青藤碱对脂多糖诱导的神经细胞环氧化酶-2表达的影响

目的 :了解青藤碱 (sinomenine ,Sin)对PC 12细胞增殖和表达环氧化酶-2 (COX-2 )及合成前列腺素E₂(PGE₂ )的影响 ,探讨Sin对神经细胞的作用机制。方法 :采用NGF诱导培养PC-12细胞 ,用不同浓度的Sin(0,3×10^-6,30×10^-6,150×10^-6mol·L^-1)预处理后 ,加或不加脂多糖 (LPS) ,分别用³H-TdR法、竞争ELISA、半定量逆转录 聚合酶联反应 (RT-PCR)法、细胞酶联免疫法及Western blot法 ,检测PC-12细胞的增殖活性、细胞培养上清液中PGE₂ 的水平、细胞COX-2 mRNA及蛋白表达水平。同时提取各组细胞蛋白 ,测定其核转录因子NF κB活性。结果 :Sin对LPS诱导的PC-12细胞的COX-2 mRNA、蛋白表达具有不同程度的抑制作用 ,与Sin浓度呈明显正相关性,且对LPS激活的PC-12细胞的核转录因子NF-κB活性有明显的抑制作用。实验未观察到Sin对PC-12细胞增殖的影响。结论 :Sin可显著抑制LPS诱导的PC-12细胞COX-2的表达及其产物PGE₂ 的合成 ,其作用机制可能是通过Sin抑制PC-12细胞核转录因子NF-κB活性而实现。

Inhibitory effect of sinomenine on expression of cyclooxygenas e-2 in lipopolysaccharide-induced PC-12 cells

OBJECTIVE: To study the effects of sinomenine (Sin) on cell proliferation, intracellular expression of cyclooxygenase-2 (COX-2), and production of PGE2 in lipopolysaccharide-induced PC-12 cells, To explore the Sin's mechanism on nerve cell. METHOD: PC-12 cells were cultured with nerve growing factors (NGF), and pretreated with Sin at various concentrations (0, 3 x 10(-6), 30 x 10(-6), 150 x 10(-6) mol x L(-1)) for 2 hours, then with or without stimulation of lipopolysaccharide (LPS). The proliferation activity of PC-12 cells was determined by 3H-TdR incorporation, and the production of PGE2 in culture supernatants of PC-12 cells was detected with competitive ELISA. Expression of COX-2 mRNA in PC-12 cells was analyzed by semi-quantitative RT-PCR, and expression of COX-2 protein was estimated by Western blot method and cellular enzyme immunoassay. Nuclear factor-kappa B (NF-kappaB) activity in whole-cell extract of PC-12 cells was also measured by an ELISA-based method. RESULT: The data showed that Sin down-regulated the expression of COX-2 mRNA and protein, and reduced the production of PGE2 in the LPS-stimulated PC-12 cells which correlated with Sin's concentrations positively. In addition, NF-kappaB activity in LPS-stimulated cells was suppressed significantly by Sin. No inhibition of proliferation of PC-12 cells due to Sin treatment was observed. CONCLUSION: Sin mediates the down-regulation of expression of COX-2 and production of induced PGE2 in PC-12 cells by suppressing the activity of NF-kappaB.