红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

doi:10.16098/j.issn.0529-1356.2023.05.006

解剖学报 ›› 2023, Vol. 54 ›› Issue (5): 538-545.doi: 10.16098/j.issn.0529-1356.2023.05.006

红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

何可可¹ 李远迪¹文廷浩¹ 朱婕¹ 高杰^1,3 胡蓉^1,3韩锋⁴ 苏敏^1,2*

1.贵州医科大学基础医学院组织学与胚胎学教研室，贵阳 550025; 2.贵州医科大学细胞工程生物医药技术国家地方联合工程实验室，贵州省再生医学重点实验室，中国医学科学院成体干细胞转化研究重点实验室，贵阳 550025; 3.贵州医科大学转化医学研究中心，贵阳 550025; 4.贵州医科大学附属医院神经外科，贵阳 550004

收稿日期:2022-12-29 修回日期:2023-03-16 出版日期:2023-10-06 发布日期:2023-12-25
通讯作者: 苏敏 E-mail:summ30@163.com
基金资助:
国家自然科学基金;国家自然科学基金;贵州省科技支撑计划;贵州省科技支撑计划;贵州省卫健委科技基金

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6/STAT3/ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice encephalomyelitis mice

HE Ke-ke¹ LI Yuan-di¹ WEN Ting-hao¹ ZHU Jie1 GAO Jie^1,3HU Rong^1,3 HAN Feng⁴ SU Min^1,2*

1. Department of Histology and Embryology, Basic Medical College, Guizhou Medical University, Guiyang 550025, China;

2.National Joint Local Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key

Laboratory of Regenerative Medicine, Key Laboratory of Adult Stem Cell Translational Research(Chinese Academy of Medical Sciences), Guizhou Medical University, Guiyang 55025, China; 3.Translational Medicine Research Center, Guizhou Medical University, Guiyang 55025, China; 4.Department of Neurosurgery, the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

Received:2022-12-29 Revised:2023-03-16 Online:2023-10-06 Published:2023-12-25

摘要/Abstract

摘要：

目的探讨小鼠实验性自身免疫性脑脊髓炎（EAE）模型中，外源性和内源性红细胞膜相关蛋白（ERMAP）通过白细胞介素6/信号转录及转录激活因子3（IL-6/STAT3）/维甲酸相关孤核受体γt/（ROR-γt）信号通路对辅助性T细胞17（Th17）细胞分化的影响。方法流式细胞术验证不同浓度ERMAP-Ig融合蛋白功能；琼脂糖凝胶电泳鉴定ERMAP基因敲除小鼠。流式细胞术检测ERMAP-Ig融合蛋白在体外对Th17细胞分化影响。40只6周龄普通C57BL/6小鼠随机分为2组建立EAE模型：对照（control-Ig和ERMAP-Ig组，每组20只；记录临床评分；流式细胞术检测EAE小鼠体内Th17细胞分化情况。40只6周龄已鉴定的野生型和ERMAP基因敲除小鼠分为2组建立EAE模型：ERMAP+/+和ERMAP-/-组，每组20只。记录临床评分；脊髓HE和固蓝（LFB）染色并进行组织学半定量评分。流式细胞术检测IL-17A+CD4+ T细胞百分比；Western blotting检测IL-6、白细胞介素17（IL-17）、STAT3、磷酸化STAT3（p-STAT3）、ROR-γt蛋白水平表达；Real-time PCR检测IL-17、肿瘤坏死因子α（TNF-α）、IL-6、STAT3和ROR-γt的mRNA表达；ELISA检测细胞水平IL-17和TNF-α的表达。结果 1.外源性ERMAP-Ig融合蛋白抑制体内外Th17细胞分化且减轻小鼠EAE症状。2.与对照组相比，ERMAP-/-组EAE小鼠炎性浸润和脱髓鞘症状加重，Th17分泌IL-17A增加。3.内源性ERMAP基因敲除后，IL-17、TNF-α、IL-6、STAT3及ROR-γt表达均增加。差异均有统计学意义（P<0.05）。

结论 ERMAP可能通过IL-6/STAT3/ROR-γt信号通路调控Th17细胞分化，参与小鼠EAE发生发展。

关键词:

红细胞膜相关蛋白, 实验性自身免疫性脑脊髓炎, 辅助性T细胞17, 白细胞介素-6, 信号转录及转录激活因子3, 维甲酸受体相关孤核受体γt, 流式细胞术, 小鼠

Abstract:

Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein (ERMAP) on helper T cell 17(Th17) cell differentiation through interleukin 6/signal transducers and activators of transcription 3/retionoid-related orphan nuclear receptor-γt(IL-6/STAT3/ROR-γt) signal pathway in the mouse model of experimental autoimmune encephalomyelitis (EAE). Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations; Agarose gel electrophoresis was performed to identify ERMAP knockout mice. Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro. Forty 6-week-old normal C57BL/6 mice were randomly divided into 2 groups to establish EAE models, control-Ig and ERMAP-Ig groups, with 20 mice in each group; Clinical scores were recorded; Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo. Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models. Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models, ERMAP+/+ and ERMAP-/- groups, with 20 mice in each group. Clinical scores were recorded; Spinal cord HE and luxol fast blue(LFB) staining and histological semi-quantitative scoring were performed. Flow cytometry was performed to detect the percentage of IL-17A+CD4+ T cells; Western blotting was performed to detect the expression of IL-6, IL-17, STAT3, p-STAT3, and ROR-γt protein levels; Real-time PCR was performed to detect the mRNA expression of IL-17, TNF-α, IL-6, STAT3, and ROR-γt; ELISA was performed to detect the expression of IL-17 and TNF-α at the cellular level was detected by ELISA. Results 1. Exogenous ERMAP-Ig fusion protein inhibited Th17 cell differentiation and attenuated EAE symptoms in mice. 2. Compared with the control group, ERMAP-/- EAE mice showed increased inflammation and demyelination symptoms and increased Th17 secretion of IL-17A. 3. Endogenous ERMAP knockdown resulted in increased expression of IL-17, TNF-α, IL-6 STAT3, and ROR-γt. All differences were statistically significant(P<0.05). Conclusion ERMAP may regulate Th17 cell differentiation through the IL-6/STAT3/ROR-γt signaling pathway and is involved in the development of EAE in mice.

Key words:

Erythrocyte membrane-associated protein, Experimental autoimmune encephalomyelitis, Thelper cell 17, Interleukin-6, Signal transducers and activators of transcription 3, Retinoid acid receptor related orphan receptor-γt, , Flow cytometry, Mouse

中图分类号:

何可可李远迪文廷浩朱婕高杰胡蓉韩锋苏敏. 红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化[J]. 解剖学报, 2023, 54(5): 538-545.

HE Ke-ke LI Yuan-di WEN Ting-hao ZHU Jie GAO Jie HU Rong HAN Feng SU Min .

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6/STAT3/ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice encephalomyelitis mice

[J]. Acta Anatomica Sinica, 2023, 54(5): 538-545.

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红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6/STAT3/ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice encephalomyelitis mice

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