磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路和白细胞介素17在小鼠实验性自身免疫性脑脊髓炎中的表达变化

doi:10.16098/j.issn.0529-1356.2021.03.005

解剖学报 ›› 2021, Vol. 52 ›› Issue (3): 358-364.doi: 10.16098/j.issn.0529-1356.2021.03.005

磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路和白细胞介素17在小鼠实验性自身免疫性脑脊髓炎中的表达变化

李倩¹ 高杰^1,2胡蓉¹ 韩锋³李红¹ 苏敏^1*

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.贵州医科大学基础医学科学研究中心，贵阳 550025; 3.贵州医科大学附属医院神经外科，贵阳 550004

收稿日期:2020-11-06 修回日期:2021-01-29 出版日期:2021-06-06 发布日期:2021-06-06
通讯作者: 苏敏 E-mail:summ30@163.com
基金资助:
黔科合平台人才;黔科合支撑;黔科合平台人才

Expression of phosphatidylinositol 3-kinase/protein kinase B/FoxO1 signal pathway and interleukin-17 in experimental autoimmune encephalomyelitis in mice

LI Qian¹ GAO Jie^1,2HU Rong¹ HAN Feng³ LI Hong¹ SU Min^1*

1.Department of Histology and Embryology，School of Basic Medical Sciences，Guizhou Medical University，Guiyang 550025，China; 2.Research Center for Basic Sciences of Medicine, Guizhou Medical University，Guiyang 550025，China; 3.Department of Neurosurgery， the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

Received:2020-11-06 Revised:2021-01-29 Online:2021-06-06 Published:2021-06-06
Contact: SU Min E-mail:summ30@163.com

摘要/Abstract

摘要：

目的探讨磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）/FoxO1和白细胞介素17（IL-17）与自身免疫性脑脊髓炎(EAE)发病的相关机制。方法将C57BL/6小鼠60只随机分为对照组和模型组（EAE），每组30只。采用髓鞘少突胶质细胞糖蛋白（MOG_35～55）联合完全弗氏佐剂诱导建立EAE模型。观察各组小鼠的行为学评分，并于评分4分时取各组小鼠的脊髓、脾脏和外周血。HE染色及Luxol fast blue染色观察脊髓炎性细胞浸润及髓鞘脱失情况；脾细胞在体外用a-CD3和MOG_35~55分别刺激7 d，收集其上清液，ELISA检测小鼠脾细胞上清液和血清中细胞因子白细胞介素17（IL-17）和干扰素γ（IFN-γ）的含量；流式细胞术检测小鼠脾脏中CD4+IL 17+细胞的百分比；Western blotting检测IL-17及PI3K/Akt/FoxO1在小鼠脊髓中表达。结果与对照组相比，EAE组小鼠行为学评分明显高于对照组(P<0.05)；HE染色及Luxol fast blue染色结果显示，EAE组脊髓病理组织表现出炎性浸润和髓鞘脱失显著增多(P<0.05)。 ELISA结果显示，EAE组血清中及体外培养脾细胞的上清中IL-17和IFN-γ的含量明显升高。流式结果表明，EAE组小鼠脾细胞中CD4+IL 17+T细胞的百分比明显增加(P<0.05)。Western blotting 结果显示，EAE组小鼠脊髓IL-17、磷酸化Akt（p-Akt）蛋白水平明显升高，但磷酸化FoxO1（p-FoxO1）蛋白水平显著降低(P<0.05)。结论 EAE组小鼠脊髓中促炎因子IL-17分泌增加，可能与PI3K/Akt/FoxO1信号通路活化进而激活T细胞功能有关。

关键词: 实验性自身免疫性脑脊髓炎, 磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路, Luxol fast blue染色, 流式细胞术, 免疫印迹法, 酶联免疫吸附法, 小鼠

Abstract:

Objective To investigate the relevant mechanisms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/FoxO1 and interleukin-17(IL-17) in the oneset of experimental autoimmune encephalomyelitis(EAE) mice. Methods Sixty C57BL/6 mice were randomly divided into control group and model group (EAE)， 30 in each group. The EAE model was induced by myelin oligodendrocyte glycoprotein (MOG_35-55) together with complete Freund’s adjuvant. The behavioral score of each group was observed. The spinal cord，spleen and peripheral blood were obtained when the behavioral score was 4. IL-17 and interferon-γ (IFN-γ) contents in mouse serum and supernatant of splenocytes stimulated with a-CD3 and MOG_35-55，respectively，in vitro for 7 days were detected by ELISA. The percentage of CD4+IL-17+ cells in the mouse spleen was monitored via flow cytometry. The expressions of IL-17 and PI3K/Akt/FoxO1 in the spinal cord were detected by Western blotting. Results Compared with the control group, the behavioral score of the EAE group was significantly higher than that of the control group. HE staining and Luxol fast blue staining indicated that EAE spinal cord increased prominently inflammatory infiltration and demyelination(P<0.05). IL-17 and IFN-γ contents of the EAE group in serum and supernatant of splenocytes cultured in vitro increased remarkably(P<0.05). The percentage of CD4+IL-17+ T cells in splenocytes also increased markedly(P<0.05). The protein levels of IL-17 and phosphorylated Akt (p-Akt)in the spinal cord of the EAE group increased observably，while that of phosphorylated FoxO1 (p-FoxO1)decreased significantly(P<0.05). Conclusion The increased secretion of proinflammatory factor IL-17 in the spinal cord of the EAE group may be related to the activation of the PI3K/Akt/FoxO1 signaling pathway and T-cell function.

Key words: Experimental autoimmune encephalomyelitis, Phosphatidylinositol 3-kinase/protein kinase B/FoxO1 signaling pathway, Luxol fast blue staining, Flow cytometry, Western blotting, Enzyme-linked immunosorbent assay, Mouse

中图分类号:

R329

李倩高杰胡蓉韩锋李红苏敏. 磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路和白细胞介素17在小鼠实验性自身免疫性脑脊髓炎中的表达变化[J]. 解剖学报, 2021, 52(3): 358-364.

LI Qian GAO Jie HU Rong HAN Feng LI Hong SU Min . Expression of phosphatidylinositol 3-kinase/protein kinase B/FoxO1 signal pathway and interleukin-17 in experimental autoimmune encephalomyelitis in mice[J]. Acta Anatomica Sinica, 2021, 52(3): 358-364.

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磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路和白细胞介素17在小鼠实验性自身免疫性脑脊髓炎中的表达变化

Expression of phosphatidylinositol 3-kinase/protein kinase B/FoxO1 signal pathway and interleukin-17 in experimental autoimmune encephalomyelitis in mice

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