CaM抑制剂对LPS/IFN-γ诱导的RAW264.7细胞NO生成及iNOS表达的影响

Effect of calmodulin inhibitor on NO production and iNOS expression induced by LPS/IFN-γ in RAW264.7 cells

目的观察钙调蛋白(Calmodulin,CaM)抑制剂对脂多糖/干扰素γ(LPS/IFN-γ)诱导的RAW264.7细胞一氧化氮(NO)的生成及诱生型一氧化氮合酶(iNOS)表达的作用及其可能的作用机制。方法采用LPS/IFN-γ诱导RAW264.7巨噬细胞建立细胞炎症反应模型,钙调蛋白抑制剂(W-7,TFP)预处理细胞后,采用Griess试剂法测定NO释放量;采用Western blot法测定目的蛋白的表达;采用反转录聚合酶链反应法(RT-PCR)分析iNOS mRNA表达的变化。结果 CaM抑制剂可抑制LPS/IFN-γ诱导的RAW264.7细胞NO的释放(P<0.01);可抑制iNOS蛋白和mRNA的表达,早期可抑制IκBα的降解、磷酸化IKK(PIKK)和STAT1的蛋白(PSTAT1)表达。结论 CaM抑制剂可明显降低LPS诱导的RAW264.7细胞NO、iNOS蛋白和mRNA表达;可以通过抑制IκBα降解和磷酸化IKK蛋白表达而发挥抗炎作用。

Objective To investigate the effect of calmodulin（CaM） inhibitor on nitric oxide（NO） production and inducible nitric oxide synthase（iNOS） expression induced by lipopolysaccharide（LPS） and/or interferon-γ（IFN-γ） in mouse RAW264.7 macrophage cells,and to study the anti-inflammation mechanism of CaM inhibitor.Methods An inflammatory response model was established using RAW264.7 cells induced by LPS and/or IFN-γ.CaM inhibitors N-（6-aminohexyl）-5-chloro-1-naphthalenesulfonamide（W-7） and trifluoperazine（TFP） were used to pre-treat RAW264.7 cells to evaluate the effects of the CaM inhibitors.NO concentration was measured with Griess Reagent Kit.Target protein and mRNA expression was tested by Western blot and RT-PCR.Results In RAW264.7 cells induced by LPS or IFN-γ,CaM inhibitors W-7 and TFP significantly blocked NO production（P0.01） and protein and mRNA expression of iNOS（P0.01）.In RAW264.7 cells induced by LPS and IFN-γ,W-7 and TFP remarkably prevented IκBα degradation（P0.01） and phospho-IKK and phospho-STAT1 protein expression（P0.01）.Conclusion CaM inhibitors W-7 and TFP can inhibit LPS/IFN-γ-induced NO production and iNOS protein and mRNA expression in RAW264.7 cells,probably by the mechanism of suppressing IκBα degradation and phospho-IKK and phospho-STAT1 protein expression.The anti-inflammatory effects of CaM inhibitors may be associated with NF-κB and JAK-STAT1 signaling pathways.