关键词: 苦豆子, RAW 264.7细胞, 抗炎, NF-κB信号通路

Abstract:

The anti-inflammatory effect and mechanism of Sophora alopecuroides L. were explored and the inflammation were induced by lipopolysaccharide in RAW 264.7 macrophages.The water decoction extract (WDE),water ultrasonic extract (WUE),75% ethanol reflux extract (ERE) and 75% ethanol ultrasonic extract (EUE) of S. alopecuroides were prepared by water decocting,water ultrasonic,75% ethanol reflux and 75% ethanol ultrasonic.Total alkaloids extract (TAE) and total flavonoids extract (TFE) of S. alopecuroides were extracted by D101 macroporous resin adsorption method,total polysaccharides extract (TPE) of S. alopecuroides was extracted by water extraction and alcohol precipitation.The contents of TA,TF and TP were determined by acid dye colorimetry,NaNO₂-Al(NO₃)₃-NaOH spectrophotometry and phenol-sulfuric acid method,respectively.Nine different combinations of TAE,TFE,TPE were preliminarily designed by orthogonal method (1∶1∶1,1∶2∶2,1∶3∶3,2∶1∶2,2∶2∶3,2∶3∶1,3∶1∶3,3∶2∶1,3∶3∶2).LPS (1 μg/mL) induced RAW 264.7 macrophages to establish in vitro inflammatory model.Cell counting kit-8 assay was used to detect cell viability,nitrate reductase was used to detect nitric oxide release,and enzyme-linked immunosorbent assay was used to detect interleukin-1β,interleukin-6 and tumor necrosis factor alpha in cell supernatant,the expression of p65 NF-κB,p-p65 NF-κB,IκBα and p-IκBα were detected by Western blot.The results showed that the content of TA,TF and TP were 94.79%,71.59% and 73.51% after purification.Compared with the model group,WDE,WUE,ERE,EUE,TAE,TFE,TPE and their 9 combinations significantly reduced the release of IL-1β,IL-6,TNF-α and NO,and 2∶1∶2 of TAE∶TFE∶TPE had the best anti-inflammatory,which significantly reduced the phosphorylation levels of IкBα and p65 NF-κB(P<0.01).In conclusion,S. alopecuroides have anti-inflammatory effects on the inflammation of RAW 264.7 macrophages induced by lipopolysaccharide,The 2∶1∶2 of TAE∶TFE∶TPE had the best anti-inflammatory effect,which alleviated the LPS-induced inflammatory response of RAW 264.7 macrophages by inhibiting the activation of NF-κB signaling pathway and the transcription expression of downstream inflammatory mediators.

Key words: Sophora alopecuroides L., RAW 264.7 macrophages, anti-inflammatory, NF-κB signaling pathway