| ObjectiveHigh mobility group box-1 protein (HMGB1) was an important late-acting cytokine that mediates lethality of sepsis and systemic inflammation. The present study was performed to investigate the effects of HMGB1-Abox (specific antagonist of HMGB1) on expression level of interleukin (IL)-35 and T cell-mediated immunity in mice with major burns.MethodsAdult Male BALB/c mice were randomly divided into control group, sham group, burn group, and Abox treatment group, respectively. sham group, burn group and Abox treatment group were further divided into 3 subgroup equally according to the pionts of time after burn injury (24,48,72 hours).The dorsal and lateral surfaces of the mice were shaved under ether anesthesia with 15% total body surface area (TBSA) 24 hours before the burn injury. Mice in burn or Abox treatment group were exposed and immersed in 97℃ hot water through a window with the size of 15% TBSA based on the weight-TBSA formula for 6 seconds. Follow the burn injury, mice were resuscitated with sterile saline and Abox (300 μg per mouse) was given at 2 and 12 hours postburn. The hearts, livers, spleens, and lungs were homogenized after animals were sacrificed and organs were harvested at 24,48,72hours after thermal injury. Spleen mononuclear cells and T lymphocytes were obtained from parts of the spleens. Levels of HMGB1 and IL-35 in tissues, as well as cytokines including HMGB1, IL-35, IL-2, IL-4 and IFN-γ in cultural supernatant of spleen mononuclear cells or T lymphocytes were determined with ELISA. Gene expressions of p35 and EBI3, which are two constituents of IL-35, were quantified by q-PCR. The proliferation of splenic lymphocytes from various groups was measured with CCK-8 kit, after cultured in vitro for 3 days.All data were expressed as means ± S.D. and were analysed by independent-samples T test or one-way ANOVA followed by LSD test.Some datawere analysed by rank sum test or Dunnett’s testwhen data variance uneven.Results1. Treatment with Abox decreased HMGBl expression in burn miceProtein level of HMGB1 in tissues of hearts, livers, spleens, and lungs significantly increased in burn injuried mice at 24 h (P<0.01), and maintain a high level till 72 hours after burn injury. Compared with burn group at same time-point, protein levels of HMGB1 were significantly decreased after treatment with Abox in different tissues (P<0.01). HMGB1 secretion by spleen mononuclear cells was also increased in burn mice and decreased in Abox-treated mice (P<0.01).2. Treatment with Abox decreased the expression level of IL-35 in burn mice2.1 mRNA level of IL-35IL-35 is composed of IL-12a(p35) and EBI3. Therefore, we analysis the gene expression of two constituents of IL-35 (EBI3 and p35) respectively. Compared with sham group,the gene expression of p35 mRNA in each organs increased significantly at 24h after injury and most obviously in spleens,least obviously in lungs (P<0.01). the gene expression of EBI3 mRNA increased as well upon the burn injury by 24h,but most obviously in livers,also least obvious in lungs (P<0.01). The gene expression of both subunits of IL-35 were significantly decreased after treatment with Abox at 24h after injury(P<0.01),and The gene expression of both subunitsin lung tissue show no significant difference after 48h.2.2 Protein level of IL-35Compared with sham group, the protein level of p35 in hearts,livers,spleens and culture supernatant of spleen mononuclear cells increased significantly following the burn injury. most obviously in spleens (P<0.01),But lung tissue express aberrantly reduced protein levels of IL-35.Protein level of IL-35 in hearts,livers,spleens and culture supernatant of spleen mononuclear cells significantly decreased after treatment with Abox (P<0.01) while lung tissue still showed the opposite trend with this organ tissue or culture supernatant of spleen mononuclear cells.3. Abox treatment improved the immune function of mouse splenetic T lymphocytes after burn injury3.1 Expression of IFN-y and IL-4 in T lymphocytes culture supernatants:Compared with sham group, burn injury rapid highten both expression of IFN-y and IL-4 by mouse splenetic T lymphocytes(both P<0.01).reevaluation the data provide further insight into IFN-y/IL-4 ratio, the IFN-y/IL-4 ratio of burn group significantly lower than the ratio of control group(P<0.01). Whereas, the IFN-y/IL-4 ratio was reversed after treatment with Abox (P<0.01).3.2 Proliferative activity of T lymphocytes:Proliferative activity of T lymphocytes of burn group lower than sham group at 24h after injury(P<0.05), while the expression of IL-2 which promote the proliferative activity of Tlymphocytes was markedly reduced (P<0.01)as well.whereas,both the proliferative ability of T lymphocytesand expression of IL-2 (both P<0.01) were significantly restored after Abox treatment.ConclusionsSpecific inhibition of HMGB1 with Abox treatment can obviously down-regulate IL-35 expression in heart,liver,spleen, and expression level of PBMC.meanwhile, promote Th cell proliferative response as well as shifting from Th2 to Th1, in turn contributing to the amelioration of host immune dysfunction in the setting of severe burns. |
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