细胞因子信号转导抑制蛋白1在小蘖碱抑制脂多糖和γ-干扰素诱导的N9小胶质细胞激活中的作用

Role of suppressor of cytokine signaling 1 in berberine mediated-inhibition of lipopolysaccharide and interferon-γ-induced N9 microglial activation

目的探讨细胞因子信号转导抑制蛋白1(suppressor of cytokine signaling,SOCS1)在小蘖碱(berberine,BBR)抑制小胶质细胞激活中的作用。方法联合应用脂多糖(lipopolysaccharide,LPS)和γ-干扰素(interferonγ,IFN-γ)激活N9小胶质细胞模拟神经炎症。将细胞分为对照组、模拟神经炎症组(10 ng/ml LPS+10 U/ml IFN-γ)、BBR处理组(1μmol/L BBR+LPS/IFN-γ)、SOCS1-siRNA处理组(SOCS1-siRNA+BBR+LPS/IFN-γ)和乱序-siRNA处理组(SC-siRNA+BBR+LPS/IFN-γ)。孵育24 h后,采用酶联免疫吸附法检测细胞培养液内肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)和白细胞介素-6(interleukin-6,IL-6)表达水平,采用蛋白质印迹法检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和核因子κB(nuclei factor-κB,NF-κB)蛋白表达水平,采用免疫细胞化学法观察细胞形态和iNOS表达。结果与对照组相比,LPS/IFN-γ可显著增高培养基内TNF-α和IL-6含量,上调iNOS和NF-κB表达水平(P均<0.05),并增大小胶质细胞体积。1μmol/L BBR可显著抑制小胶质细胞TNF-α和IL-6释放量,降低iNOS和NF-κB表达水平(P均<0.05),并改善细胞形态。SOCS1-siRNA能显著逆转BBR对小胶质细胞激活的抑制作用(P均<0.05)。结论BBR可能通过SOCS1分子介导抑制LPS和IFN-γ对小胶质细胞的激活。

Objective To investigate the role of suppressor of cytokine signaling 1(SOCS1)in berberine(BBR)mediated-inhibition of microglial activation.Methods The combination of lipopolysaccharide(LPS)and interferon-γ(IFN-γ)activated N9 microglia to mimic neuroinflammation.The cells were divided into control group,simulated neuroinflammation group(10 ng/ml LPS+10 U/ml IFN-γ),BBR treatment group(1μmol/L BBR+LPS/IFN-γ),SOCS1-siRNA treatment group(SOCS1-siRNA+BBR+LPS/IFN-γ),and scrambled siRNA treatment group(SC-siRNA+BBR+LPS/IFN-γ).After incubation for 24 h,the expression levels of tumor necrosis factorα(TNF-α)and interleukin-6(IL-6)in cell culture medium were detected by enzyme-linked immunosorbent assay.Western blotting was used to detect the expression levels of inducible nitric oxide synthase(iNOS)and nuclear factorκB(NF-κB).Immunocytochemical method was used to observe cell morphology and iNOS expression.Results Compared with the control group,LPS/IFN-γsignificantly increased the levels of TNF-αand IL-6 in the medium,up-regulated the expression levels of iNOS and NF-κB(all P<0.05),and increased the volume of microglia.1μmol/L-BBR significantly inhibited the release of TNF-αand IL-6 from microglia,decreased the expression levels of iNOS and NF-κB(all P<0.05),and improved cell morphology.SOCS1-siRNA significantly reversed the inhibitory effect of BBR on microglia activation(all P<0.05).Conclusions BBR may inhibit LPS and IFN-γ-induced microglial activation through SOCS1 molecule.