血清蛋白质组学技术发现鹿角胶治疗维甲酸致股骨头坏死模型大鼠的机制  

Mechanism by which antler glue treats avascular necrosis of the femoral head induced by retinoic acid in rats:a serum proteomics study

作　　者：胡亚楠 王平[2,3] 杜海涛 景田园 王程程 Hu Yanan;Wang Ping;Du Haitao;Jing Tianyuan;Wang Chengcheng(School of Pharmacy,Shandong University of Traditional Chinese Medicine,Jinan 250355,Shandong Province,China;Shandong Academy of Traditional Chinese Medicine,Jinan 250014,Shandong Province,China;State Key Laboratory of Precision Testing Technology and Instruments,Tianjin University,Tianjin 300072,China)

机构地区：[1]山东中医药大学药学院,山东省济南市250355 [2]山东省中医药研究院,山东省济南市250014 [3]天津大学精密测试技术及仪器国家重点实验室,天津市300072

出　　处：《中国组织工程研究》2022年第14期2243-2251,共9页Chinese Journal of Tissue Engineering Research

基　　金：山东省自然科学基金项目(ZR2020MH386),项目负责人:王平;山东省重大科技创新工程项目(2018CXGC1310,2020CXGC010505-04),项目负责人:王平;国家重点研发计划项目(2019YFE0117800);山东省高校中药质量控制与全产业链建设协同创新中心项目(CYLXTCX2021-01;CYLXTCX2020-04),项目负责人:王平;中央引导地方项目(YDZX20203700002055),项目负责人:王平。

摘　　要：背景:课题组前期研究发现鹿角胶小分子肽富含促进骨生长的肽链,对骨髓间充质干细胞具有良好的促增殖、促成骨化作用。目前对鹿角胶防治股骨头坏死的研究仅停留在物质基础及药效学研究方面,其作用机制及作用靶点尚未明确。目的:基于蛋白质组学初步探讨鹿角胶治疗股骨头坏死的作用机制。方法:SPF级雌性Wistar大鼠36只,随机分为空白组、模型组、鹿角胶组,后2组大鼠灌胃维甲酸70 mg/kg,每日1次,连续灌胃8周,建立股骨头坏死大鼠模型;空白组大鼠不做处理。造模8周后鹿角胶组大鼠给予鹿角胶0.35 g/kg,模型组、空白组予同体积0.9%氯化钠溶液,每天灌胃1次,连续给药8周。通过骨组织病理切片检验造模是否成功及鹿角胶对股骨头的修复效果。通过非标(Label-free)蛋白质组定量技术研究各组之间的差异蛋白情况,根据GO和KEGG数据库分析对差异蛋白的生物功能及参与的信号通路进行富集;采用蛋白质互作STRING Database构建靶点蛋白相互作用图。实验方案经山东省中医药研究院动物实验伦理委员会批准。结果与结论:①骨组织病理切片结果显示,鹿角胶可有效修复股骨头坏死;②蛋白质组学共鉴定蛋白1457种,空白组与模型组、模型组与鹿角胶组共同差异蛋白共有353种,其中,在模型组升高或降低的差异蛋白,经过鹿角胶干预后明显回调的蛋白有97种;③鹿角胶治疗股骨头坏死的作用机制涉及到血液循环、能量代谢、炎症反应及免疫过程,利用差异蛋白质组学寻找的蛋白质二硫键异构酶(P4HB)、钙黏蛋白2(CDH_(2))、膜联蛋白A2(ANXA2)、枯草杆菌前蛋白转换酶/可信9型(PCSK9)、乳脂球-表皮生长因子8(MFGE8)、骨形态发生蛋白等可能是鹿角胶的重要靶点,鹿角胶可能通过调控转化生长因子β、糖酵解/糖异生、PI3K-Akt信号通路等治疗股骨头坏死。BACKGROUND:The research group has found that the small molecule peptides of antler gum are rich in peptide chains that promote bone growth,proliferation and ossification of bone marrow mesenchymal stem cells.The specific mechanism and target of antler glue in the treatment of avascular necrosis of the femoral head are still unclear,which needs further studies.OBJECTIVE:To explore the mechanism of antler glue in the treatment of femoral head necrosis based on proteomics.METHODS:Thirty-six female Wistar rats,SPF grade,were randomized into a blank group,a model group,and an antler glue group.The rats in the latter two groups were given retinoic acid 70 mg/kg once a day for 8 weeks to establish a rat model of femoral head necrosis,while the rats in the blank group were not treated.At 8 weeks after modeling,the rats in the antler glue group were given antler glue 0.35 g/kg,and those in the model and blank groups were given the same volume of 0.9%sodium chloride solution,once a day by gavage for 8 weeks.Pathological examination of bone tissue was used for verifying the successful modeling and the repair effect of antler glue on the femoral head.Non-standard(Label-free)proteome quantification technology was used to detect differential genes among groups.Biological function of differential proteins and signaling pathways involved were enriched based on GO and KEGG database analyses.STRING database was used to construct the target protein-protein interaction network diagram.The study protocol was approved by the Animal Experiment Ethics Committee of Shandong Academy of Traditional Chinese Medicine.RESULTS AND CONCLUSION:The pathological sections of bone tissue showed that antler glue could effectively repair the necrotic femoral head.A total of 1457 kinds of proteins were identified,including 353 kinds of common differential proteins between blank group and model group as well as between model group and antler glue group.In the model group,97 kinds of differential proteins that increased or decreased showed a significant

关 键 词：鹿角胶 血清 蛋白质组学 液质联用 股骨头坏死 差异蛋白 kegg通路分析 转化生长因子β 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]