衣康酸二甲酯对树突状细胞分泌促炎因子以及实验性自身免疫性葡萄膜炎小鼠辅助性T细胞17的影响  

Effects of dimethyl itaconate on secretion of pro-inflammatory factors in dendritic cells and on T helper 17 cells in mice with experimental autoimmune uveitis

作　　者：王加利 杨超 陈思思 张开朗 魏瑞华 粘红 WANG Jiali;YANG Chao;CHEN Sisi;ZHANG Kailang;WEI Ruihua;NIAN Hong(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin International Jiont Research and Development Centre of Ophthalmology and Vision Science,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、眼视光学院、眼科研究所,国家眼耳鼻喉疾病临床医学研究中心天津市分中心,天津市视网膜功能与疾病重点实验室,天津市300384

出　　处：《眼科新进展》2024年第5期346-349,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:81970793,81770901);天津市教委科技计划项目(编号:2020KJ177);天津市临床重点学科(专科)建设项目(编号:TJLCZDXKT003)。

摘　　要：目的探究衣康酸二甲酯(DMI)对树突状细胞(DCs)分泌促炎因子的作用,并初步研究其对实验性自身免疫性葡萄膜炎(EAU)小鼠光感受器间维生素A类结合蛋白1-20(IRBP_(1-20))特异性辅助性T细胞17(Th17)的影响。方法分离C57BL/6J小鼠双侧股骨和胫骨得到骨髓细胞,利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4将其定向诱导分化为骨髓来源的DCs。诱导分化6 d,将细胞分为DMI组和PBS组,分别用250μmol·L^(-1)DMI及等量的磷酸盐缓冲液(PBS)预处理3 h后,每组加入100μg·L^(-1)脂多糖(LPS)刺激24 h。采用实时荧光定量PCR(qRT-PCR)检测两组DCs中促炎因子IL-6、IL-1β和IL-23 mRNA相对表达量。采用IRBP_(1-20)、弗氏不完全佐剂及结核分枝杆菌H37RA主动免疫小鼠构建EAU模型,免疫后13 d,将分离的EAU小鼠脾脏及淋巴结T细胞与PBS或DMI处理的DCs在含有IRBP_(1-20)的培养基中共培养,并向Th17方向极化。流式细胞仪检测共培养细胞中Th17细胞比例。ELISA检测共培养上清液中IL-17水平。qRT-PCR检测共培养细胞中维甲酸相关核孤儿受体γt(RORγt)、IL-17、IL-23R和GM-CSF mRNA相对表达量。结果qRT-PCR检测结果显示,DMI组DCs中IL-6、IL-1β和IL-23 mRNA相对表达量均明显低于PBS组,差异均有统计学意义(均为P<0.05)。流式细胞仪检测结果显示,DMI组共培养细胞中Th17细胞比例明显低于PBS组,差异有统计学意义(P<0.05)。ELISA检测结果显示,DMI组共培养上清液中IL-17水平明显低于PBS组,差异有统计学意义(P<0.05)。DMI组共培养细胞中IL-17、RORγt、IL-23R和GM-CSF mRNA相对表达量均明显低于PBS组,差异均有统计学意义(均为P<0.05)。结论DMI能够抑制DCs中促炎因子IL-6、IL-1β和IL-23的表达,进而负向调控IRBP_(1-20)特异性Th17细胞反应。Objective To investigate the effects of dimethyl itaconate(DMI)on the secretion of pro-inflammatory factors in dendritic cells(DCs)and on the interphotoreceptor retinoid-binding protein(IRBP)1-20-specific T helper 17(Th17)cells in mice with experimental autoimmune uveitis(EAU).Methods Bilateral femur and tibia of C57BL/6J mice were isolated to obtain bone marrow cells,and these bone marrow cells were directionally induced with granulocyte macrophage-colony-stimulating factor(GM-CSF)and interleukin(IL)-4 to differentiate DCs.After 6 days,DCs were randomly divided into the DMI group and the phosphate-buffered saline(PBS)group.Cells in the DMI group were pretreated with 250μmol·L^(-1)DMI,and cells in the PBS group were pretreated with the same volume of PBS for 3 hours.Afterwards,100μg·L^(-1)lipopolysaccharide was added in the every group to stimulate cells for 24 hours.The relative mRNA expression levels of IL-6,IL-1β,and IL-23 in DCs were measured by quantitative real-time polymerase chain reaction(qRT-PCR).The EAU model was constructed by actively immunizing mice with IRBP_(1-20),Freund’s incomplete adjuvant,and mycobacterium tuberculosis H37RA.Thirteen days after immunization,T cells in the spleen and lymph node isolated from EAU mice were cocultured with DMI-treated or PBS-treated DCs in the medium containing IRBP_(1-20).They polarized toward Th17 cells.The percentage of Th17 cells in the cocultured cells was detected by flow cytometry.The IL-17 level in the coculture supernatant was detected by enzyme-linked immunosorbent assay(ELISA).qRT-PCR was performed to detect the relative mRNA expression levels of retinoid-related orphan receptor gamma t(RORγt),IL-17,IL-23R,and GM-CSF in the cocultured cells.Results qRT-PCR analysis revealed that the relative mRNA expression levels of IL-6,IL-1β,and IL-23 in the DMI group were significantly lower than those in the PBS group(all P<0.05).Flow cytometry analysis showed that the proportion of Th17 cells in the cocultured cells in the DMI group was significantly low

关 键 词：衣康酸二甲酯 树突状细胞 辅助性T细胞17 实验性自身免疫性葡萄膜炎 

分 类 号：R773[医药卫生—眼科]