| Objective: High mobility group box 1 protein (HMGB1) as a late-acting cytokine mediates lethality of sepsis and systemic inflammation. The present study was performed to determine the expression patterns of HMGB1 in rats with delayed resuscitation after burn injury, and to clarify the potential mechanism of postburn immunosuppression related to HMGB1 induction, in turns preventing the development of sepsis and subsequent multiple organ dysfunction syndrome following major burns.Methods: Male Wistar rats were subjected to 30% full-thickness scald injury and delayed resuscitation after 6 hours. One hundred and three rats were randomly divided into four groups as follows: ①in normal control group (n=7), animals were killed after anesthesia; ② in sham group (n=32), animals were killed on days 1, 3, 5, and 7 after sham burns (37C°); ③ in burn group (n=32), animals were resuscitated with 40ml/kg of normal saline intraperitoneally, and treated with Ringer's solution intraperitoneally at 12, 24, 36, and 48 hours after burn injury; ④ in the treatment group (n=32), animals were resuscitated with 40ml/kg of Ringer's-type balanced salt solution of Ethyl pyruate (EP) intraperitoneally, and treated with the same solution intraperitoneally at 12, 24, 36, and 48 hours after burn injury. Samples of blood and spleen were harvested aseptically. IL-2 and sIL-2R levels in serum were determined by using ELISA kits for rats. Spleen was divided into two parts: one part was detected mRNA expression of HMGBl, IL-2, and IL-2R by RT-PCR, and determined activation of NEAT with ESMA. The other part was prepared for cell culture, and the supernatants were harvested for the measurement of IL-2, IL-4, and IFN-γ bycommercial available ELISA kits for rats. Apoptosis, expression of mIL-2R and cell cycle were detected by using Flow Cytometry.Result: 1. The role of HMGB1 secretion and mRNA expression: (1) compared with sham group, the HMGB1 in serum increased dramatically on days 1-5 after burns (P < 0.05 or P < 0.01) . Treatment with EP decreased HMGB1 levels in serum on days 1-5 postburn (P < 0.05 or P < 0.01) and there was no significantly different on day 7. (2) The mRNA expression of HMGB1 was significantly elevated on days 1 and 3 (P < 0.05 ) , especially on the first day after burns, while there was no marked difference in HMGB1 mRNA expression between EP treatment group and burn group. 2. The effect of HMGB1 on the T lymphocyte proliferation, production of IL-2 and change in Th1 and Th2 cytokine profiles: (1) the proliferative response of spleen cells was depressed during 7 days after burns (P < 0.05 or P < 0.01) . The inhibition of IL-2 production was observed from 1 to 5 days after burns. At the early stage of burns (1-3 days), the expression of mIL-2R declined significantly( P < 0.05), but restored following the recovery of rats from burned insults. Treatment with EP completely restored the response on days 1,3,5, and 7 postburn (P < 0.05 ) . Compared with burn groups, the ability of IL-2 production was increased (P < 0.05 ) . (2) The amount of sIL-2R in serum increased following burns (P < 0.05) , but EP did not decreased sIL-2R levels. (3) Severe burn injury inhibited shift of spleen from G1 to G2+S stage on days 1 and 3 (P< 0.05 ) , and EP injection restored the ability(P < 0.05 ) . (4) IL-4 levels in supernatants from ConA-stimulated spleen cells were high, especially on days 1 and 3 (P < 0.05) , in present of EP, the concentration IL-4 decreased markedly (P < 0.05 ) . On the contrary, IFN-γ levels in spleen cell supernatants were significantly reduced on days 3, 5, and 7 postburn(P < 0.05) , however the impaired production of IFN-γ was restored by EP (P < 0.05) .3. Effect of HMGB1 on the T cell apoptosis, production of IL-2 and signaling pathway: (1) The apoptosis of CD3~+CD4~+T lymphocytes was increased on the first day of burns( P < 0.05 ), and reduced sharply by use of EP( P < 0.05 ).
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