基于TMT和PRM技术筛选车叶草苷干预CUMS大鼠海马组织的差异表达蛋白

doi:10.16431/j.cnki.1671-7236.2023.11.020

摘要/Abstract

摘要： 【目的】基于蛋白质组学探讨车叶草苷对慢性温和应激（chronic unpredictable mild stress，CUMS）抑郁模型大鼠海马组织的保护作用机制，为车叶草苷的开发利用提供依据。【方法】选取45只雄性SD大鼠预饲1周后，随机分为3组：空白组、模型组、车叶草苷组，每组各15只；空白组每笼饲养3只，其他组单笼饲养。试验周期为5周，于试验第3周开始，温和不可预知性刺激同时给药，车叶草苷组按0.070 g/kg BW剂量灌胃给药，空白组、模型组灌服等体积的生理盐水，每天一次。造模完成后提取大鼠海马组织总蛋白，利用同位素串联质谱标签（tandem mass tags，TMT）定量标记技术进行抑郁模型大鼠海马蛋白质组学分析，筛选潜在差异表达蛋白，借助平行反应监测（parallel reaction monitoring，PRM）技术对候选蛋白在海马组织中的表达情况进行靶向验证。【结果】筛选出车叶草苷组、模型组之间6个上调和17个下调的差异表达蛋白；GO功能和KEGG通路富集分析表明，它们主要参与神经活性配体-受体相互作用、互补和协同调节级联、胃酸分泌等信号通路；PRM成功验证缓激肽（BK）、免疫球蛋白（IGHM及ILD）、蛋白酶体激活剂复合物亚基1（PSME1）4个蛋白表达趋势与TMT定量结果一致，可能是车叶草苷调节抑郁的潜在靶标蛋白。车叶草苷通过参与调控神经活性配体-受体相互作用等信号通路的关键靶标、下调BK蛋白表达对神经元发挥直接或间接的保护作用、上调与免疫球蛋白IgG表达显著相关的PSME1蛋白水平产生免疫抑制、使免疫蛋白浓度趋于正常等途径保护海马组织。【结论】车叶草苷可能通过多途径协同作用减轻机体抑郁症状。本研究结果为深入探索CUMS模型病理机制及阐明车叶草苷调控海马组织的蛋白质或通路提供参考。

关键词: 同位素串联质谱标签(TMT); 平行反应监测(PRM); 车叶草苷; 蛋白质组学; 海马组织

Abstract: 【Objective】 This study was aimed to explore the protective mechanism of asperuloside on hippocampus in chronic unpredictable mild stress (CUMS) rats based on proteomics, and provide a basis for the development and utilization of asperuloside.【Method】 Forty-five male SD rats were chosen and randomly divided into three groups after one week pre-feed with 15 rats in each group, including the blank, model and asperuloside groups.In the blank group, 3 rats were raised in one cage, while in the other groups, one cage for one rat.The experimental period was 5 weeks, stimulated mice while medication administration starting from the 3rd week of the experiment.The rats in blank and model groups were given equal volume of saline by oral administration while in the asperuloside group was given a dose of 0.070 g/kg BW by intravenous injection once a day.The total hippocampus protein was extracted and the proteomics analysis of the hippocampus of depression rats was carried out by tandem mass tags (TMT)-LC-MS/MS technique to screen potential differential expression proteins.In addition, parallel reaction monitoring (PRM) was used to verify the expression of selected biomarkers in hippocampus.【Result】 A total of 6 up-regulated and 17 down-regulated proteins were screened between asperuloside and model groups.GO function and KEGG pathway enrichment analysis showed that the differentially expressed proteins were involved in signaling pathways such as neuroactive ligand-receptor interaction, complementary and synergistic regulation cascade and gastric acid secretion.PRM was used to verify the expression trend of bradykinin, immunoglobulin (IGHM and ILD) and proteasome activator complex subunit 1 (PSME1) were consistent with TMT quantitative results, it might be potential target proteins for the regulation of depression by asperuloside.Asperuloside could protect hippocampus through various pathways such as controlling key targets involved in regulating neuroactive ligand receptor interactions signaling pathways, exerting direct or indirect protective effect on neurons by down-regulating BK protein expression, up-regulating PSME1 protein levels significantly correlated with IgG leads to immunosuppression and normalization of immune protein concentration.【Conclusion】 Asperuloside might alleviate depression symptoms through multiple synergistic effects.The results could provide a references for further exploring the pathological mechanisms of the CUMS model and the proteins or pathways regulated by asperuloside in hippocampus.

Key words: tandem mass tags (TMT); parallel reaction monitoring (PRM); asperuloside; proteomics; hippocampus

中图分类号:

S816.41

尹丽, 卢成淑, 曾国芳, 曾诗媛, 吕佳榆, 王华坤. 基于TMT和PRM技术筛选车叶草苷干预CUMS大鼠海马组织的差异表达蛋白[J]. 中国畜牧兽医, 2023, 50(11): 4513-4525.

YIN Li, LU Chengshu, ZENG Guofang, ZENG Shiyuan, LYU Jiayu, WANG Huakun. Screening of Differentially Expressed Proteins in Asperuloside Intervention CUMS Rats Hippocampus Based on TMT and PRM Techniques[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4513-4525.

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