丹翘液对脂多糖诱导RAW264.7细胞炎症相关因子的抑制效应分析

Inhibiting Effect Analysis of Danqiao Liquid on Inflammation-related Factors Induced by LPS in RAW264.7 Cells

为研究丹翘液的体外抗炎作用及其抗炎机制,利用LPS诱导巨噬细胞建立炎症模型,MTT法检测不同浓度丹翘液对RAW264.7细胞活力影响;NO测试盒检测丹翘液对LPS诱导的NO释放量的影响;ELISA方法检测丹翘液对LPS诱导的TNF-α、IL-6和PGE2分泌的影响;RT-PCR方法检测丹翘液对LPS诱导的TNF-α、IL-6、COX-2和iNOS基因转录的影响;Western blot检测对细胞核内NF-κB p65蛋白表达的影响。结果显示丹翘液小于700μg·mL-1对细胞无毒性作用;丹翘液各剂量组(100、300、600μg·mL-1)能不同程度地抑制LPS诱导的NO、PGE2、TNF-α和IL-6的分泌;能显著抑制iNOS、COX-2、TNF-α和IL-6基因转录和细胞核内NF-κB p65蛋白表达。丹翘液的抗炎作用机制可能与抑制NF-κB通路激活,进而抑制炎症介质和炎性细胞因子的转录和表达有关。

The aim of the present study was to study the anti-inflammatory effects and possible underlying mechanisms of Danqiaoliquid in LPS-stimulated RAW264.7cells.The cytotoxicity of Danqiaoliquid was detected by MTT method.The NO kit assay was adopted to detect the effect of Danqiaoliquid on NO release from LPS-induced RAW264.7cells.ELISA was used to evaluate the production of TNF-α,IL-6and PGE2 in LPS-induced RAW264.7cells.Real-time PCR was used to detect the transcription of COX-2,iNOS,TNF-αand IL-6in LPS-induced RAW264.7cells.The protein expression of nuclear NF-κB p65 was detected by Western blot.The resultshowed that the safe medication range of Danqiaoliquid was less than 700μg·mL-1.Compared with the LPS model group,Danqiaoliquid(100,300,600μg·mL-1)could reduce the secretion of NO,PGE2,TNF-α,and IL-6in cells induced by LPS,and significantly inhibit the mRNA transcription of iNOS,COX-2,TNF-α,IL-6and the protein expression of NF-κB p65.In conclusion,this study preliminarily proves the protective effect of Danqiaoliquid on LPS-induced RAW264.7macrophages.Its action mechanism may be related to inhibit NF-κB signal pathway and the genes expressions and secretion of inflammatory mediators and cytokines.