Hmgb1-a Box Gene Expression And Its Protective Effects On Liver Injury

Objective Recent studies identify that extracellular high mobility group-1 (HMGB1) protein as a late mediator of endotoxemia and sepsis. The appearance of HMGB1 much later than TNF and 1L-1 in the inflammatory cascade suggested that HMGB1 -targeted therapies might be initiated even at late stages of acute inflammatory conditions. It was interesting that HMGB1 A-box can inhibit HMGB1 release and action,instead of initiating inflammation. But HMGB1 actual role in acute liver injury(ALI) and HMGB1 A-box protection aganist ALI have not been determined. We design such an experiment to clone and prokaryoticly express HMGB1 A-box , then explicate the protection of HMGB1 A-box against ALI.Methods1. Cloning and expression of the HMGB1 A-box gene in E.coli: HMGB1 A-box gene was amplified by nested RT-PCR from RAW264.7 cells and cloned into vector pMD-19T. After sequenced, It was subcloned into prokaryotic expressive vector pET-26b (+). Expressed recombinant plasmid pET-26b(+)-HMGB1 A-box was constructed. Having transformed E.coli BL21(DE3) and induced by IPTG for 4 hours, recombinant fusion protein His-HMGB1 A-box was expressed.2. purification and identification of HMGB1 A-box : The expressed fussion protein was purified by Ni2+-NTA affinity chromatograph. Because 6×His-labelled did not affect the protein secretion and collapse and did not disturb the structure and function of protein, so itneed not be cut out after putification.To examined whether purified protein HMGB1 A-box might inhibit TNF-α secretion induced by HMGB1, RAW264.7 cells were stimulated with A-box and HMGB1 or LPS. After 12 hours, TNF-α release in RAW264.7cells was observed by ELISA.3. HMGB1 actual role in ALI and HMGB1 A-box protection aganist ALI : ALI model was constructed by intraperitoneal injection of GalN /LPS. At the same time, ALI animals were treated with HMGB1 A-box. Then serum ALT , LPS , TNF- α levels and HMGB1 levels in liver tissure were determined at all kinds of times.Results1 .The recombinant expression plasmid pET-26b(+)-HMGB1 A-box was constructed and the His-HMGB1 A-box protein was expressed, it was about 60% of total bacterial protein.2.The purified protein was obtained by Ni²⁺-NTA affinity chromatograph. And the purified protein His-HMGB1 A-box can significantly inhibit TNF- α secretion induced by HMGB1, instead of inhibiting TNF- α secretion induced by LPS.3.HMGB1 levels of liver tissue increased significantly at 24h, then kept high levels between 48h and 72h. The relativity analysis showed that liver HMGB1 levels was correlative with liver...