[1]刘玉玲,赵璐,杨超,等.c-Myc抑制剂10058-F4对实验性自身免疫性葡萄膜炎小鼠模型Th17细胞比例及细胞因子表达的影响[J].眼科新进展,2023,43(6):429-433.[doi:10.13389/j.cnki.rao.2023.0086]
　LIU Yuling,ZHAO Lu,YANG Chao,et al.Effects of c-Myc inhibitor 10058-F4 on T helper 17 cell proportion and cytokine expression in mouse model of experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2023,43(6):429-433.[doi:10.13389/j.cnki.rao.2023.0086]
点击复制

c-Myc抑制剂10058-F4对实验性自身免疫性葡萄膜炎小鼠模型Th17细胞比例及细胞因子表达的影响/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
43卷
期数:
2023年6期
页码:
429-433
栏目:
实验研究
出版日期:
2023-06-05

文章信息/Info

Title:
Effects of c-Myc inhibitor 10058-F4 on T helper 17 cell proportion and cytokine expression in mouse model of experimental autoimmune uveitis
作者:
刘玉玲; 赵璐; 杨超; 陈思思; 魏瑞华; 粘红
300384　天津市，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
Author(s):
LIU Yuling; ZHAO Lu; YANG Chao; CHEN Sisi; WEI Ruihua; NIAN Hong
Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China
关键词:
辅助性T细胞17; 实验性自身免疫性葡萄膜炎; c-Myc抑制剂; 10058-F4
Keywords:
T helper 17 cells;  experimental autoimmune uveitis;  c-Myc inhibitor;  10058-F4
分类号:
R773
DOI:
10.13389/j.cnki.rao.2023.0086
文献标志码:
A
摘要:
目的　探讨c-Myc抑制剂10058-F4对实验性自身免疫性葡萄膜炎（EAU）小鼠模型光感受器间维生素A类结合蛋白1-20（IRBP1-20）特异性辅助性T细胞17（Th17）细胞比例及细胞因子表达的影响。
方法　取健康C57BL/6J小鼠（8~10周龄）6只，采用随机数字表法将小鼠分为正常对照组、EAU组，每组3只。EAU组小鼠腹腔内注射350 ng百日咳毒素，单后足和脊柱两侧皮下注射含150 μg IRBP1-20、0.8 mg结核分枝杆菌H37RA和弗氏不完全佐剂的乳化剂以诱导EAU模型；正常对照组小鼠不给予特殊干预。免疫后13 d，行间接检眼镜检查并通过小动物视网膜成像系统可视化检测小鼠眼底炎性病灶，HE染色观察视网膜组织病理学改变，实时荧光定量PCR（qRT-PCR）检测T细胞和眼球组织中c-Myc mRNA的表达。将EAU模型小鼠T细胞分为对照组和10058-F4组，10058-F4组T细胞加入50 μmol·L-1 10058-F4，对照组T细胞不给予药物干预。培养6 h后，洗去药物，将两组T细胞与IRBP1-20（10 mg·L-1）及骨髓来源树突细胞共培养，同时加入20 μg·L-1 白细胞介素（IL）-23（Th17细胞诱导条件）。采用qRT-PCR检测c-Myc、维甲酸相关核孤儿受体γt（RORγt）、IL-17A、IL-17F和粒细胞-巨噬细胞集落刺激因子（GM-CSF） mRNA相对表达量，ELISA检测共培养细胞上清液中IL-17含量，流式细胞仪检测共培养细胞中Th17细胞比例。
结果　本研究EAU组小鼠模型建立成功。qRT-PCR检测结果显示，EAU组小鼠T细胞、眼球组织中c-Myc mRNA相对表达量分别为1.85±0.37、2.31±0.47，较正常对照组（1.01±0.02、0.99±0.05）均明显升高，差异均有统计学意义（P=0.017、0.008）；10058-F4组T细胞c-Myc mRNA相对表达量为0.57±0.03，明显低于对照组（1.04±0.02），差异有统计学意义（P<0.001）。ELISA检测结果显示，10058-F4组共培养细胞上清液中IL-17含量明显低于对照组，差异有统计学意义（P=0.025）。流式细胞仪检测结果显示，10058-F4组共培养细胞中Th17细胞比例为（17.97±0.91）%，明显低于对照组［（22.50±0.90）%］，差异有统计学意义（P=0.004）。qRT-PCR检测结果显示，10058-F4组T细胞RORγt、IL-17A、IL-17F、GM-CSF mRNA相对表达量分别为0.52±0.11、0.51±0.06、0.58±0.15、0.75±0.03，明显低于对照组（1.01±0.01、1.00±0.01、1.02±0.02、1.01±0.02），差异均有统计学意义（均为P<0.05）。
结论　c-Myc抑制剂10058-F4可显著降低T细胞中c-Myc表达水平，抑制Th17细胞特异性转录因子RORγt表达及效应细胞因子IL-17A、IL-17F、GM-CSF的产生，负向调控IRBP1-20特异性Th17细胞免疫反应。
Abstract:
Objective　To investigate the effects of c-Myc inhibitor 10058-F4 on the proportion of interphotoreceptor retinoid-binding protein 1-20 (IRBP1-20)-specific T helper 17 (Th17) cells and expression of cytokines in the mouse model of experimental autoimmune uveitis (EAU).
Methods　Six healthy C57BL/6J mice (8-10 weeks old) were included and randomly divided into the normal control (NC) group and the EAU group using the random number table method, 3 for each group. Mice in the EAU group were injected intraperitoneally with 350 ng pertussis toxin and injected subcutaneously with an emulsion containing 150 μg IRBP1-20, 0.8 mg mycobacterium tuberculosis H37RA and Freund’s incomplete adjuvant on a hind leg and flank to induce the EAU model, while mice in the NC group were not given any special interventions. At 13 d after the immunization, fundus inflammatory lesions in EAU mice were evaluated by indirect ophthalmoscopy and visualized via retinal imaging system for small animals, hematoxylin-eosin staining was performed for histopathological examination of the retina, and quantitative real-time PCR (qRT-PCR) was used to detect the messenger ribonucleic acid (mRNA) expression of c-Myc in T cells and eyeball tissues. T cells isolated from EAU mice were divided into the control group and the 10058-F4 group, T cells in the 10058-F4 group were added with 50 μmol·L-1 10058-F4, and T cells in the control group were not given drug treatment. After cultivation for 6 h, T cells in the two groups were washed and cultured with IRBP1-20 (10 mg·L-1), bone marrow-derived dendritic cells and 20 μg·L-1 interleukin (IL)-23 (Th17 cell inducing conditions). Relative mRNA expressions of c-Myc, retinoic acid-related nuclear orphan receptor γt (RORγt), IL-17A, IL-17F and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected by qRT-PCR, the content of IL-17 in the supernatant of co-cultured cells was detected by enzyme-linked immunosorbent assay (ELISA), and the proportion of Th17 cells in the co-cultured cells was analyzed by flow cytometry.
Results　Mouse model in the EAU group was established successfully. The qRT-PCR analysis showed that the relative mRNA expressions of c-Myc in T cells and eyeball tissues in the EAU group were 1.85±0.37 and 2.31±0.47, which were significantly higher than 1.01±0.02 and 0.99±0.05 in the NC group (P=0.017, 0.008); the relative mRNA expression of c-Myc in T cells of the 10058-F4 group was 0.57±0.03, which was significantly lower than 1.04±0.02 in the control group (P<0.001). ELISA showed that the content of IL-17 in the supernatant of co-cultured cells in the 10058-F4 group was significantly lower than that in the control group (P=0.025). The results of flow cytometry showed that the proportion of Th17 cells in the co-cultured cells was (17.97±0.91)% in the 10058-F4 group, which was significantly lower than (22.50±0.90)% in the control group (P=0.004). The qRT-PCR analysis results demonstrated that the relative mRNA expressions of RORγt, IL-17A, IL-17F and GM-CSF were 0.52±0.11, 0.51±0.06, 0.58±0.15, and 0.75±0.03, respectively, in the 10058-F4 group, which were significantly lower than 1.01±0.01, 1.00±0.01, 1.02±0.02, and 1.01±0.02 in the control group (all P<0.05).
Conclusion　The c-Myc inhibitor 10058-F4 can significantly suppress the expression level of c-Myc in IRBP1-20-specific T cells, and inhibit the expression of Th17 cell-specific transcription factor RORγt and the production of pathogenic Th17-related cytokines IL-17A, IL-17F and GM-CSF, thus negatively regulating IRBP1-20-specific Th17 cell immunological response.

参考文献/References:

［1］　EGWUAGU C E,ALHAKEEM S A,MBANEFO E C.Uveitis:molecular pathogenesis and emerging therapies［J］.Front Immunol,2021,12:623725.
［2］　PRETE M,DAMMACCO R,FATONE M C,RACANELLI V.Autoimmune uveitis:clinical,pathogenetic,and therapeutic features［J］.Clin Exp Med,2016,16(2):125-136.
［3］　KIM J,CHUN J,AHN M,JUNG K,MOON C,SHIN T.Blood-retina barrier dysfunction in experimental autoimmune uveitis:the pathogenesis and therapeutic targets［J］.Anat Cell Biol,2022,55(1):20-27.
［4］　KLASKA I P,FORRESTER J V.Mouse models of autoimmune uveitis［J］.Curr Pharm Des,2015,21(18):2453-2467.
［5］　ZHONG Z,SU G,KIJLSTRA A,YANG P.Activation of the interleukin-23/interleukin-17 signalling pathway in autoinflammatory and autoimmune uveitis［J］.Prog Retin Eye Res,2021,80:100866.
［6］　GUO K,ZHANG X.Cytokines that modulate the differentiation of Th17 cells in autoimmune uveitis［J］.J Immunol Res,2021,2021:6693542.
［7］　CHEN Y H,LIGHTMAN S,CALDER V L.CD4(+) T-cell plasticity in non-infectious retinal inflammatory disease［J］.Int J Mol Sci,2021,22(17):9584.
［8］　GNANAPRAKASAM J N R,SHERMAN J W,WANG R.MYC and HIF in shaping immune response and immune metabolism［J］.Cytokine Growth Factor Rev,2017,35:63-70.
［9］　GUO Y,SUN D,ZHANG Y,YU X,FANG Y,LV C,et al.The neuropeptide cortistatin attenuates Th17 cell response through inhibition of glycolysis via GHSR1［J］.Int Immunopharmacol,2022,108:108843.
［10］　WANG R,DILLON C P,SHI L Z,MILASTA S,CARTER R,FINKELSTEIN D,et al.The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation［J］.Immunity,2011,35(6):871-882.
［11］　VERBIST K C,GUY C S,MILASTA S,LIEDMANN S,KAMINSKI M M,WANG R,et al.Metabolic maintenance of cell asymmetry following division in activated T lymphocytes［J］.Nature,2016,532(7599):389-393.
［12］　GOMEZ-RODRIGUEZ J,WOHLFERT E A,HANDON R,MEYLAN F,WU J Z,ANDERSON S M,et al.Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells［J］.J Exp Med,2014,211(3):529-543.
［13］　BANDUKWALA H S,GAGNON J,TOGHER S,GREENBAUM J A,LAMPERTI E D,PARR N J,et al.Selective inhibition of CD4+T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors［J］.Proc Natl Acad Sci U S A,2012,109(36):14532-14537.
［14］　MASSO-VALLES D,BEAULIEU M E,SOUCEK L.MYC,MYCL,and MYCN as therapeutic targets in lung cancer［J］.Expert Opin Ther Targets,2020,24(2):101-114.
［15］　CHEN J,CASPI R R.Clinical and functional evaluation of ocular inflammatory disease using the model of experimental autoimmune uveitis［J］.Methods Mol Biol,2019,1899:211-227.
［16］　MEITEI H T,LAL G.T cell receptor signaling in the differentiation and plasticity of CD4(+) T cells［J］.Cytokine Growth Factor Rev,2023,69:14-27.
［17］　CHI W,ZHU X,YANG P,LIU X,LIN X,ZHOU H,et al.Upregulated IL-23 and IL-17 in Behcet patients with active uveitis［J］.Invest Ophthalmol Vis Sci,2008,49(7):3058-3064.
［18］　SUGITA S,KAWAZOE Y,IMAI A,YAMADA Y,HORIE S,MOCHIZUKI M.Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behcet’s disease［J］.Arthritis Res Ther,2012,14(3):R99.
［19］　YOSEF N,SHALEK A K,GAUBLOMME J T,JIN H,LEE Y,AWASTHI A,et al.Dynamic regulatory network controlling TH17 cell differentiation［J］.Nature,2013,496(7446):461-468.
［20］　GHAFFARNIA R,NASROLLAHZADEH A,BASHASH D,NASROLLAHZADEH N,MOUSAVI S A,GHAFFARI S H.Inhibition of c-Myc using 10058-F4 induces anti-tumor effects in ovarian cancer cells via regulation of FOXO target genes［J］.Eur J Pharmacol,2021,908:174345.
［21］　ZHANG L,ZHANG W,SUN J,LIU K N,GAN Z X,LIU Y Z,et al.Nucleotide variation in histone H2BL drives crossalk of histone modification and promotes tumour cell proliferation by upregulating c-Myc［J］.Life Sci,2021,271:119127.
［22］　JIE Z,JINNA Z,JINGJUN Z,PENGCHENG L,FANG Y,QINYANG C,et al.Antitumor effects of 10058-F4 and curcumin in combination therapy for pancreatic cancer in vitro and in vivo［J］.J Healthc Eng,2022,2022:1620802.
［23］　TROP-STEINBERG S,AZAR Y.Is Myc an important biomarker? Myc expression in immune disorders and cancer［J］.Am J Med Sci,2018,355(1):67-75.
［24］　WEBB L M,NARVAEZ MIRANDA J,AMICI S A,SENGUPTA S,NAGY G,GUERAU-DE-ARELLANO M.NF-kappaB/mTOR/MYC axis drives PRMT5 protein induction after T cell activation via transcriptional and non-transcriptional mechanisms［J］.Front Immunol,2019,10:524.
［25］　TAN J,LIU H,HUANG M,LI N,TANG S,MENG J,et al.Small molecules targeting RORgammat inhibit autoimmune disease by suppressing Th17 cell differentiation［J］.Cell Death Dis,2020,11(8):697.
［26］　MEEHAN E V,WANG K.Interleukin-17 family cytokines in metabolic disorders and cancer［J］.Genes (Basel),2022,13(9):1643.
［27］　YASUDA K,TAKEUCHI Y,HIROTA K.The pathogenicity of Th17 cells in autoimmune diseases［J］.Semin Immunopathol,2019,41(3):283-297.
［28］　BETTELLI E,KORN T,OUKKA M,KUCHROO V K.Induction and effector functions of T(H)17 cells［J］.Nature,2008,453(7198):1051-1057.
［29］　ZHANG M,ZHOU L,WANG Y,DORFMAN R G,TANG D,XU L,et al.Faecalibacterium prausnitzii produces butyrate to decrease c-Myc-related metabolism and Th17 differentiation by inhibiting histone deacetylase 3［J］.Int Immunol,2019,31(8):499-514.
［30］　SHIOMI A,USUI T.Pivotal roles of GM-CSF in autoimmunity and inflammation［J］.Mediators Inflamm,2015,2015:568543.
［31］　KOMUCZKI J,TUZLAK S,FRIEBEL E,HARTWIG T,SPATH S,ROSENSTIEL P,et al.Fate-mapping of GM-CSF expression identifies a discrete subset of inflammation-driving T helper cells regulated by cytokines IL-23 and IL-1β［J］.Immunity,2019,50(5):1289-1304.e6.

相似文献/References:

[1]唐凯,郭大东,张莲,等. 龙胆泻肝汤对大鼠实验性自身免疫性葡萄膜炎的治疗作用[J].眼科新进展,2015,35(4):305.[doi:10.13389/j.cnki.rao.2015.0082]
　TANG Kai,GUO Da-Dong,ZHANG Lian,et al. Treatment efficacy of Longdan Xiegan Tang on rats with experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2015,35(6):305.[doi:10.13389/j.cnki.rao.2015.0082]
[2]符敏,吴伟,陆晓和,等.实验性自身免疫性葡萄膜炎T淋巴细胞亚群与Treg细胞的表达研究[J].眼科新进展,2016,36(3):227.[doi:10.13389/j.cnki.rao.2016.0061]
　FU Min,WU Wei,LU Xiao-He,et al.Changes of T-cell subpopulation and T regulatory cells in experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2016,36(6):227.[doi:10.13389/j.cnki.rao.2016.0061]
[3]唐凯,郭大东,卢秀珍,等.龙胆泻肝汤对葡萄膜炎大鼠关键炎症细胞因子表达的影响[J].眼科新进展,2017,37(7):610.[doi:10.13389/j.cnki.rao.2017.0155]
　TANG Kai,GUO Da-Dong,LU Xiu-Zhen,et al.Immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rats with experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2017,37(6):610.[doi:10.13389/j.cnki.rao.2017.0155]
[4]殷学伟,刘滨,魏慧霞,等.rno-miR-30b-5p对葡萄膜炎大鼠Notch1和Dll4基因表达的调控作用[J].眼科新进展,2019,39(1):012.[doi:10.13389/j.cnki.rao.2019.0003]
　YIN Xue-Wei,LIU Bin,WEI Hui-Xia,et al.Regulatory role of rno-miR-30b-5p inNotch1 and Dll4 geneexpressions in experimental autoimmune uveitis rats[J].Recent Advances in Ophthalmology,2019,39(6):012.[doi:10.13389/j.cnki.rao.2019.0003]
[5]殷学伟,刘滨,魏慧霞,等.龙胆泻肝汤抑制葡萄膜炎大鼠Notch信号通路活化的作用[J].眼科新进展,2019,39(7):615.[doi:10.13389/j.cnki.rao.2019.0142]
　YIN Xue-Wei,LIU Bin,WEI Hui-Xia,et al.Inhibitory role of longdanxiegan decoction in the activation of notch signaling pathway in treating experimental autoimmune uveitis rats[J].Recent Advances in Ophthalmology,2019,39(6):615.[doi:10.13389/j.cnki.rao.2019.0142]
[6]魏慧霞,殷学伟,吴姗姗,等.龙胆泻肝汤对实验性自身免疫性葡萄膜炎大鼠补体C4、MBL2表达的影响[J].眼科新进展,2020,40(2):105.[doi:10.13389/j.cnki.rao.2020.0026]
　WEI Huixia,YIN Xuewei,WU Shanshan,et al.Effect of Longdan Xiegan decoction on expression levels of C4 and MBL2 in experimental autoimmune uveitis rats[J].Recent Advances in Ophthalmology,2020,40(6):105.[doi:10.13389/j.cnki.rao.2020.0026]
[7]殷学伟,郭励劼,周梦贤,等.龙胆泻肝汤对实验性自身免疫性葡萄膜炎大鼠M1/M2巨噬细胞极化平衡的调控作用[J].眼科新进展,2021,41(5):408.[doi:10.13389/j.cnki.rao.2021.0085]
　YIN Xuewei,GUO Lijie,ZHOU Mengxian,et al.Regulatory role of Longdan Xiegan Decoction in polarization balance of M1/M2 macrophages in rats with experimental autoimmune uveitis[J].Recent Advances in Ophthalmology,2021,41(6):408.[doi:10.13389/j.cnki.rao.2021.0085]
[8]周梦贤,殷学伟,郭励劼,等.龙胆泻肝汤抑制Notch信号通路活化对实验性自身免疫性葡萄膜炎大鼠Th1、Th2细胞分化的影响[J].眼科新进展,2021,41(6):506.[doi:10.13389/j.cnki.rao.2021.0105]
　ZHOU Mengxian,YIN Xuewei,GUO Lijie,et al.Effects of Longdan Xiegan Decoction on Th1 and Th2 cell differentiation by inhibiting Notch pathway activation in experimental autoimmune uveitis rats[J].Recent Advances in Ophthalmology,2021,41(6):506.[doi:10.13389/j.cnki.rao.2021.0105]
[9]殷学伟,郭励劼,周梦贤,等.miR-30b-5p 抑制Notch信号通路活化对实验性自身免疫性葡萄膜炎的治疗作用[J].眼科新进展,2022,42(2):099.[doi:10.13389/j.cnki.rao.2022.0020]
　YIN Xuewei,GUO Lijie,ZHOU Mengxian,et al.Therapeutic effect of miR-30b-5p on experimental autoimmune uveitis by inhibiting the activation of Notch signaling pathway[J].Recent Advances in Ophthalmology,2022,42(6):099.[doi:10.13389/j.cnki.rao.2022.0020]
[10]郭晓宇,陈震,邢怡桥.短期高浓度葡萄糖摄入对小鼠实验性自身免疫性葡萄膜炎发展的作用[J].眼科新进展,2022,42(3):210.[doi:10.13389/j.cnki.rao.2022.0042]
　GUO Xiaoyu,CHEN Zhen,XING Yiqiao.Effect of short-term high glucose intake on experimental autoimmune uveitis in mice[J].Recent Advances in Ophthalmology,2022,42(6):210.[doi:10.13389/j.cnki.rao.2022.0042]
[11]钱丽丽,徐亚茹,胡芳,等.Th1、Th17细胞相关因子在实验性自身免疫性葡萄膜炎大鼠中的表达及作用[J].眼科新进展,2015,35(10):918.[doi:10.13389/j.cnki.rao.2015.0250]
　QIAN Li-Li,XU Ya-Ru,HU Fang,et al.Expression and roles of Th1,Th17 cytokines in experimental autoimmune uveitis of rats[J].Recent Advances in Ophthalmology,2015,35(6):918.[doi:10.13389/j.cnki.rao.2015.0250]
[12]王加利,杨超,陈思思,等.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[J].眼科新进展,2024,44(5):346.[doi:10.13389/j.cnki.rao.2024.0067]
　WANG Jiali,YANG Chao,CHEN Sisi,et al.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[J].Recent Advances in Ophthalmology,2024,44(6):346.[doi:10.13389/j.cnki.rao.2024.0067]

备注/Memo

备注/Memo:
国家自然科学基金项目（编号：81970793,82070929）；天津市医学重点学科（专科）建设项目（编号：TJYXZDXK-037A）
更新日期/Last Update: 2023-06-05