来氟米特调控巨噬细胞极化及其机制研究

doi:10.3969/j.issn.1004-616x.2025.02.005

癌变·畸变·突变 ›› 2025, Vol. 37 ›› Issue (2): 121-127,133.doi: 10.3969/j.issn.1004-616x.2025.02.005

来氟米特调控巨噬细胞极化及其机制研究

张瑜娇^1,2, 姜晓旭³, 白瑞萍², 黄新怡², 王钊², 吴昊², 于卫华², 刘启玲¹

1. 陕西中医药大学公共卫生学院, 陕西咸阳 712046;
2. 空军军医大学军事预防医学系军事毒理学与防化医学教研室, 特殊作业环境危害评估与防治教育部重点实验室, 陕西省自由基生物学与医学重点实验室, 陕西西安 710032;
3. 空军军医大学基础医学院计算机基础教研室, 陕西西安 710032

收稿日期:2024-09-27 修回日期:2025-03-10 出版日期:2025-03-30 发布日期:2025-04-11
通讯作者: 于卫华，刘启玲
作者简介:张瑜娇，E-mail:zyj11211584@163.com。
基金资助:
国家自然科学基金(32171231)

Regulation and mechanism of leflunomide on anti-inflammatory differentiation of macrophages

ZHANG Yujiao^1,2, JIANG Xiaoxu³, BAI Ruiping², HUANG Xinyi², WANG Zhao², WU Hao², YU Weihua², LIU Qiling¹

1. School of Public Health, Shaanxi University of Chinese Medicine, Xianyang 712046;
2. Key Lab of Hazard Assessment and Control in Special Operational Environment of Ministry of Education, Shaanxi Provincial Key Lab of Free Radical Biology and Medicine, Department of Toxicology, School of Public Health, Air Force Medical University, Xi'an 710032;
3. Computer Department of Basic Medicine School, Air Force Medical University, Xi'an 710032, Shaanxi, China

Received:2024-09-27 Revised:2025-03-10 Online:2025-03-30 Published:2025-04-11

摘要/Abstract

摘要： 目的：探讨来氟米特对巨噬细胞极化的影响及其分子机制。方法：200 mmol/L来氟米特分别处理小鼠单核巨噬细胞(RAW264.7) 0、1、3、6、12和24 h后，采用实时荧光定量PCR(qPCR)法检测细胞沉淀中抗炎因子(IL-10、Arg-1、CD206)、促炎因子(TNF-α、IL-6)以及线粒体融合基因(MFN1、MFN2)的mRNA表达水平；ELISA法检测不同时长来氟米特处理后细胞培养上清液抗炎因子含量；DCFH-DA和Mito-LX染色法检测细胞内总活性氧(ROS)和线粒体ROS水平；Mitotracker-Green染色激光共聚焦观察细胞线粒体形态；试剂盒测定细胞内氧化产物丙二醛(MDA)含量。分别采用qPCR和Western blot法测定过氧化物酶体增殖物激活受体-γ(PPAR-γ)的mRNA和蛋白表达；PPAR-γ抑制剂T0070907(40 mmol/L)预处理细胞4 h后，分别采用qPCR和Western blot法检测细胞抗炎因子的变化。结果：qPCR和ELISA试验检测结果表明，相较于对照组，来氟米特处理1、3、6、12和24 h 后细胞的抗炎因子IL-10、Arg-1、CD206的mRNA相对表达水平升高(P<0.05)；线粒体融合基因MFN1、MFN2的mRNA和蛋白表达水平升高(P<0.05)，线粒体长度增加(P<0.05)，细胞和线粒体内ROS和MDA含量降低(P<0.05)。相较于空白对照组，来氟米特组PPAR-γ的mRNA和蛋白表达升高(P<0.05)，而其抑制剂T0070907可逆转来氟米特诱导抗炎因子升高和促炎因子降低(P<0.05)。结论：线粒体融合激动剂来氟米特可通过降低ROS生成和激活PPAR-γ调控巨噬细胞极化，可能作为抗炎治疗的潜在靶点。

关键词: 来氟米特, 巨噬细胞, 抗炎, 过氧化物酶体增殖物激活受体-γ, 线粒体融合

Abstract: OBJECTIVE：To investigate the effect and molecular mechanism of leflunomide on the anti-inflammatory differentiation of macrophages. METHODS：Mouse mononuclear macrophages (RAW264.7) were treated with 200 mmol/L leflunomide for 0，1，3，6，12 and 24 h. From these cells，mRNA expression levels of anti-inflammatory factors (IL-10，Arg-1，CD206)，pro-inflammatory factors (TNF-α，IL-6) and mitochondrial fusion genes (MFN1 and MFN2) in cell pellet were detected by real-time quantitative PCR (qPCR). ELISA was used to detect the content of anti-inflammatory factors in cell supernatants after flumid treatment for different years. DCFH-DA and Mito-LX staining were used to detect levels of total reactive oxygen species (ROS) and mitochondrial ROS. Mitotracker-Green staining laser confocal was used to determine cell mitochondrial morphology. Kits were used to determine content of malondialdehyde (MDA)，an intracellular oxidation product. The mRNA and protein expressions of peroxisome proliferator-activated receptor-γ (PPAR-γ) genes were determined by qPCR and Western blot，respectively. PPAR-γ inhibitor T0070907 (40 mmol/L) were pretreated for 4 h，and the changes of anti-inflammatory factors were detected by qPCR and Western blot，respectively. RESULTS：Results from the qPCR and ELISA analyses showed that the relative mRNA expression levels of anti-inflammatory factors IL-10，Arg-1 and CD206 treated with leflunomide were significantly higher than those in the control group (P<0.05). Expression of mitochondrial fusion genes MFN1 and MFN2 increased，mitochondrial length increased，and contents of ROS and MDA in cells and mitochondria decreased (P<0.05). Compared with the blank control group，mRNA and protein expression of PPAR-γ in the leflunomide group increased，and its inhibitors T0070907 reversed the increase of anti-inflammatory factors and the decrease of pro-inflammatory factors induced by leflunomide (P<0.05). CONCLUSION：The mitochondrial fusion agonist leflunomide may be useful for anti-inflammatory therapy by reducing ROS production and activating PPAR-γ to regulate the anti-inflammatory differentiation of macrophages.

Key words: leflunomide, macrophages, anti-inflammatory, peroxisome proliferator-activated receptor-γ, mitochondrial fusion

中图分类号:

R114

张瑜娇, 姜晓旭, 白瑞萍, 黄新怡, 王钊, 吴昊, 于卫华, 刘启玲. 来氟米特调控巨噬细胞极化及其机制研究[J]. 癌变·畸变·突变, 2025, 37(2): 121-127,133.

ZHANG Yujiao, JIANG Xiaoxu, BAI Ruiping, HUANG Xinyi, WANG Zhao, WU Hao, YU Weihua, LIU Qiling. Regulation and mechanism of leflunomide on anti-inflammatory differentiation of macrophages[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2025, 37(2): 121-127,133.

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来氟米特调控巨噬细胞极化及其机制研究

Regulation and mechanism of leflunomide on anti-inflammatory differentiation of macrophages

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