Anti-inflammatory Effects Of Daphnetin On Fibroblast-like Synoviocytes Of CIA Rats Induced By TNF-α Based On NF-κB Signaling Pathway

Objectives:To investigate the effect of daphnetin(DAP)on the inflammatory response of fibroblast-like synoviocytes(FLS)induced by TNF-α in CIA rats,and to elucidate the molecular mechanism of NF-κB signaling pathway in reducing inflammatory response.Methods:1.The experiment was divided into four groupsAddition to the FLS group,all were induced with 10 ng/ml TNF-α for 30 min:FLS group(cell control)Model Group(TNF-α+FLS--Inflammation Model Group)DAP group(TNF-α+FLS+DAP--experimental group)TP group(TNF-α+FLS+TP--positive drug group)2.Effect of Daphnetin in Synoviocytes of CIA Rats induced by TNF-α on NF-κB Signal Pathway(1)Western blot was used to detect the effect of daphnetin on NF-κB signaling pathway P65,P-P65 protein expression and phosphorylation of synoviocytes induced by TNF-α in CIA rats.(2)The effect of Daphnetin on the nuclear expression of P65,P-P65 in synoviocytes of CIA rats induced by TNF-α was observed by immunofluorescence.(3)Real-time PCR was used to detect the effect of daphnetin on the expression of IL-6,TGF-β,ICAM-1 and IL-10 mRNA in synoviocytes of CIA rats induced by TNF-α.(4)ELISA was used to detect the effect of Daphnetin on the secretion of IL-6,TGF-β,ICAM-1 and IL-10 in culture supernatants of synoviocytes from CIA rats induced by TNF-α.Result:1.In the protein level assay,the protein level of P-P-65 in the NF-κB signaling pathway in the model group was higher than that in the non-TNF-α-induced FLS group.The difference was statistically significant.A comparison between the daphne group and the model group showed that the NF-κB signaling pathway protein P-P-65 protein level in FLS cells induced by the addition of daphnetin was lower than that of FLS cells induced by TNF-α without daphnetin.The difference was statistically significant.2.Observe in the nuclear expression,it was found that TNF-α induced the expression of P-P-65 in the NF-κB signaling pathway was higher compared with the non-TNF-α induced FLS.In comparison between the daphne group and the model group,it was found that the intranuclear expression of the NF-κB signaling protein P-P-65 added with Daphnetin was lower in comparison to the group without the addition of daphnetin.3.At the transcriptional level,the mRNA relative expression levels of NF-κB signaling-related cytokines IL-6,TGF-β,ICAM-1 in TNF-α-induced model group were higher than those without TNF-α.The difference was statistically significant.The mRNA relative expression levels of IL-6,TGF-β,and ICAM-1 in the NF-κB signaling pathway in the DAP group were lower compared with model group.However,the relative mRNA expression of IL-10 was higher than that model group without daphnetin.The difference was statistically significant..4.In the cell supernatant,the secretion of NF-κB signaling pathway related cytokines IL-6,TGF-β,and ICAM-1 in the model group induced by TNF-α was higher than that in the FLS group not induced by TNF-α.The difference was statistically significan.The secretion of IL-6,TGF-β,and ICAM-1 of NF-κB signaling pathway in DAP group was lower than model group.There was statistical significance;however,the secretion of IL-10 was higher than that model group.The difference was statistically significant.Conclusion:1.Daphnetin(20μg/ml)can significantly reduce the phosphorylation of P-65 and P-P-65 protein in synoviocytes of CIA rats induced by TNF-α.2.Daphnetin(20μg/ml)can significantly reduce the expression of P-65 and P-P-65 in nucleus of synoviocytes induced by TNF-α in CIA rats.3.Daphnetin(20μg/ml)significantly reduced the expression of IL-6,TGF-β,ICAM-1 mRNA in synoviocytes of CIA rats induced by TNF-α,increase the expression of IL-10 mRNA in synoviocytes of CIA rats.4.Daphnetin(20μg/ml)can significantly reduce the secretion of IL-6,TGF-β,and ICAM-1 in culture supernatants of synoviocytes from CIA rats induced by TNF-α and increase the secretion of IL-10.Level.In summary,TNF-α can induce the activation of NF-κB signaling pathway in FLS cells,and adding daphnetin can reduce the phosphorylation of P65 in the NF-κB signaling pathway,regulate the secretion of related inflammatory factors,and reduce the inflammatory response.