Preventive Effect Of Recombinant Human HMGB1 Protein On Sepsis In Mice By Active Immunization

Background:Cytokines play key roles in the process of sepsis, among them TNF-a and IL-1β are the prominent early pro inflammatory mediators. However, clinical efforts directed towards TNF-a and IL-1β were proved to be in vain for the treatment of sepsis, one of probable reasons for such failure is that these targets provide a very short therapeutic window due to their very short half-lives in the serum of patients. More than a decade ago, HMGB1 was recognized as a late pro inflammatory mediator responsible for the severity of sepsis, and since then it has been emerging as a more promising new target than those early pro inflammatory mediators. At present, strategies that trying to inhibit HMGB1 expression and releasing have been employed by many investigaters and a lot of positive results have been achieved. In this study, we tried to investigate the protective potential of active immunization with HMGB1 protein on a mouse model of sepsis.Methods:Using routine molecular biological techniques, human HMGB1 (hHMGBl) cDNA was cloned into a plasmic vector, and the expression of HMGB1 protein in a E.coli expression system was induced by lactose. Bal b/c mice were immunized with purified human recombinant HMGB1. The antiserum titer of mice was determined by indirect ELISA. Anti-HMGB1 antiserum from mouse with stabilized titer was used for the following Western blotting. The protective eflfect of active immunization against sepsis was assessed in a septic mouse model induced by cecal ligation and puncture (CLP). The morphological changes of vital organs of septic mice were determined by H&E staining. The level of HMGB1 in mouse serum was measured with a commercial HMGB1 ELISA kit.Results:Human HMGB1 cDNA was successfully constructed to the plasmic vector pET28a in frame with a His-tag. After induction by lactose and purification with Ni-beads, soluble rhHMGB1 protein was obtained. The plateau of antiserum titer was confirmed both by indirect ELISA and Western blotting. Upon CLP challenge, mice pre-immunized with rhHMGB1 showed greatly improved outcomes as compared to those in control group, including increased survival rate, alleviated organ damages and decreased serum HMGB1 level.Conclusions:Active immunization with recombinant human HMGB1 protein renders mice great potency to resist the challenge of experimental sepsis, which might provide a new avenue for the prevention and treatment of sepsis.