机构地区:[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
出 处:《癌变.畸变.突变》2025年第2期121-127,133,共8页Carcinogenesis,Teratogenesis & Mutagenesis
基 金:国家自然科学基金(32171231)。
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。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-γinhibitor 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-γin the leflunomide group increased,and its inhibitors T0070907 reversed the increase of antiinflammatory 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-γto regul
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
分 类 号:R114[医药卫生—卫生毒理学]
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