基于蛋白质组学结合分子对接分析贵州天麻对肝脏CYP450酶的影响

Effects of Gastrodia elata Bl.on liver CYP450 enzymes based on proteomics-binding molecular docking

目的应用蛋白质组学结合液相色谱和质谱技术,鉴定和定量分析大鼠肝脏细胞色素P450(cytochromeP450,CYP450)酶系蛋白的表达情况。方法取SD大鼠16只分为正常组和天麻组,给药7天后,取肝脏组织提取蛋白,应用同位素标记相对和绝对定量(isobaric tag for relative and absolute quantitation,iTRAQ)试剂标记后进行质谱鉴定,使用蛋白质组学定量软件对二级质谱数据进行分析。通过注释、可视化和综合发现数据库(the database for annotation,visualizationand integrated discovery,DAVID)对靶点进行基因本体论和通路富集分析。采用相互作用分析核心靶点,酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)检测血清和肝脏中核心靶点的表达情况,基于分子对接分析天麻化学成分与核心靶点的结合情况。结果与正常组比较,天麻可以上调10个CYP450酶系蛋白的表达(Cyp11a1,Cyp11b1,Cyp21a1,Cyp2b2,Cyp2b15,Cyp8b1,Cyp17a1,Cyp4a2,Cyp2b1,Cyp2c13),下调Cyp7a1和Cyp51a1酶系蛋白的表达(P<0.05)。这些蛋白主要参与类固醇激素生物合成、花生四烯酸代谢等(P<0.01)。相互作用分析结果发现Cyp17a1和Cyp7a1为核心靶点。ELISA检测结果发现,与正常组相比,天麻组大鼠肝脏中CYP17A1活性显著升高(P<0.05),CYP7A1活性显著降低(P<0.05)。分子对接分析发现天麻醚苷、枸橼酸、4-(4′-羟苄氧基)苄基甲醚、胡萝卜苷与CYP17A1有较好的结合,天麻素、天麻醚苷、4-(4′-羟苄氧基)苄基甲醚、胡萝卜苷与CYP7A1有较好的结合。结论本研究进一步了解天麻及其化学成分对CYP450酶系的影响,为扩大天麻制品的临床应用提供依据。

Objective To identify and quantitatively analyze the expression of CYP450 in rat liver by proteomics combined with liquid chromatography and mass spectrometry.Methods 16 SD rats were divided into normal group and Gastrodia elata Bl.(GE)group.After 7 days of administration,liver tissue was extracted and labeled with iTRAQ reagent for mass spectrometry,and the secondary MS data were analyzed using proteomics quantitative software.The GO and pathway enrichment analysis of the targets were performed by DAVID.The expression of core targets by ELISA in serum and liver was analyzed,and the binding of GE and core targets were analyzed based on the analysis and docking.Results Compared with normal group,GE can up-regulate the expression of 10 CYP450 enzymes(Cyp11 a1,Cyp11 b1,Cyp21 a1,Cyp2 b2,Cyp2 b15,Cyp8 b1,Cyp17 a1,Cyp4 a2,Cyp2 b1 and Cyp2 c13),down-regulate the expression of Cyp7 a1 and Cyp51 a1 enzyme proteins(P<0.05).These proteins are mainly involved in steroid hormone biosynthesis and arachidonic acid metabolism(P<0.01).The results of interaction analysis identified Cyp17 a1 and Cyp7 a1 as core targets.The ELISA test results found that CYP17 A1 activity in the liver of GE rats was significantly increased(P<0.05)and CYP7 A1 activity was significantly decreased(P<0.05).Molecular docking analysis found that gastrodioside,citric acid,4-(4′-hydroxybenzyloxy)benzyl methyl ether and daucosterol could bind CYP17 A1 well,while gastrodin,gastrodioside,4-(4′-hydroxybenzyloxy)benzyl methyl ether and carotenoside had good binding to CYP7 A1.Conclusion This study further understands the influence of GE and its chemical composition on the CYP450 enzyme system,in order to provide a basis for expanding the clinical application of GE products.