手掌参醇提取物对染尘大鼠早期肺组织蛋白谱的影响

Effects of Gymnadenia conopse achohol extract on early protein profiles in lung tissue of rats exposed to silica

目的应用比较蛋白质组学方法分析手掌参醇提取物对染尘大鼠早期肺组织蛋白表达的影响，以寻找低毒有效的矽肺防治药物并初步探讨手掌参干预后的分子机制。方法随机将Wistar大鼠分为染尘组和手掌参干预组，每组4只，气管暴露法给予1ml SiO2混悬液（50mg／ml）建立大鼠模型。染尘后第2天，干预组给予手掌参醇提取物按0．8g/100g（0．8ml／100g）的量每天灌胃，染尘组给予蒸馏水2ml每天灌胃共14d处死大鼠。取肺组织，提取总蛋白，双向凝胶电泳（two—dimensional gel electrophoresis，2-DE）结合基质辅助激光解吸电离飞行时间质谱（matrix—assisted laser desorption／ionization time—of-flight mass spectrometry，MALDI-TOF-MS）分析鉴定差异蛋白。免疫印迹（Westernblot）法验证差异蛋白在肺组织中的表达。结果染尘组大鼠肺泡隔水肿增厚，淋巴细胞、巨噬细胞及少数中性粒细胞浸润，伴有较多的成纤维细胞和平滑肌细胞增生。胶原纤维聚集，肺泡结构破坏，出现斑片状纤维化，细胞性结节的数量多、体积大。手掌参干预组肺组织的变化与染尘组基本相似，但病理改变较之减轻。鉴定出组织蛋白酶D前体、硫氧还蛋白过氧化物酶-1（peroxiredoxin-1，Prx-1）和SECl4类蛋白33种表达差异的蛋白。与染矽尘组相比，手掌参干预组中组织蛋白酶D前体、Prx-1表达下调，SECl4类蛋白3表达上调，差异均有统计学意义（P〈0．01）。用Westernblot法分析显示，手掌参干预组大鼠肺组织中Prx-1表达为（0．26±0．02），明显低于染矽尘组（0．35±0．04），差异有统计学意义P〈0．01）。结论手掌参醇提取物可能多环节干预肺纤维化的形成，而组织蛋白酶D前体、Prx-1和SEC14类蛋白3可能是手掌参醇提取物起作用的相关蛋白。

Objective To analyze the early expression differences of lung tissue proteins in rats exposed to silica using comparative proteomics method, to explore the effects of Chinese traditional medicine （ Gymnadenia conopse alcohol extract, GcAE ） on silicosis （50 mg/ml ）. Methods Adult male Wistar rats were randomly divided into silica-treated group and GeAE-treated group, four rats a group. The rats were exposed to silica by intratraeheal （IT） instillation of 1 ml silica suspension for 24 h. After exposure, the rats in GeAE- treated group were intragastric administration with 0.8 ml GeAE （0.8ml/100g a day） and the rats in silica-treated group were intragastric administration with 2 ml sterilized saline a day for 14 days. Then all rats were sacrificed and lung tissues were collected. The total proteins were separated by means of two-dimensional gel eleetrophoresis （2-DE） and the differentially expressed proteins were identified using matrix-assisted laser desorp- tion/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. Western blotting was used to validate the expression of certain candidate proteins in lung tissues. Results Obvious pathological changes of lung could be observed in silica-treated group, such as the thicken of interalveolar septum, which was infiltrated with lymphocytes, macrophages and a few neutrophils with the proliferation of fibroblasts and smooth muscle cells. The accumulation of collagen, the destruction of alveolus structure and the more dotted fibrosis or granuloma could also be found. However, the pathological changes of lung in GcAE-treated group were lighter than those of silica-treated group. Thirty three differentially expressed proteins were identified, including cathepsin D precursor, peroxiredoxin-1 （Prx-1 ） and SEC 14-like protein 3. Compared with silica-treated group, cathepsin D precursor and Prx-1 were significantly downregulated in GcAE-treated group, and SEC l d-like protein 3 was significantly upregulated （P〈0.01）. The results of western blot indicated that the expression level of Prs-1 in GcAE-treated group was 0.26±0.02, which was significantly lower than that （0.35±0.04） in silica-treated group （P〈0.01） Conclusion GcAE may inhibit the progress of silicosis in the early period and cathepsin D precursor, SEC14- like protein 3 and Prx-1 may participate in this process.