The LPS-induced RAW264. 7 cells inflammation model was used as a carrier to investigate the in vitro anti-inflammation effects of Jingfang n-butanol extraction(JFNE) isolated fraction A and explore its preliminary anti-inflammation mechanism by observing the regulatory effect on PI3 K/AKT signaling pathway and NF-κB pathway. The RAW264. 7 cells inflammation model was established by stimulating with LPS for 12 h. After 3 h pre-treatment with fraction A,the contents of interleukin-6(IL-6),interleukin-1β(IL-1β) and tumor necrosis factor(TNF-α) in the supernatant of RAW264. 7 cells inflammation model were determined by ELISA and the contents of NO in supernatant were assayed by Griess. Reverse transcription-polymerase chain reaction(RT-PCR) method was used to determine the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammatory cells,and the expression levels of phosphorylated and total PI3 K/AKT protein,NF-κB p50,p65,p-p65,p105 protein in cells were determined via Western blot. In addition,LC-MS and database were used to identify the possible chemical constituents in fraction A. The results showed that fraction A could significantly reduce the release levels of NO,IL-6,IL-1β and TNF-α in the supernatant and the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammation model cells(P<0. 05 or P<0. 01) and significantly inhibit the phosphorylation expression levels of PI3 K and AKT protein and mRNA expressions(P<0. 05 or P<0. 01). Moreover,fraction A could significantly reduce the levels of NF-κB p50,p-p65 and i NOS protein,as well as NF-κB1,Rela mRNA expressions in RAW264. 7 cells,and increase the expression of CHUK gene.A total of 196 compounds were identified from fraction A in the composition analysis,and isoobtusilactone,5-O-methyl-vismitol,emebel(embelin) and prim-O-glucosylcimifugin showed high contents. The results all above showed that fraction A had a certain antiinflammatory effect in LPS-induced RAW264. 7 inflammation model cells,and its anti-inflammatory effects may be related to its regulatory effect on the activation of PI3 K/AKT signaling pathway and NF-kappa B signaling pathway. In addition,emblin may be its effective anti-inflammation chemical composition.
Keywords: Jingfang n-butanol extraction; LPS; NF-κB; PI3K/AKT; RAW264.7 cells; fractions A.