目的研究莱菔硫烷(SFN)对脂多糖(LPS)诱导的血管内皮细胞ECV304中COX-2和iNOS mRNA表达的影响及转录机制。方法以体外培养的血管内皮细胞ECV304为研究模型;采用实时定量PCR(real-time PCR)测定COX-2 mRNA的表达;采用RT-PCR测定细胞中iNOS mRNA的表达;采用Western blotting测定细胞浆中IκB-α蛋白表达;采用免疫荧光杂交法测定NF-κB核内转位情况。结果Real—time PCR结果表明,5~20μmol/L SFN能明显降低脂多糖诱导的COX-2 mRNA的表达,抑制率分别为24%、16%和30%;RT-PCR结果显示,SFN对LPS诱导的iNOS mRNA表达也有显著的抑制作用;SFN抑制LPS诱导的IκB-α蛋白降解及NF—κB核内转位。结论SFN能抑制LPS诱导的血管内皮细胞炎症反应,提示SFN对心血管疾病具有预防作用。
Objective To investigate the effect of sulforaphane on the expression of COX-2 and iNOS mRNA induced by LPS in vascular endothelial cells. Methods Human vascular endothelial ECV 304 cells were cultured in vitro. COX-2 mRNA was detected by real-time reverse transcription polymerase chain reaction (PCR);iNOS mRNA was measured by RT-PCR; The expression of IκB-α protein in cytoplasm was determined by Western blotting analysis; Nuclear translocation of NF-κB was tested by hybridization. Results 5, 10, 20 μmol/L sulforaphane decreased the expression of COX-2 mRNA by 24%, 16% and 30% by realtime PCR method. Also sulforaphane significantly suppressed the expression of iNOS from the result of RT-PCR. Sulforaphane upregulated the expression of IκB-α protein, an inhibitor of NF-κB, reduced by LPS. Sulforaphane inhibited the nuclear translocation of NF-κB stimulated by LPS in ECV304 cells. Conclusion SFN inhibited the inflammation of ECV304 cells induced by LPS, suggesting its preventive role in cardiovascular diseases.