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目的 :探讨牛珀至宝微丸对蛋白激酶 C(PKC)调控 (L PS)诱导单核巨噬细胞诱导型一氧化氮合酶(i NOS)基因表达的影响。方法 :用 PKC活性测定法观察 L PS对 PKC的激活作用 ,用 Griess还原法测定一氧化氮 (NO)的生成 ,用基因重组技术构建 i NOS荧光素酶报告基因载体 ,用基因转染及报告基因检测等方法研究牛珀至宝微丸调控 L PS刺激 RAW2 6 4 .7细胞对 i NOS启动子活性的诱导作用及其与 PKC的关系。结果 :牛珀至宝微丸可负调节 L PS刺激 RAW2 6 4 .7细胞引起的 PKC磷酸化激活和膜移位 ,并可延长 PKC抑制剂 H 7下调 NO作用时间。荧光素酶报告基因实验结果显示 ,牛珀至宝微丸可抑制 L PS刺激诱导的 i NOS启动子的转录活性 ,并可增强 H 7和钙离子通道阻滞剂维拉帕米的作用。结论 :牛珀至宝微丸抑制 L PS刺激 RAW2 6 4 .7细胞所致的 NO生成增加 ,其机制之一是量效与时间两方面抑制 PKC激活和细胞内钙增加 ,从而影响 i NOS启动子的转录活性
Objective: To investigate the effect of Niupo Zhibao Pellet on the expression of inducible nitric oxide synthase (i NOS) gene induced by protein kinase C (PKC) (L PS) in monocyte-macrophages. Methods: The PKC activity assay was used to observe the activation of PKC by L PS. The production of nitric oxide (NO) was measured by Griess reduction method. The i NOS luciferase reporter gene vector was constructed by gene recombination technology and transfected and reported by gene. The gene detection method was used to study the effect of Niupo Zhibao Pellet on the induction of iNOS promoter activity by LPS-stimulated RAW2 64.7 cells and its relationship with PKC. Results: Niupo Zhibao Pellets could negatively regulate the activation of PKC phosphorylation and membrane translocation induced by LPS in RAW2 6 4 .7 cells, and prolong the time of NO downregulation by PKC inhibitor H 7 . The luciferase reporter assay results showed that Niupo Zhibao Pellets inhibited the transcriptional activity of the iNOS promoter induced by LPS stimulation and enhanced the action of H 7 and the calcium channel blocker verapamil. CONCLUSION: Niupo Zhibao Pellet inhibits the increase of NO production induced by LPS-stimulated RAW2 64.7 cells. One of its mechanisms is the inhibition of PKC activation and the increase of intracellular calcium in both dose- and time-dependent manners, thereby affecting the iNOS promoter. Transcriptional activity