2'-Hydroxy-5'-Methoxyacetophenone Attenuates The Inflammatory Response BV-2 And RAW264.7 Cells

Seahorse has been used as a traditional medicine in Southeast Asian countries for a long time.Recently,seahorse has been exhibited various biological activities,including anti-oxidative,anti-fatigue,promoting cell differentiation,neuro-inflammatory protection and anti-inflammatory effects.Therefore,based on previous reports and works,in this study,we investigated the anti-inflammatory and anti-oxidative effects of paeonol analogue,2'-Hydroxy-5'-Methoxyacetophenone(2H5M)from Hippocampus kuda,in LPS-induced BV-2 and RAW264.7 cells.And the methods and results were presented as followed:(1)MTT assay was used to determine whether 2H5 M has cytotoxicity on BV-2 and RAW264.7 cells,and the cell concentration and dimension were measured by Countstar Automated cell counter.And the results showed that the tested concentrations(10,20,50,100,and 200 ?M)of 2H5 M has no cytotoxicity on two kinds of cells;(2)Griess Reaction System was employed to estimate the inhibitory effect of 2H5 M on NO production.2H5 M could significantly inhibit the NO concentration in a dose-dependent manner(3)Cells pretreated with 2H5 M and then stimulated with LPS,and the concentration of TNF-? was assessed using Elisa.The results showed 2H5 M inhibited the TNF-? level in a dose-depented manner.(4)The reactive oxygen species(ROS)level in LPS-induced cells was detected by DCFH-DA fluorescent probe,which showed 2H5 M could scavenge the ROS.(5)The protein expressions of nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)were tested by Western Blot analysis,which showed 2H5 M could inhibited the protein levels of iNOS and COX-2.(6)MAPK signaling pathway investigated by Western Blot analysis,and as a consequence,2H5 M could surpress the phosphorylation of ERK rather than p38 and JNK.(7)The phosphorylation levels of p65 and I?B? of NF-?B signaling pathway were also down-regulated by 2H5 M studied by Western Blot analysis.(8)Cell immunofluorescence was used to detect the inhibitory of 2H5 M on nuclear translocation of NF-?B p65 and the result indicated that 2H5 M could inhibit the translocation of NF-?B p65.(9)2H5M weakened NF-?B DNA bing activity determined by EMSA in a dose-dependent manner.(10)Molecular docking study was utilized to simulate and clarify the interaction between 2H5 M and NF-?BCollectively,these results indicated that 2H5 M possesses anti-inflammatory effects and it may be as a candidate molecule for the development of the therapeutic strategy against inflammatory disorders in the future.