用的分子机制。

Objective To investigate the protein basis and molecular mechanism of combination therapy with epigallocatechin gallate（EGCG）, taurine, and genistein on CCl4-induced liver fibrosis in rats using isobaric tags for relative and absolute quantitation（i TRAQ）. Methods The SD rats were randomly divided into normal control, model, and combination therapy（taurine 200 mg/kg ＋ EGCG 30 mg/kg ＋ genistein 20 mg/kg） groups. The rats in the model and combination therapy groups were induced by ig administration of 50% CCl4 for 14 weeks, and the rats in the normal control group were given peanut oil. The combination therapy was started at week 9 for 6 weeks. HE staining of liver tissue was performed to evaluate histopathologic changes. The high-abundance proteins in the serum of model and combination therapy groups were depleted by Proteo Miner Protein Enrichment Kit. Proteins differentially expressed in the serum of those two groups were identified with i TRAQ coupled with LC-ESI-MS/MS technology and analyzed with bioinformatics tools. The expression of representative differential protein（Txn1） was validated by ELISA. Results A total of 359 proteins with the confidence coefficient above 95% were identified, of which 78 were differential expressed proteins, including 51 up-regulated ones and 27 down-regulated ones. The serum level of Txn1 increased significantly in the combination therapy group compared with the model group（P〈0.05）. Conclusion The combination therapy can alleviate the progression of liver fibrosis effectively by affecting multiple biological processes. The regulation of anti-oxidant defense system and coagulation cascade pathway may be the molecular mechanisms of the combination therapy against liver fibrosis.