Cloning, Expression And Functional Study Of The Human High Mobility Group Box 1 Protein

Objective and SignificanceSepsis is defined as systemic inflammatory response syndrome (SIRS) induced by infection, and it is a common complication of severe injury, burn, hypoxia, reperfusion injury and surguries. With the development of the illness, sepsis may result in septic shock and multiple organ dysfunction syndrome (MODS), which is a difficult problem of surgery department and one of important reasons causing death. Within recent 10 years, along with advancement of research on inflammation response mechanisms, we have understood further about the essence of sepsis and its pathological process. We've made it clear that sepsis, severe sepsis, septic shock and MODS is a dynamic process reflecting pathophysiological changes of the body and changes of deterioration of illness, and sepsis is the sequence of systemic inflammatory response deteriorating. Severe injuries can induce early inflammatory response, however, because of the cascade effect of many inflammatory mediators, inflammatory response may enlarge, even out of control, and result in systemic inflammatory response. Many years ago, we thought that "early inflammatory mediators", such as TNF-a and IL-1, are "core factors" causing MODS and death, however, there is no notable clinical effects while using TNF-a receptor antagonist and IL-1 receptor antagonist. In the later stage of sepsis, the symptoms keep deteriorating and the patients die, but the level of TNF-a and IL-1 remains normal,which indicates there may be some late inflammatory mediators taking part in pathophysiological process in the late stage of sepsis.In 1999, Wang, et al, first reported that as a potential late mediator, high mobility group box-1 protein(HMGBl) took part in the pathological process of sepsis, and HMGBl is an important late inflammatory mediator of lethal effect of LPS. Henceforth, scientists focused on the correlation between HMGBl and sepsis increasingly. More and more reports have demonstrated that in severe inflammations, HMGBl can be expressed in cytoplasm. On the study of endotoxemia of rats, scientists found levels of HMGBl in serum increased notably after LPS stimulation for 16 to 32 hours, and rats died after injection of recombinant HMGBl. Through the studies in vitro, HMGBl can induce macrophages and neutrophils to produce many inflammatory mediators, such as TNF-a, IL-la> IL-1I^ IL^ IL-8> monocyte inflammatory protein-la (MlP-la) and MIP-16. Full-length HMGBl or B-box of HMGBl can induce human umbilic vein endothelial cell (HUVEC) to upregulate the levels of several adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, and release IL-8 and granulocyte colony stimulating factor (G-CSF), which has dose-dependent relation.At present, it is still in the early research stage that HMGBl can be as a new "late" mediator of sepsis, and there are still many confusing questions. For example, whether HMGBl indeed participate in the occurrence and development of systemic inflammatory response? What is the signal transduction pathway by which HMGBl activates inflammatory cells? What is the clinical significance and essential correlation between changes of HMGBl and severe injury-induced sepsis? Whether it helps to prevent and treat sepsis and MODS with the intervention ofHMGBl------a potential "late" mediator? Resolving these above problems canprovide new theoretical interpretation of mechanisms of sepsis and MODS, and break a new path of prevention and treatment.Based on the above study background, through recombinant HMGBl, in endotheliums, which are important cells participating in inflammation response, weobserved the effect of HMGBl on the production of inflammatory cytokines, such as IL-6, IL-8 and MCP-1. We also studied whether HMGBl could activate endotheliums to release inflammatory cytokines with the cooperation of LPS, which will provide a base for studying pathophysiological effect and regulation mechanisms of HMGBl.Methods1. Cloning of HMGBl gene: Through polymerase chain reaction (PCR), we amplified the code gene of HMGBl, and then cloned to the vector pET14bMCS, and identify the recombinant plasmid through PCR, enzyme digestion and DNA sequencing.2. Prokaryotic expression and purification of His-HMGBl fusion protein: The positive clone was then transferred to E. coli BL21(DE3). The His-HMGBl fusion protein was then expressed induced by IPTG, and then we purified the HMGBl protein by means of Ni-NTA resin. After dialysis and sterilization, the purified HMGBl protein was stored at -20°C.3. We used different doses of His-HMGBl protein to stimulate HUVEC, with the final concentration of 3 ^g/ml, 15 jug/m\ and 75 ^g/ml, and collected cultured supernatant 24 hours later; and examined 11 cytokines/chemokines by LiquiChip system, including GMCSF, IFN-y, IL-10, IL-12, IL-1B, IL-2, IL-4, IL-6, IL-8, TNF-a and MCP-1.4. We used different doses of His-HMGBl protein to stimulate HUVEC, with the final concentration of 3 ^g/ml, 15 fig/ml and 75 ^g/ml, and collected cultured supernatant 24 hours later; and we used His-HMGBl protein (15 jugjml) to stimulate HUVEC, collected cultured supernatant at different time point, and examined correlative cytokines/chemokines by LiquiChip system, including IL-6, IL-8 and MCP-1.5. We used different doses of His-HMGBl protein (3 ^g/ml, 15 ^g/ml and 75 ^g/ml)and LPS (10 ng/ml) to co-stimulate HUVEC, and collected culturedsupernatant 24 hours later; and examined correlative cytokines/chemokines by LiquiChip system, including IL-6, IL-8 and MCP-1.Results1. After accurate identification of cloned His-tagged HMGB1 expression vector through enzyme digestion and sequencing, we purified His-HMGBl fusion protein by means of Ni-NTA affinity chromatography.2. HUVEC were stimulated by different doses of HMGB1 protein, and we found that, only three cytokines (IL-6, IL-8 and MCP-1) increased along with the increased doses of HMGB1 protein.3. HUVEC were stimulated by different doses of HMGB1 protein, and we found that, the release of these three cytokines/chemokines, including IL-6,IL-8,MCP-1, increased along with the increased doses of HMGB1 protein. There is statistic significance between the stimulated group and the control.4. We respectively examined the level of these three cytokines/chemokines after 1 h, 3 h, 6 h, 12 h and 24 h, and we found the secretion of the cytokines increased with time prolonging.5. Through the experiment of His-HMGBl on the cytokines/chemokines production by LPS, we found that, there is the strongest enhancement effect of IL-6 and IL-8 when the dose of His-HMGBl is 75 /ug/ml; whereas, the strongest effect of MCP-1 appears when the dose of His-HMGBl is 15 jug/ml.Conclusion1. We successfully cloned, expressed and purified His-HMGBl protein, which provides essential materials for the next studies on His-HMGBl protein.2. The three cytokines/chemokines, including IL-6, IL-8 and MCP-1, can be upregulated by His-HMGBl protein, there is time-response relationship and time-concentration relationship between HMGB1 and the three cytokines /chemokines.3. His-HMGBl protein significantly enhances the production of cytokines /chemokines by LPS.These above results provide a base for studying pathophysiological effect and regulation mechanisms of HMGBl.