金铁锁基于NF-κB信号通路的体外抗炎作用机制研究

Study on the Anti-inflammatory Effects of Psammosilene tunicoides and its Triterpenoid Compounds

目的金铁锁具有较好的抗炎镇痛的药理作用,但对金铁锁的抗炎作用及机制尚不明确。本研究旨在通过LPS诱导的RAW264.7细胞炎症模型对金铁锁进行抗炎作用及机制研究。方法采用LPS诱导RAW264.7细胞炎症模型,在给予金铁锁三萜类化合物干预后,采用ELISE试剂盒检测细胞上清液中NO、PGE_(2)的水平,PCR检测细胞中COX-2,i NOS,IL-6,TNF-αm RNA的表达,用western blot分别检测细胞总蛋白、细胞质及细胞核内NF-κB信号通路相关蛋白(IKKβ、photo-IκBα、NF-κBp65、photo-NF-κBp65)的表达情况,考察金铁锁三萜类化合物的抗炎作用机制。结果金铁锁能明显降低RAW264.7细胞活化后细胞上清液中NO和PGE_(2)的水平,对细胞上清液中NO和PGE_(2)的水平无明显影响,能明显降低对RAW264.7细胞活化后COX-2、i NOS m RNA的表达水平;金铁锁能明显抑制IKKβ的活化,抑制p-IκBα蛋白的降解,同时也能降低总蛋白中p-NF-κB p65的表达量,从而抑制细胞质内NF-κB p65向核内的转移。结论金铁锁具有良好的抗炎镇痛作用,能够抑制炎症介质的释放及酶的基因表达,改善炎症部位的病变情况,其抗炎机制与抑制NF-κB信号通路的激活有关。

Objective It has been reported that Psammosilene tunicoides has agood pharmacological effect of antiinflammatory and analgesic,but the anti-inflammatory effect and mechanism of Psammosilene tunicoides is not clear.Therefore,the aim of this study was to investigate the anti-inflammatory effect and mechanism of the Psammosilene tunicoides and its triterpenoid compounds through RAW264.7 cell inflammatory model inducedby LPS.Methods Using RAW264.7 cell inflammation modelinduced by LPS,the level of NO and PGE_(2) in cell supernatant was detected by ELISE Reagent and the expression of COX-2,i NOS,IL-6,TNF-αm RNA in cells was detected by PCR and the expression of NF-κB signaling pathway related proteins(IKKβ,photo-lκBα,NF-κBP65,photo-NF-κBp65)in total protein,cytoplasm and nucleus of cells were detected by westerning blotting.Thusthe anti-inflammatory mechanism of triterpenoids compounds of Psammosilene tunicoides was investigated.Results Triterpenoid compounds from Psammosilene tunicoides(QA,GYP)can significantly reduce the level of NO and PGE_(2) in the supernatant of RAW264.7 cells after activation,while TSPT have no significant effect on the level of NO and PGE_(2) in the supernatant of the cells.The results showed that the expression of COX-2 and i NOS m RNA in RAW264.7 cells was significantly decreased by the triterpenoids compounds from Psammosilene tunicoides.The results were consistent with the quantitative results of PGE_(2),which was consistent with the quantitative results of PGE_(2) and NO.Different components had different effects on the release level of inflammatory mediators and the expression of synthase m RNA.The expression of NF-κBp65 within the cells decreased.The expression of NF-κBp65 in the nucleus increased,the expression of p-NF-κBp65 in the total protein increased,and the expression of p-NF-κBp65 in the total protein was increased.After the intervention of the triterpenoids compounds,the activation of IKKβand the degradation of p-IκBαprotein were inhibited,and the expression of p-NF-κBp65 in the total protein was also decreased,thus inhibiting the transfer of NF-κBp65 to the nucleus in the cytoplasm.Conclusion We have studied the antiinflammatory effect and mechanism of Psammosilene tunicoides and its triterpenoids compounds vitro.They have good antiinflammatory and analgesic effects,and can inhibit the release of inflammatory mediators and the gene expression of enzymes.The anti-inflammatory mechanism are related to the inhibition of activation of NF-κB signaling pathway.