菊苣酸介导外泌体对巨噬细胞免疫抑制的影响

doi:10.16431/j.cnki.1671-7236.2023.10.036

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (10): 4223-4230.doi: 10.16431/j.cnki.1671-7236.2023.10.036

菊苣酸介导外泌体对巨噬细胞免疫抑制的影响

白莉霞¹, 刘希望¹, 杨亚军¹, 秦哲¹, 李世宏¹, 葛闻博¹, 许笑¹, 冯晨婧^1,2, 李剑勇¹

1. 中国农业科学院兰州畜牧与兽药研究所, 农业农村部兽用药物创制重点实验室, 甘肃省新兽药工程重点实验室, 兰州 730050;
2. 天津农学院动物科学与动物医学学院, 天津市农业动物繁育与健康养殖重点实验室, 天津 300384

收稿日期:2023-05-17 出版日期:2023-10-05 发布日期:2023-09-26
通讯作者: 李剑勇 E-mail:lijy1971@163.com
作者简介:白莉霞,E-mail:bailx552369@163.com
基金资助:
甘肃省青年科技基金计划(21JR7RA031)；中国农业科学院基本科研业务费专项所级统筹项目(1610032021013)

Effect of Cichoric Acid Mediated Exosomes on Macrophage Immunosuppression

BAI Lixia¹, LIU Xiwang¹, YANG Yajun¹, QIN Zhe¹, LI Shihong¹, GE Wenbo¹, XU Xiao¹, FENG Chenjing^1,2, LI Jianyong¹

1. Key Laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Pharmaceutical Development of Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China;
2. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China

Received:2023-05-17 Online:2023-10-05 Published:2023-09-26

摘要/Abstract

摘要： 【目的】建立巨噬细胞免疫抑制模型,探究菊苣酸(cichoric acid,CA)对磷酰胺氮芥(phosphoramide mustard,PM)诱导巨噬细胞免疫抑制的作用及调控机制,为将CA开发为临床防治免疫抑制性疾病的新型药物提供理论依据。【方法】以巨噬细胞源性外泌体为研究对象,采用ExoQuick免疫沉淀试剂盒提取外泌体,用透射电镜(transmission electron microscopy,TEM)、纳米颗粒跟踪分析技术(nanoparticle tracking analysis,NTA)、Western blotting鉴定外泌体形态、粒径大小与浓度,以及表面标志蛋白CD63、HSP90的表达。同时,采用浓度为3 μmol/L 的PM诱导巨噬细胞24 h,建立免疫抑制模型后,给予低、中、高剂量CA(25、50、100 μmol/L)进行干预,通过CCK-8测定细胞活力、中性红检测细胞吞噬能力及NTA测定外泌体粒径大小与浓度,研究CA介导外泌体增强免疫的作用机制。【结果】提取的巨噬细胞源性外泌体杯状形态完整,粒径在30~200 nm之间,标志蛋白表达清晰,具有良好的生物活性。由CCK-8、中性红及NTA测定结果可见,CA在3.12~400 μmol/L浓度范围内对巨噬细胞无毒性作用;与PM模型组相比,低、中、高剂量CA可极显著提高免疫抑制巨噬细胞活力及其吞噬能力(P＜0.01),极显著促进外泌体的分泌(P＜0.01)。【结论】本研究结果表明,CA可能通过调控巨噬细胞源性外泌体分泌,增强其细胞活力与吞噬能力激活巨噬细胞,揭示了CA增强免疫的潜在作用机理。

关键词: 菊苣酸; 巨噬细胞; 外泌体; 磷酰胺氮芥; 免疫抑制

Abstract: 【Objective】 The macrophage immunosuppression model was established in vitro to investigate the effect and regulatory mechanism of cichoric acid (CA) on macrophage immunosuppression induced by phosphoramide mustard (PM),which would provide a theoretical basis for the development of CA as a new drug for clinical prevention and treatment of immunosuppressive diseases.【Method】 The macrophage-derived exosomes were extracted by ExoQuick immunoprecipitation kit.The morphology,particle size and concentration of exosomes as well as the expressions of surface marker proteins CD63 and HSP90 were identified by transmission electron microscopy (TEM),nanoparticle tracking analysis (NTA) and Western blotting.Meanwhile,macrophages were induced by PM with a concentration of 3 μmol/L for 24 h,the immunosuppression model was established,then low,medium and high doses of CA (25,50,100 μmol/L) were given for intervention.CCK-8 was used to measure cell viability,neutral red was used to measure phagocytosis ability,and NTA was used to measure exosome particle size and concentration,so as to study the mechanism of CA mediated exosomes to enhance immunity.【Result】 The extracted macrophage derived exosomes had intact cup shape,particle size between 30-200 nm,clear expression of marker proteins,and good biological activity.According to the results of CCK-8,neutral red,and NTA measurements,CA had no toxic effect on macrophages in the concentration range of 3.12-400 μmol/L.Compared with PM model group,low,medium and high doses of CA could extremely significantly improve the activity and phagocytosis of immunosuppressive macrophages (P＜0.01),promote the secretion of exosomes observably (P＜0.01).【Conclusion】 CA might activate macrophages by regulating the secretion of macrophage-derived exosomes,enhancing the cell viability and phagocytosis ability,revealing the potential mechanism of CA in enhancing immunity.

Key words: cichoric acid; macrophages; exosomes; phosphoramide mustard; immunosuppression

中图分类号:

S859.79

白莉霞, 刘希望, 杨亚军, 秦哲, 李世宏, 葛闻博, 许笑, 冯晨婧, 李剑勇. 菊苣酸介导外泌体对巨噬细胞免疫抑制的影响[J]. 中国畜牧兽医, 2023, 50(10): 4223-4230.

BAI Lixia, LIU Xiwang, YANG Yajun, QIN Zhe, LI Shihong, GE Wenbo, XU Xiao, FENG Chenjing, LI Jianyong. Effect of Cichoric Acid Mediated Exosomes on Macrophage Immunosuppression[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4223-4230.

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菊苣酸介导外泌体对巨噬细胞免疫抑制的影响

Effect of Cichoric Acid Mediated Exosomes on Macrophage Immunosuppression

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