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-Ke; LI Yuan-Di; WEN Ting-Hao; ZHU Jie; GAO Jie; HU Rong; HAN Feng; SU Min

doi:10.16098/j.issn.0529-1356.2023.05.006

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Acta Anatomica Sinica ›› 2023, Vol. 54 ›› Issue (5) : 538-545. DOI: 10.16098/j.issn.0529-1356.2023.05.006

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*

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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

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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 https://doi.org/10.16098/j.issn.0529-1356.2023.05.006

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