| OBJECTIVE: To investigate the influence of bum serum on the TNF-α,HMGB1 and IL-10 secretion dynamics in mice macrophages line RAW264.7.METHODS: RAW264.7 cells were stimulated with normal serum and burn serum (0%,5%,10%,20%) for 24 hours respectively. Another, RAW264.7 cells were stimulated with 20% normal serum and burn serum for 4 h, 12 h, 24h respectively. The induced TNF-αand IL-10 from RAW264.7 cells were measured by ELISA. The expression of HMGB1 in RAW264.7 cells was observed by Western-blot.RESULTS: The secretion of TNF-αfrom RAW264.7 cells treated with burn serum with 0%,5%,10%,20% for 24 hours was (0.06±0.02 ng/ml), (0.14±0.03 ng/ml), (0.16±0.04 ng/ml), (0.21±0.02 ng/ml) respectively(P＜0.05). The secretion of TNF-αin RAW264.7 cells treated with burn serum(20%) was 0.16±0.05 ng/ml), (1.24±0.08 ng/ml), (0.87±0.07 ng/ml) for indicated durations. The expression of HMGB1 significantly increased in RAW264.7 cells treated with burn serum for indicated durations, Western-blot analysis demonstrated that①small quantity of HMGB1 expressed in RAW264.7;②Use normal serum of SD rat to interfere RAW264.7, the expression of HMGB1 fluctuate around the standard level;③Use burning serum of SD rat to interfere RAW264.7, the expression of HMGB1 rise in 3 hour, then come down to the standard level. In the 18 hour, it reached the peak, in 24 hour it still remain the high level. The secretion of IL-10 from RAW264.7 cells treated with burn serum with 0%,5%,10%,20% for 24 hours was (38.48±3.90 pg/ml), (900.34±2.57 pg/ml), (934.72±3.62 pg/ml), (944.38±2.86 pg/ml) respectively. The secretion of TNF-αin RAW264.7 cells treated with burn serum (20%) was (798.44±4.88pg/ml), (944.46±2.67 pg/ml), (1744.21±3.79 pg/ml)for indicated durations. Treatment of RAW264.7 macrophages line with bum serum induced a time-dependent increase in IL-10 protein.CONCLUSION: Bum serum can markedly increased the release of TNF-α,HMGB1 and IL-10 in mouse macrophages RAW264.7.The effect of Heat Shock Response on burn rats and on TNF-α, HMGB1 and IL-10 induced by burn serumOBJECTIVE: To study the protective effect of heat shock response (HSR) on rats and the bum serum induced expression of TNF-α, HMGB 1 and IL-10.METHODS: Sixty SD rats were employed in the study and were randomly divided into normal control groups, burn groups and HSR groups with 20 rats in each group. The survival rate and organ injury in lung and liver were observed in each groups. Macrophages collected from heat shock treated and untreated RAW264.7 were stimulated with burn serum. Total RNA was extracted and RT-PCR assay was performed to detect the expression level of TNF-αmRNA and IL-10mRNA. Western-blot analysis was employed to detect the release of HMGB1.RESULTS: Burn serum stimulation obviously increased the mRNA level of TNF-αand HMGB1 in macrophages of RAW264.7 mice, which was inhibited by heat shock pretreatment. Induction of IL-10 mRNA was observed in cells treated with burn serum for 1 h and lasted at least up to 24h. Bum serum stimulation decreased obviously the mRNA level of IL-10 in macrophages of RAW264.7 mice, which was up-regulated by heat shock pretreatment. The survival rate was 60% in HSR groups and 40% in burn groups. The injury in lung and liver were reduced in HSR groups than that in bum groups.CONCLUSION: HSR inhibits the expression of TNF-αmRNA and HMGB1 protein in macrophages induced by bum serum, and up-regulated the expression of IL-10 mRNA. HSR could ameliorate the organ injury and increased the survival rate in bum rats. OBJECTIVE: To observe the effect of heat shock factor-1 (HSF-1) on the burn serum-induced gene expression of TNF-α, HMGB1 and IL-10 in macrophage cells RAW264.7.METHODS: To construct the vector of pcDNA3.1/HSF1, Western-blot demonstrated that over expression of HSF1 protein in RAW264.7 macrophages transfected with pcDNA3.1/HSF1. Total RNA was extracted and RT-PCR assay was performed to detect the expression level ofTNF-αmRNA, HMGB lmRNA and IL-10mRNA. in RAW264.7 cells treated with burn serum.RESULTS: The expression of HSF1 in RAW264.7 cells treate with burn serum was observed by Western-blot and RT-PCR. The expression of HSF1 in RAW264.7 cells treated with burn serum for 24 hours were increased. Western-blot analysis demonstrated the expression of HSF 1 in RAW264.7 cells. compared with the expression of HSF1 in 12 hours, that was slightly decreased in 24 hours. RT-PCR testified the expression of TNF-αmRNA and HMGB1 mRNA and IL-10mRNA in RAW264.7 cells treated with burn serum for 24 hours, TNF-αmRNA and HMGB1 mRNA were increased in RAW264.7 cells treated with burn serum for 24 hours, But the over expression of HSF1 could reduce the expression of TNF-αmRNA, HMGB1mRNA; At the same time, IL-10mRNA ncreased slightly in RAW264.7 cells treated with burn serum for 24 hours, The over expression of HSF1 could up-regulate the IL-10mRNA significantly.CONCLUSION: The activated HSF-1 could down-regulates the expression of TNF-αmRNA and HMGBlmRNA and up-regulates the expression of IL-10mRNA. OBJECTIVE: To explore the transcription regulation molecular mechanism of HSF-1 up-regulates the expression of IL-10 mRNA.METHODS: To analyzed the murine IL-10 promoter in RAW264.7 macrophages line by MatInspector program to search for heat shock element(HSE). The murine IL- 10 promoter region(—668/+64bp)was prepared by PCR amplification of RAW264.7 genomic DNA with specific primers, purified PCR products of IL-10 promoter were end-labeled with biotin. Electrophoretic mobility shift assay(EMSA) was employed to analyze the binding activity of HSF1 and HSE. To constructed the luciferase reporter vectors, the DNA fragments were inserted into luciferase plasmid pGL-3Basic to form plasmid (HSE-WT). Mutated murine IL-10 promoter was prepared by PCR amplification of wild-type plasmid DNA with site-directed mutated primers.The mutated DNA fragments were also ligated into pGL-3Basic to form plasmid (HSE-Mut). The mutation sites were confirmed by DNA sequencing. All of the plasmids for transfection were purified by using the NucleoBond endotdxin-free plasmid purification kit. Cells were transfected with plasmids by lipofection according to the manufacturers instructions. Luciferase activity was measured by the luciferase assay system.RESULTS: Electrophoretic mobility shift assay(EMSA) demonstrated a increase in HSF1 binding activity with DNA, which showed the binding of HSFI to the HSE on the promoter of IL-10 gene(-376bp～-369bp). Luciferase assay system demonstrated that the luciferase activityies are decreased in mutated sequence of the IL-10 promoter (34.23±2.14) than in wild type (110.09±5.48).CONCLUSION: HSF-1 may up-regulated the expression of IL-10 by binding of HSF-1 to the HSEs on the promoter of IL-10 gene and enhanced IL-10 transcription activity.
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