蛋白激酶C对脂多糖诱导的诱导型一氧化氮合酶基因表达的调控作用

Regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by protein kinase C

目的 探讨脂多糖 (LPS)诱导单核 巨噬细胞诱导型一氧化氮合酶 (iNOS)基因表达的分子机制。方法 用LPS刺激诱导RAW2 64 .7细胞 ,观察其对蛋白激酶C(PKC)的激活 ,用Griess还原法测定PKC抑制剂对LPS诱导的一氧化氮 (NO)生成作用 ,并用逆转录PCR(RT PCR)研究其对iNOS基因表达的影响 ;构建iNOS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LPS刺激RAW2 64 .7细胞对iNOS启动子活性的诱导作用及PKC对其影响。结果 LPS刺激RAW2 64 .7细胞引起PKC磷酸化激活和膜移位 (P <0 .0 1 ) ;LPS刺激RAW2 64 .7细胞 1 2h后即可明显诱导NO生成 (30 .1μmol L± 5 .4μmol L ,P <0 .0 1 )和iNOS基因表达 ;PKC抑制剂H 7可抑制NO的生成 (P <0 .0 1 )和iNOS基因表达 ;LPS刺激诱导iNOS启动子的转录活性 [(2 5 .3± 4 .6)相对荧光素酶活性单位 ,P <0 0 1 ] ,用H 7和钙离子通道阻滞剂异搏定均可抑制其转录活性 (P <0 .0 1 )。结论 LPS刺激RAW2 64 .7细胞 ,通过引起细胞内钙增加和PKC激活诱导iNOS基因表达 ,使NO生成增加 。

Objective To explore the molecular mechanism of inducible nitric oxide synthase (iNOS) gene expression induced by lipopolysaccharide (LPS) in monocytes or macrophages. Methods Luciferase report gene vector pGL2iNOSLuc driven by murine iNOS promoter region was constructed using gene recombination technique. RAW264.7 cells were cultured and transfected with luciferase report gene vector pGL2iNOSLuc. Twenty four hours later, PKC inhibitor H 7, calcium channel blocker verapamil, was added into the culture. LPS or PKC activator PMA was added 2 hours later to stimulate the cells. The activity of β galactosidase and luciferase was examined and the relative luciferase activity, representing the iNOS transactivity, was calculated. Modified Griess method was used to measure the concentration of NO - 2/NO - 3in the culture so as to evaluate the effect of PKC inhibitor on NO production induced by LPS. RT PCR was used to study the effect of LPS on iNOS gene expression in RAW264.7 cells. Results Thirty minutes after stimulation of RAW264.7 cells by LPS, the activity of PKC in cytomembrane increased and the activity of PKC in cytoplasm decreased significantly in comparison with the baseline levels ( P <0.01). The NO concentration was 30.1 μmol/L±5.4 μmol/L 12 hours after the stimulation of LPS on RAW264.7 cells, and was much more higher 24 and 48 hours after, all significantly higher than before (all P <0.01). The NO concentration in the supernatant with H 7 at any time point was significantly lower than those in corresponding samples without H 7 (all P <0.01). After stimulation of LPS and PMA, the relative luciferase activity was 25.3±4.6,significantly higher than the baseline level ( P <0.01). H 7 and verapamil significantly inhibited the LPS induced iNOS transactivity (both P <0.01). iNOS mRNA expression was increased greatly, and H 7 significantly inhibited this expression 12, 24, and 48 hours after LPS stimulation. Conclusion In RAW264.7 cells, LPS stimulation increases intracellular calcium and activates PKC, thus inducing iNOS gene expression that contributes to the production of NO, which is an important signaling mechanism of NO production in septic shock.