中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (31): 5022-5027.doi: 10.12307/2023.521

• 骨科植入物相关基础实验 Basic experiments of orthopedic implant • 上一篇    下一篇

血清TMT标记定量蛋白质组学分析鹰嘴豆芽异黄酮对骨质疏松模型大鼠的作用

郑靖杰1,2,黄金勇2,吐尔逊江·达地汗2,高彦华3,李志强4,马海蓉1   

  1. 新疆医科大学第一附属医院,1临床医学研究院,2骨科中心,新疆维吾尔自治区乌鲁木齐市  830011;3中国科学院新疆理化技术研究所,新疆维吾尔自治区乌鲁木齐市  830011;4新疆医科大学动物实验中心,新疆维吾尔自治区乌鲁木齐市  830011
  • 收稿日期:2022-07-28 接受日期:2022-09-02 出版日期:2023-11-08 发布日期:2023-01-31
  • 通讯作者: 马海蓉,博士,研究员,硕士生导师,新疆医科大学第一附属医院临床医学研究院,新疆维吾尔自治区乌鲁木齐市 830011
  • 作者简介:郑靖杰,男,1990年生,新疆维吾尔自治区石河子市人,新疆医科大学在读硕士,主要从事骨与关节损伤研究。
  • 基金资助:
    国家自然科学基金项目(81860746,82060411),项目负责人:马海蓉

Quantitative proteomic analysis of serum TMT markers for the effect of isoflavones extracted from chickpea sprouts in osteoporosis rat models

Zheng Jingjie1, 2, Huang Jinyong2, Tuerxunjiang·Dadihan2, Gao Yanhua3, Li Zhiqiang4, Ma Hairong1   

  1. 1Clinical Medicine Institute, 2Department of Orthopedics, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China; 3Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Xinjiang Urumqi 830011, Uygur Autonomous Region, China; 4Experimental Animal Center , Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China 
  • Received:2022-07-28 Accepted:2022-09-02 Online:2023-11-08 Published:2023-01-31
  • Contact: Ma Hairong, PhD, Researcher, Master’s supervisor, Clinical Medicine Institute, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • About author:Zheng Jingjie, Master candidate, Clinical Medicine Institute, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; Department of Orthopedics, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, Nos. 81860746 and 82060411 (both to MHR)

摘要:


文题释义:

TMT标记定量:提取样品中的总蛋白并酶解为多肽,多肽N-term以及末端Lysine侧链基团的游离氨基能够与TMT标签进行共价结合,从而使每条多肽都带上至少一个TMT标签;不同TMT标签具有相同的分子质量,但是却有不同的同位素分布,通过检测标记的丰度,测算该多肽在对应样品中的相对含量。TMT最高可同时标记 16 (8) 组不同的生物样品,显著提高了实验通量,降低了定量误差,可发现更细微的生物学差异。
鹰嘴豆芽异黄酮:由鹰嘴豆种子发芽后通过大孔树脂吸附、乙醇提取、洗脱、干燥等工艺等获得,主要包括鹰嘴豆芽素A、芒柄花素、芒柄花苷和鹰嘴豆芽素-7-葡萄糖苷等异黄酮活性成分。

背景:在既往研究中发现鹰嘴豆芽异黄酮对于雌激素缺乏导致的骨质疏松症有防治作用,但其作用机制未做进一步研究。
目的:通过血清差异表达蛋白表达的生物信息学分析,探讨鹰嘴豆芽异黄酮对去势大鼠骨质疏松的防治机制。
方法:27只10周龄雌性SD大鼠,分为模型组、鹰嘴豆芽异黄酮给药组和假手术组,每组各9只。模型组和鹰嘴豆芽异黄酮给药组大鼠以双侧卵巢切除方式建立骨质疏松症模型。造模1周后,鹰嘴豆芽异黄酮给药组大鼠予以鹰嘴豆芽异黄酮300 mg/(kg·d)灌胃,假手术组和模型组予以等体积的生理盐水灌胃,1次/d。连续灌胃3个月后,应用串联质谱标记(TMT)定量蛋白质组学技术对各组实验动物血清进行蛋白质组学分析,筛选出差异蛋白,并进行GO功能和KEGG通路分析。

结果与结论:①鹰嘴豆芽异黄酮给药组同模型组对比可筛选出21个差异蛋白,其中包括信号传导通路蛋白及脂代谢相关蛋白等;②差异蛋白通过GO功能富集分析发现其多与脂质的代谢及转运调控相关;③差异蛋白通过KEGG通路富集分析发现胆固醇代谢和蛋白酶体显著富集,其可能通过参与Wnt-β-catenin、RANKL/RANK/OPG等重要通路,调节成骨细胞和破骨细胞分化;④结果提示,鹰嘴豆芽异黄酮干预血清中多种脂蛋白及信号通路蛋白的表达,可能通过多种信号通路调控共同作用,发挥防治骨质疏松的功能。

https://orcid.org/0000-0002-8401-9010(郑靖杰)

中国组织工程研究杂志出版内容重点:人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程

关键词: 骨质疏松, 鹰嘴豆芽异黄酮, TMT技术, 蛋白质组, 脂代谢, 信号通路

Abstract: BACKGROUND: In previous studies, isoflavones extracted from chickpea sprouts have preventive and therapeutic effects on osteoporosis caused by estrogen deficiency. However, the underlying mechanism has not been clarified.
OBJECTIVE: To explore the mechanisms of isoflavones extracted from chickpea sprouts to prevent and treat osteoporosis in ovariectomized rats based on proteomics analysis of differentially expressed proteins in serum.  
METHODS: Twenty-seven 10-week-old female Sprague-Dawley rats were randomized into model, chickpea sprout isoflavone, and sham operation groups, with nine rats in each group. Animal models of osteoporosis were prepared by bilateral ovariectomizy in the model and chickpea sprout isoflavone groups. One week after modeling, the chickpea sprout isoflavone group was given 300 mg/kg isoflavones extracted from chickpea sprouts per day by gavage, while the sham operation and model groups were given the same volume of saline by gavage, once a day. After 3 months of continuous administration, serum samples from experimental groups were extracted for proteomic analysis. Tandem mass tag technology was used to screen the differentially expressed proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were also performed. 
RESULTS AND CONCLUSION: (1) Compared with the model group, 21 differentially expressed proteins were found in the isoflavone group, including signal transduction pathway related proteins and lipid metabolism related proteins. (2) GO functional enrichment analysis showed that the differentially expressed proteins were mostly related to the regulation of lipid metabolism and transport. (3) The KEGG enrichment analysis showed the differentially expressed proteins were significantly enriched in cholesterol metabolism and proteasome pathways, which may regulate the differentiation of osteoblasts and osteoclasts via important pathways such as Wnt-β-catenin and RANKL/RANK/OPG. (4) To conclude, isoflavones extracted from chickpea sprout are involved in the expression of numerous lipoproteins and signaling pathway proteins in serum and can play a role in the prevention and treatment of osteoporosis through various signaling pathways.

Key words: osteoporosis, isoflavones extracted from chickpea sprouts, TMT technique, proteomic analysis, lipid metabolism, signaling pathway

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