藜芦人参配伍对大鼠肝功能及肝组织蛋白质表达的影响

Effect ofVeratrum nigrum and ginseng combination on liver function and differential proteomics in rats

目的利用差异蛋白质组技术,探讨藜芦人参配伍对肝功能及蛋白质表达的影响。方法大鼠雌雄各半,ig给予藜芦0.27 g·kg-1、藜芦0.09 g·kg-1+人参0.24 g·kg-1、藜芦0.27 g·kg-1+人参0.71 g·kg-1、藜芦0.81 g·kg-1+人参2.13 g·kg-1,每天1次,连续8周。收集血清及肝组织。采用全自动生化仪测定谷草转氨酶(GOT)、谷丙转氨酶(GPT)的水平,并对肝组织进行常规HE染色观察组织病理变化。通过差异凝胶电泳技术(DIGE)分离大鼠肝组织总蛋白,基质辅助激光解吸电离时间飞行串联质谱及数据库查询鉴定差异表达蛋白质。结果血生化指标检测结果表明,只有藜芦0.81 g·kg-1+人参2.13 g·kg-1检测到GOT没有升高的趋势;而GPT明显升高(P<0.05)。病理学结果显示,正常对照、藜芦0.27 g·kg-1、藜芦0.09 g·kg-1+人参0.24 g·kg-1及藜芦0.27 g·kg-1+人参0.71 g·kg-1组大鼠肝组织无明显病理变化;藜芦0.81 g·kg-1+人参2.13 g·kg-1有2例(8.7%)样本分别出现肝窦扩张淤血及淋巴细胞浸润的病理改变。藜芦人参合用前后大鼠肝组织差异蛋白质经DIGE进行分离后,成功获得了分辨率高、重复性好的双向凝胶电泳图谱;经DeCyder6.5软件分析获得差异>1.5倍的蛋白质点共30个;质谱鉴定去冗余后得到4种差异蛋白质,其中二硫键异构酶A3前体和碳酸酐酶Ⅲ明显降低;谷氨酸脱氢酶1及氨甲酰磷酸合成酶1明显升高。结论藜芦0.81 g·kg-1和人参2.13 g·kg-1合用可能影响大鼠肝内与pH自稳、蛋白折叠及氨基酸代谢相关酶的表达。

OBJECTIVE To explore the toxicity of V.nigrum-ginseng combination for rat liver function and protein expression. METHODS Rats were ig given V.nigrum 0.27 g·kg-1, veratrum nigrum 0.09 g·kg-1＋ginseng 0.24 g·kg-1, V.nigrum 0.27 g·kg-1＋ginseng 0.71 g·kg-1, V.nigrum 0.81 g·kg-1＋ginseng 2.83 g·kg-1 combination, once daily, for 8 weeks. All rats were sacrificed after 8 weeks, Serum was collected on the 8th day after administration. The level of serum glutamic oxaloacetic transminase （GOT） and glutamic pyruvic transminase （GPT） was measured using the automatic biochemical analyzer. And the histopathological changes of liver tissue were observed by HE staining. The total protein maps of the tumor tissue separated were observed and analyzed by two-dimensionl gel electrophoresis. The differentially expressed proteins were digested and identified by MALDI TOF/TOF-MS and database search. RESULTS GPT showed remarkably significant increase （P〈0.05） only in V.nigrum 0.81 g·kg-1＋ginseng 2.13 g·kg-1 group, and GOT did not obviously change. A noticeable lymphocytic infiltration and sinus hepaticus congestion were observed in 2 （8.7%） liver tissue samples in V.nigrum 0.81 g·kg-1＋ginseng 2.13 g·kg-1 group. Two dimensional gel electrophoresis patterns of liver differential expression proteins with high resolution and reproducibility were successfully obtained using DIGE. Four proteins were successfully identified via peptide mass print using MALDI-TOF-MS. Compared with blank control and V.nigrum groups, the protein disulfide-isomerase A3 precursor and carbonic anhydrase Ⅲ were significantly down-regulated; the glutamate dehydrogenase and carbamoyl-phosphate synthetase 1, mitochondrial significantly increased. CONCLUSION V.nigrum-ginseng combination affects liver protein expression involved in pH regulation/homeostasis, protein folding and amino acid metabolism.