AAV-mediated Gene Transfer Of HMGB1 Antibodies And Antibody Therapy Targeting LPS And HMGB1 For The Treatment Of Sepsis In Mice

Part OneObjective To investigate the therapeutic effect of Adeno-Associated Virus(AAV)-mediated high mobility group box-1protein(HMGB1)antibody gene transduction in mice with sepsis.Methods Sixty female C57 rats in the weight range of 18-22 g were randomly divided into 2batches of 10 rats in each of 3 groups,and they are named the intramuscular AAV-HMGB1 antibody group(a H for short),the intramuscular AAV-Control-Ig G group(CI for short),and the intramuscular saline group(NS for short).The a H group was injected intramuscularly with 60 μl of AAV-anti-HMGB1 virus in the left rectus femoris muscle.The CI group was injected with 60 μl AAV-Control-Ig G virus in the left rectus muscle and the NS group was injected with 60 μl saline in the left rectus muscle,and all mice were subjected to cecal ligation and perforation(CLP)surgery 4 weeks later to create sepsis mice.After the surgery,serum levels of inflammatory factors IL-6,IL-1β and TNF-α were measured by the mice blood sampling obtained from the internal canthus vein.the 14-d survival rate of the mice was observed,and sections were made from heart,liver,lung,kidney and small intestine organs to observe the presence of organ pathological changes.Results The use of AAV-HMGB1 antibody transduction to produce HMGB1 antibody had a protective effect against sepsis in mice(90% of septic mice survived until day 14post-modeling in the a H group,while only 60% and 50% of mice survived on day 14post-modeling in the NS group and the CI group,respectively).The tissue inflammatory cell infiltration has been reduced and the concentration of serum inflammatory factors IL-6,IL-1β and TNF-α were also significantly lower(P<0.05)in the mice in the a H group compared with the control NS and CI groups.The therapy had a significant protective effect on lung tissue,but not on other organs.Conclusion AAV-mediated gene transduction of HMGB1 antibody can neutralize HMGB1 in serum by high expression of HMGB1 antibody,thus reducing the level of inflammatory factors in septic mice,improving pathological changes in vital organs and possibly reducing mortality in septic mice,which is expected to be a new strategy for the treatment of sepsis.Part TwoObjective To elucidate the binding power of the bifunctional fusion protein CRISPLD2-anti-HMGB1 to LPS and HMGB1,to explore the effect of this protein on the activation of TLR4 signaling pathway and the expression of related inflammatory factors,and to study the effect of this protein on the protection of organ function and survival in septic mice.Methods The CRISPLD2-anti-HMGB1 bifunctional fusion protein was demonstrated to bind to LPS and HMGB1 using the PULL-DOWN and Bia CORE techniques,and detailed binding and dissociation KD(M)values were measured;Raw264.7 cells were stimulated with LPS(1 μg/ml)and HMGB1(1 μg/ml),and then anti-HMGB1(1μg /ml)and CRISPLD2-anti-HMGB1 bifunctional fusion protein(1μg /ml)were added to each group to investigate the expression of P-P38,P-PJNK1/2,P-PERK1/2 in NF-κB and MAPK of TLR4 signaling pathway and the expression of extracellular inflammatory factors IL-6 and TNF-α concentrations were influenced by HMGB1 antibody and bifunctional protein.After successful modeling,the mice were treated with anti-HMGB1(50 mg/kg)and CRISPLD2-anti-HMGB1 bifunctional fusion protein(50 mg/kg),and the biochemical indexes of liver and kidney function were measured after 24 h.The survival of the mice was observed,and the survival curve was plotted for 14 d.Organ pathology specimens were collected to observe the organ protective effect of bifunctional protein.The organ protection effect was observed.Results In vitro,the protein can bind to LPS,HMGB1 and LPS-HMGB1 complexes and can reduce LPS and HMGB1-induced inflammatory responses in Raw264.7 cells and decrease the levels of key inflammatory pathway proteins P38,PJNK1/2,PERK1/2 phosphorylation levels.In vivo,the protein reduced ALT,AST,CREA and LDH serum levels in septic mice,protected liver function,and reduced lung injury in septic mice,and potentially provided renal protection,which may improve the survival rate of septic mice.Conclusion CRISPLD2-anti-HMGB1 bifunctional fusion protein can effectively inhibit the inflammatory response induced by LPS and HMGB1 via TLR4 inflammatory signaling pathway and is more effective in treating septic mice.