CD200Fc reduces TLR4-mediated inflammatory responses in LPS-induced rat primary microglial cells via inhibition of the NF-κB pathway

Objective Based on recent information, CD200Fc, a CD200R1 agonist, could attenuate the inflammatory response of microglial cells in autoimmune diseases and neuro-degeneration. However, the exact molecular mechanisms responsible for the anti-inflammatory activity of CD200Fc in microglial cells have not been elucidated. In the present study, we investigated the anti-inflammatory effects and the molecular mechanisms of CD200Fc in lipopolysaccharide (LPS)-stimulated rat primary microglial cells.Methods The cell viability was measured by MTT assay. The LPS-induced cytokines release (IL-1 beta, IL-6, TNF-alpha, iNOS, MCP-1, and COX-2) was monitored by ELISA or real-time PCR, while NF-kappa B-related signals (MyD88, p-TAK1, TRIF, p-TBK1, p-IRF3, p-I kappa B, and NF-kappa B-P65) were assessed by real-time PCR, western blot and/or Immunofluorescent staining.Results CD200Fc and/or LPS exerted no significant cytotoxicity on microglial cells. LPS reduced the CD200R1 expression in microglial cells, and this effect was attenuated by CD200Fc. In addition, CD200Fc inhibited LPS-induced expression of TLR4 and its adapter molecules (MyD88 and p-TAK1, TRIF, p-TBK1, and p-IRF3), and abolished its interactions with MyD88, TAK1, and TRIF in microglial cells. CD200Fc also attenuated LPS-induced protein expression of p-I kappa B and NF-kappa B-P65 translocation to nucleus in microglial cells. Moreover, CD200Fc suppressed the LPS-induced release of inflammatory mediators in microglial cells, including IL-1b, IL-6, TNF-alpha, iNOS, MCP-1, and COX-2.Conclusion These results indicated that CD200Fc displayed an anti-inflammatory effect in LPS-induced microglial cells by blocking TLR4-mediated NF-kappa B activation.

目的 基于近期的信息，CD200Fc作为一种CD200R1激动剂，能够减轻自身免疫性疾病和神经退行性变中小胶质细胞的炎症反应。然而，CD200Fc在小胶质细胞中抗炎活性的确切分子机制尚未阐明。在本研究中，我们研究了CD200Fc在脂多糖（LPS）刺激的大鼠原代小胶质细胞中的抗炎作用及其分子机制。 方法 采用MTT法测定细胞活力。通过酶联免疫吸附测定（ELISA）或实时荧光定量聚合酶链反应（real - time PCR）监测LPS诱导的细胞因子释放（白细胞介素 - 1β、白细胞介素 - 6、肿瘤坏死因子 - α、诱导型一氧化氮合酶、单核细胞趋化蛋白 - 1和环氧化酶 - 2），同时通过实时荧光定量聚合酶链反应、蛋白质印迹法和/或免疫荧光染色评估核因子 - κB相关信号（髓样分化因子88、磷酸化转化生长因子β激活激酶1、Toll样受体相关的干扰素激活因子、磷酸化TANK结合激酶1、磷酸化干扰素调节因子3、磷酸化IκB和核因子 - κB - P65）。 结果 CD200Fc和/或LPS对小胶质细胞无明显细胞毒性。LPS降低了小胶质细胞中CD200R1的表达，而CD200Fc减弱了这种作用。此外，CD200Fc抑制LPS诱导的Toll样受体4及其接头分子（髓样分化因子88和磷酸化转化生长因子β激活激酶1、Toll样受体相关的干扰素激活因子、磷酸化TANK结合激酶1和磷酸化干扰素调节因子3）的表达，并消除其在小胶质细胞中与髓样分化因子88、转化生长因子β激活激酶1和Toll样受体相关的干扰素激活因子的相互作用。CD200Fc还减弱了LPS诱导的小胶质细胞中磷酸化IκB的蛋白表达以及核因子 - κB - P65向细胞核的转位。此外，CD200Fc抑制了LPS诱导的小胶质细胞中炎症介质的释放，包括白细胞介素 - 1β、白细胞介素 - 6、肿瘤坏死因子 - α、诱导型一氧化氮合酶、单核细胞趋化蛋白 - 1和环氧化酶 - 2。 结论 这些结果表明，CD200Fc通过阻断Toll样受体4介导的核因子 - κB激活，在LPS诱导的小胶质细胞中显示出抗炎作用。