Total Saponin Of Dioscorea Inhibit The Expression Of Inflammatory Factors Via TLR4/NF-κB Signaling Pathway In THP-1 Derived Macrophages

OBJECTIVE:To investigate the effect of total saponin of Dioscorea（TSD）on Toll-like receptor 4/nuclear transcription factor-κB（TLR4/NF-κB）signaling pathway in THP-1derived macrophages,and to explore the anti-inflammatory mechanism of TSD.METHODS:Phorbol-12-myristate-13-acetate（PMA）was used to induce the transformation of THP-1monocytes into macrophages,constructing an in vitro macrophage model,and morphologically observing the morphological characteristics of macrophages and the expression of surface specific protein CD11b.MTT assay was used to detect the effects of different concentrations of TSD on cell viability and proliferation.The experiment was divided into control group,MSU group,TSD low,medium,high(1,3,10μg·m L^-1)group and colchicine(0.2μg·m L^-1)group.In the concentration group,except for the control group and the MSU group,the other groups were pretreated for 24 hours and then stimulated by 400μg·m L^-1MSU for 6 hours（except for the control group）.The levels of inflammatory factors TNF-αand IL-1βwere detected by ELISA;RT-q PCR method was used to detect the expression levels of TLR4,NF-κB and pro-IL-1βm RNA;Western Blot method was used to detect TLR4,My D88 and NF-κB protein expression;Nuclear shift of NF-κBp65 was detected by immunofluorescence.（2）The cells were induced to differentiate as above,then the cells were divided into control group,LPS group,LPS+TSD-H group and LPS+colchicine group and to further study the regulatory effect of TSD on the TLR4/NF-κB inflammatory pathway stimulated by LPS in THP-1 derived macrophages.After 24 hours of TSD-H and colchicine treatment,all groups except the control group were added with a final concentration of 2μg·m L^-1LPS to stimulate for 6 h.The detection methods and indicators of inflammatory factors,m RNA and protein are the same as above.（3）Effect of TSD on pathway-specific blocker TAK-242 or PDTC intervention of TLR4/NF-κB signaling pathway in THP-1derived macrophages.The cells were divided into control group,MSU group,TSD-H group,and TAK-242 group,PDTC group,TAK-242+TSD-H group,PDTC+TSD-H group,except for the control group,MSU group and TSD-H group,the remaining groups were added with inhibitors to intervene for 30 minutes.Except for the control group,MSU and inhibitor group,the rest of the groups were added TSD for 24 hours,then 400μg·m L^-1MSU co-stimulated for 6h（except the control group）.The detection methods and indicators of inflammatory factors,m RNA and protein are the same as above.RESULTS:（1）100ng·m L^-1PMA induced THP-1 cells for 24h,the cells changed from suspension to adherence,and the cell morphology was fusiform,with pseudopods protruding,and the expression of cell surface specific protein CD11b significantly increased（P<0.01）,indicating that THP-1 monocytes successfully induced differentiation into macrophages.TSD had almost no effect on cell proliferation at 0-32μg·m L^-1（P>0.05）.After treatment with 400μg·m L^-1MSU for 6h,TLR4,NF-κB and pro-IL-1βm RNA expression increased（P<0.01）,TLR4,My D88 and NF-κB protein expressions were significantly up-regulated（P<0.01）,and TNF-αand IL-1βsecretion increased（P<0.01）.TSD pre-treatment can significantly reduce TLR4,NF-κB and pro-IL-1βm RNA expression（P<0.01）,down-regulate TLR4,My D88 and NF-κB protein expressions,and decrease TNF-αand IL-1βsecretion（P<0.05;P<0.01）.NF-κBp65 fluorescence increased in the nucleus,suggesting that NF-κB translocation into nuclear activation.（2）Compared with the LPS group,the TSD-H group and the colchicine group can significantly inhibit the expression of TLR4,NF-κB and pro-IL-1βm RNA（P<0.01）,increase the expression of key proteins TLR4,My D88 and NF-κB and downstream inflammatory factors TNF-αand IL-1βsecretion levels（P<0.01）.（3）Compared with the MSU group,the expression of TLR4,NF-κB and pro-IL-1βm RNA decreased in the TSD-H group and the inhibitor group（TAK-242 or PDTC）（P<0.01）,reduced the expression of TLR4,My D88 and NF-κB protein and the secretion of downstream inflammatory factors TNF-αand IL-1β（P<0.01）;Compared with the inhibitor group（TAK-242 or PDTC）,TAK-242+TSD-H group and PDTC+TSD-H group further reduced TLR4,NF-κB and pro-IL-1βm RNA expression（P<0.01）,TLR4,My D88 and NF-κB protein expression and inflammatory factors TNF-αand IL-1βsecretion levels decreased（P<0.01）.CONCLUSIONS:TSD may down-regulate the expression of TLR4,NF-κB and pro-IL-1βm RNA and TLR4,My D88 and NF-κB protein through the TLR4/NF-κB inflammation pathway,reduce the secretion of inflammatory factors TNF-αand IL-1β,and exert anti-inflammatory effect.