Anti-inflammatory Mechanism of Shenfu Injection by Regulating the Combination of Nuclear HMGB1 and NF-κB Pathway
To explore the anti-inflammatory effect and potential mechanism of Shenfu injection(SFI)on lipopolysaccharides(LPS)induced RAW264.7 cells.Inflammatory cell model in wild type RAW264.7 cells were induced by using LPS.Pro-inflammatory factors(including iNOS,IL-1 β and TNF-α)were tested by using RT-qPCR,WB and ELISA to evaluate the anti-inflammatory effect of SFI.HMGB1+(over-expressing HMGB1)-RAW264.7 cells and HMGB1m(mutation of nuclear localization sites to prevent the extra-nuclear migration)-RAW264.7 cells were constructed to explore the potential mechanism.After stimulating by LPS,the level of HMGB1,TNF-α,IL-1β and iNOS were tested by RT-qPCR and WB,and the translocation of HMGB1 were tested by immunofluorescence in wild type,HMGB1+and HMGB1 m-RAW264.7 cells.Then mammalian two-hybrid approach and co-immunoprecipitation method were carried out to detect the binding rates among HMGB1,P65 and P50.The results indicate that the expression of iNOS,1L-1 β,and TNF-α in LPS induced RAW264.7 cells were inhibited by SFI(P＜0.01).The extracellular migration of HMGB1 was inhibited also by SFI,then resulting in the high expression of HMGB1 in the nucleus.the tolerance of RAW264.7 cells to LPS was increased by the high expression of HMGB1 in the nucleus.The TNF-α and IL-1β of HMGB1+-RAW264.7 cells induced by LPS(1 μg/mL)were both increased,which was reversed by SFI.The TNF-α and IL-1 β of HMGBlm-RAW264.7 cells had no difference with control group.SFI increased the binding rate of HMGB1 to P65 and HMGB1 to P50 in the nucleus(P＜0.01),and reduced the binding rate of P50 to P65.The results show that the above results suggested that the inflammatory response of LPS induced RAW264.7 cell was inhibited by SFI.Its pathway of action may be through inhibiting the translocation of HMGB1,competitively binding to P65 and P50,as well as reducing the formation of P65-P50 dimers.
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