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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 effects of calmodulin (CaM) inhibitor on the production of nitric oxide (NO) and inducible nitric oxide synthase (NOS) in RAW 264.7 cells induced by lipopolysaccharide / interferon γ (LPS / iNOS) expression and its possible mechanism of action. Methods RAW264.7 macrophages were induced by LPS / IFN-γ to establish a model of inflammatory reaction. Pretreatment of cells with calmodulin inhibitor (W-7, TFP) followed by Griess reagent assay was used to determine NO release. Western blot The expression of iNOS mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results CaM inhibitor could inhibit the release of NO from LPS / IFN-γ-induced RAW264.7 cells (P <0.01), inhibit the expression of iNOS protein and mRNA, inhibit the degradation of IκBα, phosphorylate IKK (PIKK) and STAT1 Of protein (PSTAT1) expression. Conclusion CaM inhibitors can significantly reduce the LPS-induced NO, iNOS protein and mRNA expression in RAW264.7 cells. It can exert anti-inflammatory effects by inhibiting IκBα degradation and phosphorylating IKK protein.