目的 建立脂多糖( LPS)诱导的小鼠单核巨噬细胞(RAW264.7)炎症模型,探究新型苯并噁唑酮衍生物4-(5′-二甲氨基)-萘磺酰氧基苯并噁唑酮( W3D)的抗炎活性及其对TLR4-MyD88-NF-κB通路的调控作用.方法 MTT法测定化合物W3D对细胞活力的影响;LPS与不同浓度的化合物W3D共同作用RAW264.7细胞后,ELISA法测定细胞上清液中TNF-α、IL-6、IL-1β、COX-2 的含量,Western blot法检测IL-6、TLR4、MyD88、IRAK4、NF-κB的蛋白表达;实时荧光 定量PCR法检测细胞中TLR4、MyD88、IL-6 mRNA的表达.结果 化合物W3D对LPS诱导的RAW264.7细胞培养液中炎症因子TNF-α、IL-6、IL-1β的分泌有明显的抑制作用,但对COX-2无抑制活性;可明显下调TLR4、MyD88、IL-6 的蛋白与mRNA的表达,抑制IRAK4磷酸化和NF-κB的入核活化.结论 化合物 W3D 可通过调控 TLR4-MyD88-IRAK4-NF-κB信号通路,抑制 TNF-α、IL-6、IL-1β 等炎症因子的释放而发挥抗炎活性.%Aim To investigate the effect of the novel benzoxazolone derivative 4-( 5′-dimethylamino )-naph-thalenesulfonyl-2 ( 3H )-benzoxazolone ( W3D ) on TLR4-MyD88-NF-κB signaling pathway in LPS-in-duced RAW264.7 cells. Methods The cell viability was detected by MTT assay, and the contents of TNF-α, IL-6, IL-1β and COX-2 in the cell supernatant were analyzed using ELISA methods. The protein ex-pression of IL-6, TLR4, MyD88, p-IRAK4 and NF-κB were investigated by western blot analysis, and the mRNA expressions of TLR4, MyD88 and IL-6 were an-alyzed by RT-PCR. Results W3D could obviously in-hibit the production of TNF-α, IL-6 and IL-1β in LPS- induced RAW264.7 cell supernatant, but it had no effect on the release of COX-2. Compared with the model group, the expressions of TLR4, MyD88 and IL-6 were decreased significantly in a dose dependent manner. Meanwhile, the expressions of p-IRAK4 and nucleus of NF-κB were decrease in W3D treated group compared with the model group. Conclusion The no-vel compound W3D could inhibit the release of the in-flammatory mediators through the regulation of TLR4-MyD88-NF-κB signaling pathway.
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