The Effect Of Oleanolic Acid On Tumor Necrosis Factor Alpha-induced Inflammatory Cytokines Expression And The Related Mechanism In Synovial Cells

Objective: to investigate the effect of oleanolic acid on tumor necrosis factor alpha-induced inflammatory cytokines production and the related mechanism in synovial cells,which will lay a theoretical basis for the application of oleanolic acid in rheumatoid arthritis.Methods: 1.Cultured and passaged the fibroblasts synovial cells in vitro.2.Experimental methods: the activity of synovial cells was determined by 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT).RT-PCR was used to detect the mRNA expression of Interleukin-6(Interleukin-6,IL-6)and Interleukin-1β(Interleukin-1β,IL-1β).Western blot was used to detect the protein expression of p38 MAPK and NF-κB.ELISA was used to detect the levels of IL-6 and IL-1β in cell supernatant.3.Experimental groups: according to the research purpose,the experiment is divided into three parts:(1)to detect the effect of oleanolic acid on the basic condition of synovial cells,the cells can be divided into six groups:control group and different concentration of oleanolic acid group(5 μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L);(2)to detect the effect of oleanolic acid on TNF-α-induced inflammatory cytokines release,the cells were divided into five groups: control group,the TNF-α group and TNF-α +different concentrations oleanolic acid group(5μmol/L,10μmol/L,20μmol/L);(3)to test p38 MAPK and NF-κB protein expression,the cells were divided intofive groups: control group,the TNF-α group,TNF-α+ different concentrations oleanolic acid group(5μmol/L,10μmol/L,20μmol/L);(4)to detect the effect of the inhibitors of p38 MAPK and NF-κB on the levels of inflammatory cytokines,the cells were divided into 5 groups: control group,the TNF-α group,TNF-α+oleanolic acid(20μmol/L)group,TNF-α+ the inhibitor of p38 MAPK group,TNF-α+ the inhibitor of NF-κB group.Results: 1.5μmol/L-20μmol/L Oleanolic acid has no effect on the activity of synovial cells(P > 0.05).However,when the concentration is more than 40μmol/L,Oleanolic acid can inhibit the activity of synovial cells(P < 0.05).2.Compared with control group,TNF-α significantly promote the secretion of IL-6 and IL-1β,statistically significant difference(P < 0.05).3.Compared with the TNF-α group,oleanolic acid can inhibit TNF-α-induced mRNA expression of IL-6 and IL-1β in concentration dependence(P < 0.05).4.Compared with control group,TNF-αcan promote the phosphorylated p38 MAPK,statistically significant difference(P < 0.05);Compared with the TNF-α group,oleanolic acid can inhibit p38 MAPK phosphorylation induced by TNF-α in concentration dependence(P< 0.05).5.Compared with control group,the TNF-α can promote the protein expression of NF-κB in nucleus,statistically significant difference(P < 0.05);Compared with TNF-α group,oleanolic acid can inhibit the nucleoprotein expression of NF-κB induced by TNF-α(P < 0.05).6.When added the p38 MAPK blocker SB203580 or NF-κB inhibitors Bay11-7082,the secretion of IL-6 and IL-1β decreased significantly compared with control group(P <0.05).Conclusions: 1.TNF-α can promote the expression of IL-6 and IL-1β,and promote the phosphorylated p38 MAPK and NF-κB nuclear transfer in synovial cells.2.Oleanolic acid can inhibit TNF-α-induced expression of inflammatory cytokines(IL-6 and IL-1β),and suppress TNF-α-induced phosphorylation of p38 MAPK and NF-κB nuclear transfer in synovial cells.3.Oleanolic acid suppressed the expression of inflammatory cytokines by inhibiting p38 MAPK and NF-κB signaling pathway.