Zhang Yu, Wang Yingzi, Li Shaojing, Zhang Xiuting, Li Wenhua, Luo Shengxiu, Sun Zhenyang, Nie Ruijie
1College of Traditional Chinese Pharmacy, Beijing University of Chinese Medicine, North Third Ring Road, Number 11, Chaoyang District, Beijing, 100029 People's Republic of China.
2Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Dong cheng District, Dongzhimen Neixiang Street on the 16th, Beijing, 100700 People's Republic of China.
Proteome Sci. 2018 Apr 17;16:8. doi: 10.1186/s12953-018-0136-6. eCollection 2018.
L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet.
In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways.
A number of differentially expressed proteins that may be associated with intestinal inflammation were identified. They mainly constituted by part of the cell. The expression sites of them located within cells and organelles. G protein and Eph/Ephrin signal pathway were controlled jointly by SEP and SE. After processing, the extraction of SEP were mainly reflected in the process of cytoskeleton, glycolysis and gluconeogenesis.
These findings suggest that SE induced an inflammatory response, and activated the Interleukin signaling pathway, such as the Ang/Tie 2 and JAK2/ STAT signaling pathways, which may eventually contribute to injury result from intestinal inflammatory, while SEP could alleviate this injury by down-regulating STAT1 and activating Ang-4 that might reduce the inflammatory response. Our results demonstrated the importance of Ang-4 and STAT1 expression, which are the target proteins in the attenuated of SE after processing based on proteomic investigation. Thus iTRAQ might be a novel candidate method to study scientific connotation of hypothesis that the attenuated of SE after processing expressed lower toxicity from cellular levels.
中药狼毒常用于治疗水肿、腹水、便秘、闭经和疥疮。中药实践中常用的另一种狼毒是狼毒霜，它是通过去除狼毒种子中的油（即炮制）获得的，已知其能缓解腹泻。然而，其降低肠道毒性的机制尚未得到明确研究。
在本研究中，采用相对和绝对定量的等压标签（iTRAQ）结合液相色谱 - 串联质谱（LC - MS/MS）蛋白质组学方法，研究狼毒对肠道代谢相关蛋白质表达的影响，以阐明狼毒炮制潜在的减毒机制。鉴定了用狼毒（SE）、狼毒霜（SEP）和大戟因子1（EFL）处理后肠道中的差异表达蛋白（DEP）。进行了包括基因本体（GO）分析、通路分析和网络分析在内的生物信息学分析，以通过腹泻中的蛋白质网络分析减毒机制的关键代谢途径。进行蛋白质印迹法验证所选蛋白质及相关通路。
鉴定出许多可能与肠道炎症相关的差异表达蛋白。它们主要由部分细胞组成。其表达位点位于细胞和细胞器内。G蛋白和Eph/ Ephrin信号通路由SEP和SE共同控制。炮制后，SEP的提取物主要反映在细胞骨架、糖酵解和糖异生过程中。
这些发现表明，SE诱导炎症反应，并激活白细胞介素信号通路，如血管生成素/ Tie 2和JAK2/STAT信号通路，这可能最终导致肠道炎症引起的损伤，而SEP可以通过下调STAT1和激活血管生成素 - 4来减轻这种损伤，这可能会减少炎症反应。我们的结果证明了血管生成素 - 4和STAT1表达的重要性，它们是基于蛋白质组学研究炮制后狼毒减毒的靶蛋白。因此，iTRAQ可能是一种新的候选方法，用于从细胞水平研究炮制后狼毒毒性降低这一假说的科学内涵。
