南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (01): 35-.doi: 10.12122/j.issn.1673-4254.2019.01.06

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雷帕霉素通过上调TGF-β/smad信号通路减轻小鼠的实验性自身免疫性脑脊髓炎

李振飞，聂玲玲，陈丽萍，孙雅菲，郭力   

  • 出版日期:2019-01-20 发布日期:2019-01-20

Rapamycin alleviates inflammation by up-regulating TGF-β/Smad signaling in a mouse model of autoimmune encephalomyelitis

  • Online:2019-01-20 Published:2019-01-20

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摘要： 目的观察雷帕霉素对实验性自身免疫性脑脊髓炎（EAE）小鼠的治疗作用，并研究相关机制。方法建立C57BL/6小鼠 EAE模型，免疫成功后将小鼠分为试验对照组，雷帕霉素小剂量组（0.3 mg/kg/d）和雷帕霉素大剂量组（1 mg/kg/d）。应用Knoz 评分观察小鼠的临床评分，免疫组化法观察IL-17浸润情况，流式细胞仪观察外周Treg细胞的分化情况，ElISA法观察小鼠细胞 因子的变化情况，Western blot法观察p-smad2和p-smad3表达情况。结果大剂量雷帕霉素可以明显改善EAE小鼠的神经功能 缺损评分，在发病初期，高峰期及缓解期大剂量雷帕霉素组评分分别为（0.14±0.38），（0.43±1.13）和（0.14±0.37），而实验对照组 分别为（1.14±0.69），（2.14±1.06）和（2.2±0.75）；大剂量雷帕霉素组中可IL-17炎性细胞在在中枢神经系统的浸润为（43±1.83）， 而实验对照组为（153.5±7.02）；大剂量雷帕霉素可以抑制IL-12，IFN-γ，IL-17和IL-23炎性细胞因子同时诱导IL-10和TGF-β抗 炎性细胞因子；大剂量雷帕霉素组外周Treg 细胞为（10.17±0.68），较实验对照组（3.52±0.32）明显增多（P<0.05）；雷帕霉素和 TGF-β具有协同免疫抑制作用，体外实验发现雷帕霉素和TGF-β共同作用淋巴细胞后Treg细胞为（13.66±1.89），较单用雷帕霉 素（6.23±0.80）或单用TGF-β（4.87±0.85）均明显增多（P<0.05）；雷帕霉素可以上调p-smad2 和p-smad3 的表达，并呈剂量依赖 关系。结论雷帕霉素通过上调p-smad2 和p-smad3表达来促进Treg细胞分化，进而改善EAE的神经功能缺损评分。

Abstract: Objective To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. Methods An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low- dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p-smad3 were investigated using Western blotting. Results High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 vs 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 vs 3.52 ± 0.32). In the in vitro experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. Conclusion Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.