中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (11): 1994-2002.doi: 10.4103/1673-5374.259623

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

抑制核因子κB信号通路表达：是Atsttrin部分减轻脂多糖介导神经炎症的作用途径

  

  • 出版日期:2019-11-15 发布日期:2019-11-15
  • 基金资助:

    中国国家自然科学基金项目（81572191，81601067）

Atsttrin reduces lipopolysaccharide-induced neuroinflammation by inhibiting the nuclear factor kappa B signaling pathway

Lian Liu 1, 2 , Yuan Qu 3 , Yi Liu 1 , Hua Zhao 1 , He-Cheng Ma 1 , Ahmed Fayyaz Noor 4 , Chang-Jiao Ji 5 , Lin Nie 1 , Meng Si 1 , Lei Cheng 1   

  1. 1 Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
    2 Department of Orthopedics, Qilu Children’s Hospital of Shandong University, Jinan, Shandong Province, China
    3 Department of Hand Surgery, the Second Hospital of Jilin University, Changchun, Jilin Province, China
    4 Department of Chemistry, University of Massachusetts Lowell, Lowell, MA, USA
    5 Department of Orthopedics, the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province, China
  • Online:2019-11-15 Published:2019-11-15
  • Contact: Meng Si, spine007@163.com; Lei Cheng, PhD, 17865153925@163.com.
  • Supported by:

    This work was supported by the National Natural Science Foundation of China, No. 81572191 (to LC) and 81601067 (to HZ).

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摘要：

在中枢神经系统中，颗粒蛋白前体与神经炎症小鼠模型中的神经元存活密切相关，并且能减轻炎症反应。Atsttrin是由3个颗粒蛋白前体片段组成的工程化蛋白，且在一些条件下具有与颗粒蛋白前体相似的功能。最近有研究认为Atsttrin在多种关节炎小鼠模型中可作为抗炎蛋白，能防止关节炎发展。然而Atsttrin是否在神经炎症中也能发挥作用尚不可知。(1)实验对侧脑室注射1μL脂多糖（10μg/μL）建立神经炎症模型，以免疫组化检测体内野生型和颗粒蛋白前体基因敲除小鼠脑组织中炎性相关细胞因子的表达；接下来在颗粒蛋白前体基因敲除小鼠侧脑室注射1μL脂多糖（10μg/μL）前7d，每3d腹腔注射Atsttrin 2.5mg/kg，行免疫组化、实时PCR和酶联免疫吸附法测量脑组织中的炎性因子蛋白和基因情况；此外在体外培养的星形胶质细胞以0，100，300ng/mL脂多糖刺激前，行Atsttrin（200ng/mL）干预24h，以免疫荧光、实时PCR和酶联免疫吸附法检测星形胶质细胞中炎性细胞因子蛋白和基因的表达水平以及核因子κB信号通路相关蛋白的表达情况。(2)结果显示，脂多糖干预后，野生型小鼠脑组织以及体外培养的星形胶质细胞中颗粒蛋白前体的表达均明显增加；且颗粒蛋白前体基因敲除小鼠脑组织中促炎细胞因子肿瘤坏死因子α、白细胞介素1β和诱导型一氧化氮合酶蛋白和基因表达均明星增加；而Atsttrin注射后，颗粒蛋白前体基因敲除小鼠脑组织中肿瘤坏死因子α、白细胞介素1β、基质金属蛋白酶3和诱导型一氧化氮合酶蛋白和基因表达均显著下降；且Atsttrin还能降低体外培养的星形胶质细胞中环氧化酶2、诱导型一氧化氮合酶和基质金属蛋白酶3mRNA的表达，降低上清液肿瘤坏死因子α和白细胞介素1β的浓度；同时Atsttrin明显降低脂多糖诱导的颗粒蛋白前体基因敲除小鼠脑组织和体外培养的星形胶质细胞中p-IκBα的表达，并降低星形胶质细胞中NF-κB2的表达。(3)结果表明，Atsttrin可能成为神经炎症治疗的潜在目标，且其作用部分通过抑制核因子κB信号通路表达而实现。实验于2015-02-10经中国山东大学齐鲁医院动物伦理委员会批准，批准号为KYLL-2015(KS)-088。

orcid: 0000-0002-6880-6313(Lian Liu)

关键词: 颗粒蛋白前体, Atsttrin, 神经炎症, 炎性细胞因子, 脂多糖, 侧脑室注射, 星形胶质细胞, 核因子κB信号通路, 颗粒蛋白前体, 颗粒蛋白前体基因敲除小鼠, 脑脊液, 神经再生

Abstract:

Progranulin is closely related to neuronal survival in a neuroinflammatory mouse model and attenuates inflammatory reactions. Atsttrin is an engineered protein composed of three progranulin fragments and has been shown to have an effect similar to that of progranulin. Atsttrin has anti-inflammatory actions in multiple arthritis mouse models, and it protects against further arthritis development. However, whether Atsttrin has a role in neuroinflammation remains to be elucidated. In this study, we produced a neuroinflammatory mouse model by intracerebroventricular injection of 1 μL lipopolysaccharide (10 μg/μL). Atsttrin (2.5 mg/kg) was administered via intraperitoneal injection every 3 days over a period of 7 days before intracerebroventricular injection of 1 μL lipopolysaccharide (10 μg/μL). In addition, astrocyte cultures were treated with 0, 100 or 300 ng/mL lipopolysaccharide, with 200 ng/mL Atsttrin simultaneously. Immunohistochemistry, enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction were performed to examine the protein and mRNA levels of inflammatory mediators and to assess activation of the nuclear factor kappa B signaling pathway. Progranulin expression in the brain of wild-type mice and in astrocyte cultures was increased after lipopolysaccharide administration. The protein and mRNA expression levels of tumor necrosis factor-α, interleukin-1β and inducible nitric oxide synthase were increased in the brain of progranulin knockout mice after lipopolysaccharide administration. Atsttrin treatment reduced the lipopolysaccharide-induced increase in the protein and mRNA levels of tumor necrosis factor-α, interleukin-1β, matrix metalloproteinase-3 and inducible nitric oxide synthase in the brain of progranulin knockout mice. Atsttrin also reduced the expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase 3 mRNA in lipopolysaccharide-treated astrocytes in vitro, and decreased the concentration of tumor necrosis factor α and interleukin-1β in the supernatant. Furthermore, Atsttrin significantly reduced the levels of phospho-nuclear factor kappa B inhibitor α in the brain of lipopolysaccharide-treated progranulin knockout mice and astrocytes, and it decreased the expression of nuclear factor kappa B2 in astrocytes. Collectively, our findings show that the anti-neuroinflammatory effect of Atsttrin involves inhibiton of the nuclear factor kappa B signaling pathway, and they suggest that Atsttrin may have clinical potential in neuroinflammatory therapy. The study was approved by the Animal Ethics Committee of Qilu Hospital of Shandong University, China (approval No. KYLL-2015(KS)-088) on February 10, 2015.

Key words: nerve regeneration, progranulin, Atsttrin, neuroinflammation, inflammatory cytokines, lipopolysaccharide, intracerebroventricular injection, astrocyte, nuclear factor kappa B signaling pathway, progranulin knockout mouse, cerebrospinal fluid, neural regeneration