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

Nan Fang Yi Ke Da Xue Xue Bao. 2019 Jan 30;39(1):35-42. doi: 10.12122/j.issn.1673-4254.2019.01.06.
[Article in Chinese]


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.

Conclusions: 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.

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。

Keywords: Smad2; Smad3; Treg cells; experimental autoimmune encephalomyelitis; multiple sclerosis.

MeSH terms

  • Animals
  • Anti-Inflammatory Agents / administration & dosage
  • Anti-Inflammatory Agents / therapeutic use*
  • Cell Differentiation* / drug effects
  • Encephalomyelitis, Autoimmune, Experimental / drug therapy*
  • Encephalomyelitis, Autoimmune, Experimental / metabolism*
  • Interferon-gamma / metabolism
  • Interleukins / metabolism
  • Lymphocytes / cytology
  • Mice
  • Mice, Inbred C57BL
  • Sirolimus / administration & dosage
  • Sirolimus / therapeutic use*
  • Smad Proteins / metabolism*
  • T-Lymphocytes, Regulatory / cytology*
  • T-Lymphocytes, Regulatory / drug effects
  • Transforming Growth Factor beta / metabolism*
  • Up-Regulation*


  • Anti-Inflammatory Agents
  • Interleukins
  • Smad Proteins
  • Transforming Growth Factor beta
  • Interferon-gamma
  • Sirolimus

Grant support