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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 study the combination of epigallocatechin-3-gallate (EGCG), taurine and genistein with different antifibrotic effects and to study the combination of isotope labeled relative and absolute quantification (iTRAQ) The Protein Basis and Molecular Mechanisms of Hepatic Fibrosis in Rats. Methods SD rats were divided into control group, model group and combination group. The model group and combination group were treated with 50% CCl4 solution respectively. The control group was given peanut oil for 14 weeks. From the 9th week, the combination group was treated with ig administration (taurine 200 mg / kg + EGCG 30 mg / kg + genistein 20 mg / kg) for 6 weeks. The serum and liver samples of rats were collected, and the liver tissues were observed for pathological changes by HE staining. The high abundance protein was removed from the model group and the serum from the combined drug group by HPLC. The iTRAQ reagent was labeled and separated by liquid chromatography. Mass spectrometry identification, relative quantification and bioinformatics analysis, and the representative serum differential protein Txn1 was verified by ELISA. Results The mass spectrometry identified 359 proteins with a confidence level above 95%. Among them, 78 proteins were differentially expressed, 51 proteins were up-regulated and 27 genes were down-regulated. Compared with the model group, the level of Txn1 in the serum of the combination group was significantly increased (P <0.05). Conclusion Combination therapy can achieve the effect of anti-liver fibrosis through the comprehensive regulation of multiple proteins and pathways. The regulation of anti-oxidative defense system and the activation pathway of blood coagulation cascade may be the molecular mechanism of combined anti-liver fibrosis effect.