Erbin蛋白在GTS-21激活胆碱能抗炎通路中的调控作用

Erbin Plays a Regulatory Role in the GTS-21 Activating Cholinergic Anti-inflammatory Path to Inhibit the Muramyl Dipeptide-Induced Inflammatory Response

目的:探讨Erbin蛋白在GTS-21激活胆碱能抗炎通路抑制胞壁酰二肽(MDP)诱导的炎症反应中的调控作用。方法:将正常培养的RAW264.7细胞分为4组:空白对照组(NS组),MDP组(M组):MDP(10μg/ml),GTS-21组(G组):MDP(10μg/ml)+α7nAChRs特异激动剂/GTS-21(50μg/ml),Erbin shRNA干扰组(R组):MDP(10μg/ml)+GTS-21(50μg/ml)+Erbin shRNA。在MDP刺激后1,6,24h时间点提取标本,每个时间点10个样本。Western检测Erbin的表达,免疫荧光检测Erbin的免疫定位,凝胶迁移率实验(EMSA印迹)检测NF-κB活化,ELISA检测TNF-α、IFN-γ等促炎性细胞因子水平。结果:MDP刺激后,Erbin表达升高,NF-κB活性增强,TNF-α、IFN-γ水平显著增加;与M组相比,G组Erbin表达明显升高(P<0.05),NF-κB活性降低(P<0.05),TNF-α、IFN-γ水平均下降(P<0.05),与G组相比,R组Erbin表达明显下降(P<0.05),NF-κB活性增强(P<0.05),TNF-α、IFN-γ水平均升高(P<0.05)。结论:Erbin蛋白是激活胆碱能抗炎通路抑制MDP诱导的炎症反应中关键调控蛋白,起负调控作用。

Objective： To investigate the regulatory role of Erbin protein in the GTS-21 activating cholinergic anti-inflammatory path to inhibit the muramyl dipeptide（MDP）-induced inflammatory response. Methods： Normal cultured RAW264.7 cells were cultured in 12-well culture plates during the logarithmic growth phase. We divided the experimental cells into four groups： control group （group NS）, MDP group （group M, 10 μg/ml MDP）, GTS-21 group （group G, 10μg/ml MDP ＋ 50 μg/ml α7 nAChRs specific agonist/GTS-21）, and Erbin shRNA interference group （group R, 10 μg/ml MDP ＋ 50μg/ml GTS-21＋ Erbin shRNA）. We extracted 10 specimens at the point of 1, 6, 24 h respectively after MDP stimulation. Western blotting and immunofluorescence mi- croscopy were used to detect the expression and the immune position of Erbin protein. EMSA was used to detect the activation of NF-κB and ELISA was used to measure the pro-inflammatorycytokine levels. Results： After the stimulation of MDP, the content of Erbin, NF-κB, TNF-α, and IFN-γ increased in group M. Compared with group M, the content of Erbin increased and NF-κB, TNF-α and IFN-γ decreased in group G （all P〈0.05）. In contrast, compared with group G, the content of Erbin decreased but NF-κB, TNF-α, and IFN-γ increased （all P〈0.05） in group R. Conclusion： Erbin is the key regulatory protein to inhibit the inflammatory response induced by MDP through activating cholinergic anti-inflammatory pathway. It is likely to work by the negative regulation of NLR2/RICK signaling pathway.