The Role Of HMGB1in Ischemia/Reperfusion Injury After Lung Transplantation

Part I Changes of HMGB1expression after lung transplantation in ratsObjective: To investigate the changes of HMGB1expression in lung tissue andHMGB1level in serum after rat lung transplantation.Methods:42rats were randomly divided into control group (n=6),post-transplantati-on2h group (n=12), post-transplantation6h group (n=12) and post-transplantation12h group (n=12). The experimental orthotopic lung (left) transplantation model inrats was established by cuff technique. The expression of HMGB1mRNA wasquantitatively detected by real-time RT-PCR, and HMGB1protein levels by Westernblot and immunofluorescence staining, in lung tissue. HMGB1, TNF-α, IL-1β andIL-6in serum were analysed using ELISA.Results: HMGB1mRNA and HMGB1protein expression in lung tissue significantlyincreased2h after lung transplantation (P<0.01), and were more elevated followingpost-transplantation hours. The levels of HMGB1, TNF-α, IL-1β, and IL-6in serumwere markedly increased at each time point after lung transplantation (P<0.01).Conclusion: HMGB1expression significantly changes after lung transplantation inrats, and HMGB1may participate in lung ischemia/reperfusion injury after lungtransplantation.Part Ⅱ The role of HMGB1in ischemia/reperfusion injury after lung transplan-tationObjective: To investigate the effect of recombinant high mobility group box1(HMGB1) on lung damage in mice and anti-HMGB1antibody on reducing ischemia/reperfusion injury in transplant lung. Methods: The BALB/c mice were randomly divided into control group,10μgHMGB1group and100μg HMGB1group,5mice each group. Each mouse in10μgHMGB1group and100μg HMGB1group was intraperitoneally injected10μg and100μg recombinant HMGB1, respectively. The experimental orthotopic lung (left)transplantation model in rats was established by cuff technique, and the rats wererandomly divided into transplantation control group,0.1mg anti-HMGB1antibodytreatment group and1.0mg anti-HMGB1antibody treatment group,5rats each group.Each rat in0.1mg anti-HMGB1antibody treatment group and1.0mg anti-HMGB1antibody treatment group was intraperitoneally injected0.1mg and1.0mg anti-HMGB1antibody, respectively. Wet/dry weight ratio of lung tissue, gene expression ofcytokines (TNF-α, ICAM-1, IL-6) in lung tissue, lung histopathologic changes, andarterial blood gas were examined12h after the treatment of HMGB1or antibody.Results: The reduced PaO2, increased PaCO2and wet/dry weight ratio, enhancedmRNA expression of TNF-α, ICAM-1and IL-6, and lung histopathologic changeswere showed, after the mice were injected with recombinant HMGB1especially100μg. The other indexes, except ICAM-1mRNA expression, were found to antibodydose-dependently meliorate, after the rats with lung transplantation were treated withanti-HMGB1antibody.Conclusion: HMGB1participate in lung ischemia/reperfusion injury after lung trans-plantation. HMGB1may be a potential therapeutic target for ischemia/reperfusioninjury of transplant lung.Part Ⅲ HMGB1induces alveolar macrophages to release proinflammatorycytokinesObjective: To investigate the effect of HMGB1on the extracellular release of proin-flammatory cytokines in alveolar macrophages, and its possible mechanism.Methods: The concentration of several cytokines (TNF-α, IL-1β, IL-6) in the culture medium was determined after NR8383rat alveolar macrophages were induced byrecombinant HMGB1protein with1,10,100and1000μg/L. The inhibitory effect ofToll-Like Receptor4(TLR4) antibody (10mg/L) was observed on the release ofthese cytokines in HMGB1-induced alveolar macrophages. The concentration of TNF–α, IL-1β and IL-6was determined by ELISA.Results: Recombinant HMGB1dose-dependently increased the levels of TNF-α,IL-1β and IL-6in the culture medium of alveolar macrophages. The levels of TNF-α,IL-1β and IL-6in the culture medium reached to their peaks at6h,12h and24h,respectively, after alveolar macrophages were induced by100μg/L HMGB1. TLR4antibody partly inhibited HMGB1-induced release of TNF-α and IL-1β (P0.05), andshowed nonsignificant effect on IL-6release (P>0.05).Conclusion: HMGB1can induces alveolar macrophages to release TNF-α, IL-1β andIL-6, which may be involved with TLR4.Part Ⅳ Clinical significance of serum HMGB1determination after lungtransplantationObjective: To investigate the change of serum HMGB1level after lung transplantati-on and its clinical significance.Methods:21patients were divided into stable group (n=10), acute rejection group(n=5) and infected group (n=6) according to the complications after lung transplanta-tion. Serum HMGB1level was determinated in19healthy cases and21patients at thepreoperative day,1st day,3rd day and7th day after lung transplantation by ELISA.Results: Serum HMGB1level was markedly increased after lung transplantation.Serum HMGB1level of acute rejection group was significantly higher than that ofstable group at7th day (P0.01). Serum HMGB1level of infected group was signify-cantly higher compared with stable group and acute rejection group at3rd day and7thday. Conclusion: Serum HMGB1level was significantly elevated after lung transplantati-on. The determination of serum HMGB1is significant to the laboratory diagnosis ofcomplications after lung transplantation.