ABSTRACT
Objective: To study the influence of HMGB1-Abox on the expression of HMGB-1 in RAW264.7 cells stimulated by lipopolysaccharide(LPS)and its inhibition on the secretion of inflammatory factor.Methods: Use cloning vector to make up prokaryotic plasmid PET28a-HMGB1-Abox, which included the gene fragment of the A box of HMGB1, and recombinant vector was further transformed into Escherichia coil strain BL21(DE3),the recombinant plasmid was induced by isopropylthiogalactoside(IPTG)to express target protein.The protein was purified by Ni-column.We tested the effect of the different concentration of rHMGB1-Abox on the viabihty of RAW264.7 cells stimulated by LPS using CCK-8.Experimental group(EG)was treated with rHMGB41-Abox and control group(CG)was deh with PBS.TNF-α and IL-1β levels were detected by the enzyme linked immunosorbent assay(ELISA).Western blot and immunofluorescence staining method were used to compare the expression of HMGB1 in RAW264.7 cells with the experiment group and control group.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the variation tendency of HMGB1-mRNA.All procedures were manipulated at 2 \ 6 \ 12 \ 24 \ 48 h after treatment.Results: The recombinant prokaryotic expression vector PET28a-HMGB1-Abox was successfully constructed and mouse HMGB1-Abox protein about 14 kD was successfully expressed.There was a positive correlation between the viability of RAW264.7 cells and the concentration of protein and acting time.Compared to CG, TNF-α and IL-1β levels in EG declined significantly.In EG, Western blot and Immunofluorescence staining showed that the expression of HMGB1 protein was decreased and the expression of HMGB1-mRNA was reduced markedly and delayed.Conclusion: The rHMGB1-Abox could reduce expression and secretion of HMGB1 in RAW264.7 cells stimulated by LPS significantly,thereby to prevented the promotion of HMGB1 on pro-inflammatory mediator and inhibit the expression and secretion of inflammatory factors,which has some anti-inflammatory action.