Anti-inflammatory Effect And Mechanism Of Maslinic Acid On LPS-Induced RAW264.7 Cells

Objective: An inflammation model in mouse macrophage cell line RAW264.7 was induced by lipopolysaccharide(LPS),then the anti-inflammatory effect was examined and the mechanism of maslinic acid(MA)was explored on this model.Methods: 1.LPS dose screening: RAW264.7 cells in good condition were inoculated into 96-well cell culture plates by 1×105/well and cultured to logarithmic growth phase.Raw264.7 cells was treated with different concentrations of LPS(0,50,100,500,1000,1500,2000,5000 ng / m L)for 24 h,the viability of each group cells was detected by MTT.The cells were also seeded in 24-well cell culture plates,cultured overnight.After adding different concentrations of LPS(0,500,1000,1500,2000,4000 ng / m L)for 24 h,NO concentration of each group was measured.2.MA dose and action time screening: RAW264.7 cells were cultured,seeded in 96-well cell culture plates at 1×105/well,cultured for 12 h,and different concentrations of MA(0,2.5,5,10,15,20,25,30,35,50 ?mol/L)were added,MTT assay was used to detect the viability of each group cells.The LPS(1000 ng/m L)induced RAW264.7 cells inflammatory model was constructed,the control group,LPS group(model group),LPS+ MA(5,10,15,20,25,30 ?mol/L)group were set and the NO level of each group was assayed.The work time of MA was selected with the groups of the control group,LPS group(model group),LPS+MA(15?mol/L,preaction 2,1,0 h)group.3.Anti-inflammatory effect and mechanism of MA on LPS-stimulated RAW264.7 cell were explored as well.Setting control group,model group,LPS+MA(5,10,15,20 ?mol/L)group,under microscope(40 times)the cells were subjected to epigenetic detection.The m RNA levels of IL-6,IL-1?,TNF-?,i NOS,COX-2 and STAT3 in each group were detected by RT-PCR.The STAT3 protein phosphorylation level was detected by Western Blot.Setting control group,model group,MA(20?mol/L)alone group,LPS+MA(10,15,20?mol/L)group,the i NOS,COX-2 expression and JAK1 protein Phosphorylation level were detected by Western Blot.Results: 1.LPS stimulated cells,LPS had no significant effect on cell viability at 1000 ng/m L,and NO concentration in RAW264.7 cells increased significantly at 1000 ng/m L.2 After stimulation with different concentrations of MA,the cell viability of MA at 35?mol/L began to decrease significantly.The safe concentration of MA was within 0-30?mol/L.LPS(1000ng/m L)induced RAW264.7 cells and preaction with different concentrations of MA.MA at 10-20 ?mol/L can inhibit LPS-induced inflammation.MA(15 ?mol/L)was pretreated at different times,and MA was induced by LPS at preaction 2 h.,the protective effect of RAW2654.7 cell inflammation was better.In the apparent test,compared with the control group,the antenna angle of the model group was significantly increased.After MA addition,the antennae of RAW264.7 cells decreased significantly.The effect of MA on the expression level of IL-6,IL-1?,TNF-?,i NOS,COX-2 and STAT3 in m RNA level was detected by RT-PCR.The MA-protective group was significantly lower than the model group.Compared with the model group,i NOS,COX-2 protein expression and JAK1,STAT3 protein phosphorylation level in MA-treated groups decreased significantly.Conclusion:1.In LPS-induced RAW264.7 cell inflammation model,MA can reduce the expression of NO and IL-6,IL-1?,TNF-?,i NOS,COX-2 at m RNA level.MA show some protect effect in the LPS-induced RAW264.7 cell.2.In the inflammation model of RAW264.7 cells induced by LPS,MA can effectively reduce STAT3 expression at m RNA level.Meanwhile the phosphorylation level of JAK1 and STAT3 were significantly decreased.Therefore,MA may play an anti-inflammatory effect on RAW264.7 through STAT3 signaling pathway.It can provide some basis for anti-inflammatory mechanism of maslinic acid in the future.