Preliminary Study On The Effects Of Daphnetin On MAPK Signaling Pathway In Synoviocytes Of CIA Rat Model Induced By TNF-α

Objectives:To observe the effect of Daphnetin(DAP)on MAPK signaling pathway in synovial cells of CIA rats induced by TNF-a,and lay the foundation for exploring the molecular mechanism of Daphnetin’s anti-rheumatic inflammation.Methods:1.Screening the best concentration of daphnetin on synovial cells of CIA rats.The effects of different concentrations of daphnetin(2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μtg/ml、60μg/ml)on the survival rate of synovial cells of CIA rats were observed by CKK-8 assay.Screening out the best drug concentration.2.Regulatory effects of daphnetin on MAPK signaling pathway in synovial cells of CIA rats induced by TNF-a.Experimental cells(except C group)were induced with lOng/ml TNF-a for 30min,grouped as follows:Model:synovial cells of CIA rat+TNF-α(model group)DAP:synovial cells of CIA rat+TNF-α+DAP(daphnetin group)TP:synovial cells of CIA rat+TNF-α+TP(positive drug control group)C:synovial cells of CIA rat(cell control group)After co-culture for 48 hours:(1)Real-time PCR was used to detect the effect of daphnetin on the expression of IL-6,TGF-β,MMP-3 and MMP-13 mRNA in synovial cells of CIA rats induced by TNF-a;(2)ELISA was used to detect the effect of daphnetin on the secretion of IL-6,TGF-β,MMP-3 and MMP-13 in supernatant of synovial cells of CIA rats induced by TNF-a;(3)Cell immunofluorescence was used to observe the effect of daphnetin on the expression of p-p38、p-ERK1/2、p-JNK in synovial cells of CIA rats induced by TNF-a;(4)Western blot was used to detect the effect of daphnetin on the expression and phosphorylation level of p-p38/p38、p-ERK1/2/ERK1/2、p-JNK/JNK protein of MAPK signaling pathway in synovial cells of CIA rats induced by TNF-α;Results:1.The effect of daphnetin on the survival rate of synovial cells of CIA rats at the concentration of 2.5μgml～30μg/ml for 48h had no significant effect on the survival rate of synovial cells.The highest survival rate at 20 μg/ml.2.Effects of daphnetin on IL-6,TGF-β,MMP-3 and MMP-13 mRNA expression in synovial cells of CIA rats induced by TNF-a:The expression of IL-6,TGF-β,MMP-3 and MMP-13 mRNA in the DAP group was significantly lower than that in the Model group(P<0.05).There was no significant difference between the TP groups(P>0.05).3.Effects of daphnetin on IL-6,TGF-β,MMP-3 and MMP-13 secretion in culture supernatant of synovial cells of CIA rats induced by TNF-a:The levels of IL-6,MMP-3,MMP-13 and TGF-β in DAP group were significantly lower than that in Model group(P<0.05),and there was no significant difference between TP group(P>0.05),and the secretion of MMP-3 and TGF-β was close to that of group C(P>0.05).4.Effects of daphnetin on expression of p-p38、p-ERK1/2、p-JNK in synovial cells of CIA rats induced by TNF-α:The p-p38、p-ERK1/2、p-JNK proteins were labeled as green fluorescence in each experimental group.Observed by fluorescence microscopy showed that the fluorescence intensity of Model group was significantly higher than that of group C,and the expression in nucleus was significantly increased;The fluorescence intensity of p-p38,p-ERK1/2 and p-JNK in DAP group was significantly lower than that in model group,and the expression of p-p38,p-ERK1/2 and p-JNK was significantly decreased.5.Effects of daphnetin on the expression and phosphorylation level of p-p38/p38、p-ERK1/2/ERK1/2、p-JNK/JNK protein of MAPK signaling pathway in synovial cells of CIA rats induced by TNF-a:The expression level of p-p38,p-ERK1/2 and p-JNK protein in DAP group was significantly lower than that in Model group(P<0.05),the expression ratio of p-p38/p38、p-ERK1/2/ERK1/2、p-JNK/JNK was also significantly lower than that of Model group(P<0.05),and the expression ratio of p-ERK1/2/ERK1/2、p-JNK/JNK was significantly lower than that of C and TP group(P<0.05).Conclusion:1.The effect of daphnetin on the survival rate of synovial cells of CIA rats at the concentration of 2.5μg/ml～30μg/ml for 48h had no significant effect on the survival rate of synovial cells.The highest survival rate at 20 μg/ml.2.The expression of IL-6,TGF-β,MMP-3 and MMP-13 mRNA in synovial cells of CIA rats induced by TNF-a was significantly decreased by daphnetin(20μg/ml).3.The levels of IL-6,TGF-β,MMP-3 and MMP-13 in supernatant of synovial cells of CIA rats induced by TNF-a were significantly decreased by daphnetin(20μg/ml).4.Daphnetin(20μg/ml)significantly reduced the expression of p-p38、p-ERK1/2、p-JNK in the nucleus of synovial cells of CIA rats induced by TNF-a.5.Daphnetin(20μg/ml)significantly reduced the phosphorylation level of p38,ERK1/2 and JNK protein in synovial cells of CIA rats induced by TNF-α.In summary,daphnetin can regulate the MAPK signaling pathway,affect the activation of MAPK signaling pathway to reduce the expression of inflammatory factors to play the role of anti-rheumatic inflammation.