Effects Of Sophoridine On Lipopolysaccharide Activated Of NF-κ B Signal Pathway I In RAW264.7 Macrophage

Objective: To investigate the effects of Sophoridine on the expression of critical signal molecule IKKβ, IκBα, NF-κB and downstream inflammatory cytokine TNF-αof NF-κB pathway in RAW264.7 cell induced by lipopolysaccharide (LPS),and to explore the pharmacological mechanism of Sophoridine anti-endotoxin.Methods: Cultured RAW264.7 macrophages LPS stimulated RAW264.7 cells, established inflammatory reaction model,experiments divided into control group, sophoridine control group, LPS model group 1, sophoridine intervention group, LPS model group 2, pretreatment sophoridine group, sophoridine premixed group. After each group disposed of 5, 30, 60, 120minutes, respectively, for cells and cell culture medium. Use Western Blot technology to detect protein express of the IκBα,pIκBα,NF-κB P65 and RT-PCR to detect the mRNA express of the IKKβ,IκBα,NF-κB P65 and Radioimmunoassay (RIA) was used to detect the TNF-αcontents of cell supernatant.Results (1)Sophoridine itself has no influence on every indexes of RAW264.7 macrophage. IKKβ, NF-κB P65 mRNA and protein expression and pIκBα, TNF-αexpression at each time points in LPS model group1 were significantly higher than the control group (P < 0.01). IκBαmRNA and protein expression in macrophage were lower than control group (P < 0.01), and detection indexes with longer duration of LPS stimulation increase or decrease gradually, untill to120min without decline or rise. IKKβ, NF-κB P65 mRNA and protein expression and pIκBα, TNF-αexpression at each time points in Sophoridine intervention group were lower than LPS model group1 at the same time points, and with longer duration of Sophoridine in each indicators increased to decline untill 120min. Compared with TNF-αexpression in Sophoridine intervention group at each time points, 120min were significantly lower than 5min, it shoes that the role of Sophoridine time dependent, but they were higher than control group at each time points(P < 0.01or P < 0.05). However, IκBαmRNA and protein expression in Sophoridine intervention group from 30min to 120min were significantly higher than LPS model group1 at the same time points, but still lower than control group(P < 0.01or P < 0.05). They have significantly difference to compare with TNF-αlevel at each time points in Sophoridine intervention group(P < 0.01). It shows that effect of Sophoridine has time dependency.? (2) IKKβ, NF-κB P65 mRNA and protein expression and pIκBα, TNF-αexpression at each time points in LPS model group2 were significantly higher than control group (P< 0.01). IκBαmRNA and protein expression in macrophage were lower than control group (P < 0.01), and with longer duration of LPS stimulation increase or decrease gradually, untill to 120min without decline or rise in above indexes. Sophoridine pretreatment group at each indexes expression trends compared with LPS model group 2 similar trend. But IKKβ, NF-κBP65 mRNA and protein expression and pIκBα, TNF-αexpression in Sophoridine pretreatment group were Significantly lower than LPS model group 2 at the same time points(P < 0.01or P < 0.05), and each they higher than control group(P < 0.01or P < 0.05). IκBαmRNA and protein expression in Sophoridine pretreatment group Since 30min were Significantly higher than LPS model group2 at the same points until 120min, and they were Significantly lower than control groups(P < 0.01or P < 0.05). (3) IKKβ, NF-κB P65 mRNA and protein expression and pIκBα, TNF-αexpression in Sophoridine premixed group were lower than LPS model group2 at the same points (P < 0.01or P < 0.05). and they were higher than control groups at the same points (P < 0.01or P < 0.05). IκBαmRNA and protein expression in Sophoridine premixed groupateach were lower than LPS model group 2 at the same time points, but they were higher than contro lgroups(P < 0.01or P < 0.05), and above indexes at each time points in Sophoridine premixed group showed attachment are in flat trends.Conclusion (1) Inhibiting of Sophoridine of NF-κB signaling pathway of IKKβmRNA expression induced by lipopolysaccharide in RAW264.7 macrophages, promoted mRNA and protein of IκB expression, reduced the phosphorylation of IκB degradation, downregulating the mRNA and protein of NF-κB P65 expression, downstream inflammatory factors TNF-αrelease. It shows Sophoridine anti-endotoxin mechanism relation with contol NF-κB signaling pathway . (2) Three administration style of Sophoridine all down-regulated expression of IKKβ,IκBα,NF-κB P65 mRNA and/or protein and TNF-αlevels, and up-regulated expression of IκBαmRNA and protein in RAW264.7 macrophages activated by LPS, but their time-effect curves were various with different administration style. It shows that Sophoridine may play the role of anti-endtoxin about prevention and treatment. (3)The effect of Sophoridine anti-endtoxin has time dependency. (4)There is no statistically difference of test data between sophoridine group and control group, which proved sophoridine15.63 mg.L-1, would not effects RAW264.7 cells itself and NF-κB pathway.