Erbin在胞壁酰二肽诱导小鼠巨噬细胞炎性反应中的作用

Role of Erbin in muramyl dipeptide-induced inflammatory responses in macrophages of mice

目的评价ErbB2相互作用蛋白(Erbin)在胞壁酰二肽(MDP)诱导小鼠巨噬细胞炎性反应中的作用。方法利用CRISPR/CAS9编辑技术构建Erbin基因敲除型RAW264.7巨噬细胞系(Erbin-/-RAW264.7),体外培养野生型RAW264.7巨噬细胞,采用随机数字表法将两类细胞分为2组(n=16),分别为RAW264.7组和RAW264.7+MDP组、Erbin-/-RAW264.7组和Erbin-/-RAW264.7+MDP组。各MDP组采用10μg/ml MDP孵育6 h,随后采用免疫荧光法测定NF-κB p65表达,采用ELISA法测定培养液TNF-α和IL-6浓度。结果与RAW264.7组比较,RAW264.7+MDP组培养液TNF-α和IL-6浓度升高(P<0.05),NF-κB p65向核内移动,红色荧光面积增加;与RAW264.7+MDP组和Erbin-/-RAW264.7组比较,Erbin-/-RAW264.7+MDP组培养液TNF-α和IL-6浓度升高(P<0.05),NF-κB p65向核内移动更加明显,红色荧光面积增加。结论Erbin可通过抑制NF-κB p65活性,在MDP诱导小鼠巨噬细胞炎性反应中发挥抗炎作用。

Objective To evaluate the role of ErbB2-interacting protein(Erbin)in muramyl dipeptide(MDP)-induced inflammatory responses in the macrophages of mice.Methods Erbin gene knockout RAW264.7 cell line(Erbin-/-RAW264.7)was constructed by CRISPR/CAS9 gene-editing technology.RAW264.7 cells were cultured in vitro.Each type of cells was divided into 2 groups(n=16 each)by a random number table method:RAW264.7 group,RAW264.7 plus MDP group,erbin-/-RAW264.7 group,and erbin-/-RAW264.7 plus MDP group.In each MDP group,cells were incubated with 10μg/ml MDP for 6 h,then immunofluorescence was used to determine the expression of nuclear factor kappa B(NF-κB)p65,and the concentrations of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in the culture medium were determined by enzyme-linked immunosorbent assay.Results Compared with RAW264.7 group,the concentrations of TNF-αand IL-6 in the culture medium were significantly increased(P<0.05),NF-κB p65 moved to the nucleus,and the red fluorescence area was increased in RAW264.7+MDP group.Compared with RAW264.7+MDP group and Erbin-/-RAW264.7 group,the concentrations of TNF-αand IL-6 in the culture medium were significantly increased(P<0.05),NF-κB p65 moved more markedly to the nucleus,and the red fluorescence area was increased in Erbin-/-RAW264.7+MDP group.Conclusion Erbin inhibits MDP-induced inflammatory responses in macrophages through inhibiting the activity of NF-κB p65 in mice.