Effect And Mechanism Of High Mobility Group Box 1 In Actue Hepatic Failure

Objective:To construct the total length high mobility group box 1 and high mobility group box 1-A box expression vectors, then got the his-tagged recombinant mouse HMGB1(rHMGB1) and the his-tagged recombinant mouse HMGB1-Abox (rHMGB1-Abox).Methods:Total RNA was extracted from liver tissue of mice, as the template for a polymerase chain reaction (PCR). Two primers were synthesized to amplify the entire coding sequence and to inset a BamH I site and a Xol I (Hand III)site. PCR products were ligated into a pMD18-T vector and confirmed by restriction digestion and sequencing. Using DNA Ligation kit, the BamH I (?)Xol I(Hand III) fragment of the pMD-18T-HMGB1 was inserted into the BamH I and Xol I(Hand III) sites of the pET-28a vector (containing a his6 tag), then got the resulting vectors, pET-28a-HMGB1 and pET-28a-HMGB1-Abox. Protein expressions were induced by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG). Purification of two proteins was achieved by affinity chromatography using a Ni-NTA column and elution with buffer containing 200mM (50 mM) imidazole.Results:The pET-28a-HMGB1 and the vector pET-28a-HMGB1-Abox were confirmed by restriction digestion and sequencing. The molecular mass of purified HMGB1 and HMGB1-Abox determined by SDS-PAGE were expected based on the theoretical value of his-tagged rHMGB1 and rHMGB1-Abox.Conclusion:The prokaryotic expression plasmids of total length HMGB1 and HMGB1-Abox were successfully constructed; the rHMGB1 and rHMGB1-Abox were achieved with high purity. Objective:To evalute the effect of rHMGB1 on increasing the inflammatory response in vitro and in vivo.Methods:In vitro test:We tested the effect of the different concentration of rHMGBl on the viability of RAW264.7 cells with or without LPS by using CCK-8. Experimental group (EG) 1 was treated with rHMGBl and LPS; EG 1'and control group (CG) were dealed with rHMGBl and LPS respectively. The blank group used PBS alone. TNF-a and IL-1p levels were detected by ELISA.West blot and Immunofluorescence staining method was used to compare the expression of HMGB1 in RAW264.7 cells with the four groups. RT-PCR was used to detect the variation tendency of HMGB1-mRNA. All procedures were manipulated at 2\6\12\24\48h after treatment. In vivo test:All mice were divided into four groups:D-Gal and LPS were used to make the animal model of acute hepatic failure as CG. Addition of rHMGB1 was EG 1 and EG 1'animals were administrated with rHMGB1 protein alone. The blank group was treated with 0.9% sodium chloride solution alone. We detected the variation tendency of HMGB1-mRNA, the expression of HMGB1 in mouse hepatocytes and serum levels of TNF-a and IL-1βat six time points:2,4,6,8,12,24h after intraperitoneal injections.Results:In virto, there was an inverse correlation between the viability of RAW264.7 cells and the concentration of protein and acting time. Compared to the blank group, TNF-a and IL-1p levels in the other three groups were increased significantly. In EG, West blot and Immunofluorescence staining showed the expression of HMGB1 protein was increased and the expression of HMGB1-mRNA was higher markedly. EG 1'and CG had no differences significantly in all the tests. In vivo, we found that after addition of rHMGB1 in acute hepatic failure mice, the destroy of liver tissue, the expression of HMGB1-mRNA, the release of HMGB1 in liver tissues and the serum levels of TNF-a and IL-1p were much higher than those of the group without rHMGB1-Abox (P< 0.05), and there were significant difference between the normal mice and the mice which injected with LPS\ D-Gal and rHMGB1-Abox (P< 0.05).Conclusion:We conclude that rHMGBl had the same structure and function with the native HMGB1, and addition of rHMGB1 could activate the cytokines and intranuclear HMGB1 to release and increase the inflammatory response in vitro and in vivo.Objective:To evalute the effect of rHMGB1-Abox on decreasing the inflammatory response in vitro and in vivo.Methods:In vitro test:We tested the effect of the different concentration of rHMGB1-Abox on the viability of RAW264.7 cells stimulated by LPS using CCK-8. Experimental group (EG) was treated with rHMGB1-Abox and control group (CG) was dealed with PBS. TNF-a and IL-lβlevels were detected by ELISA.West blot and Immunofluorescence staining method was used to compare the expression of HMGB1 in RAW264.7 cells with the experiment group and control group. RT-PCR was used to detect the variation tendency of HMGB1-mRNA. All procedures were manipulated at 2\6\12\24\48h after treatment. In vivo test:We used D-Gal and LPS to make the animal model of acute hepatic failure in mouse. Used rHMGBl-Abox as the experimental group, we detected the variation tendency of HMGB1-mRNA, the expression of HMGB1 in mouse hepatocytes and serum levels of TNF-a and IL-1βat six time points:2,4,6,8,12,24h after intraperitoneal injections.Results:In virto, there was a positive correlation between the viability of RAW264.7 cells and the concentration of protein and acting time. Compared to CG, TNF-a and IL-1p levels in EG declined significantly. In EG, West blot and Immunofluorescence staining showed the expression of HMGB1 protein was decreased and the expression of HMGB1-mRNA was reduced markedly and delayed. And in vivo, we found that after addition of rHMGB1-Abox in acute hepatic failure mice, the destroy of liver tissue, the expression of HMGB1-mRNA, the release of HMGB1 in liver tissues and the serum levels of TNF-a and IL-1p were much lower than those of the group without rHMGBl-Abox (P< 0.05), and there were significant difference between the normal mice and the mice which injected with LPS\D-Gal and rHMGBl-Abox (P< 0.05).Conclusion:We conclude that rHMGB1-Abox had the same structure and function with the native HMGB1-Abox, could interdict the cytokines and intranuclear HMGB1 to release and decrease the inflammatory response in vitro and in vivo.