Changes Of The Glucocorticoid Receptor Expression And Regulation Effects Of Diazepam-ketamine On Severely Burned Mice

Objective Post-trauma stress disorder (PTSD) was one of the most important and initial factors leading to systemic damages such as systemic inflammation response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), which could result in the main cause of death after trauma. It was reported that the glucocorticoid receptor (GR) played a key role in regulation of stress and inflammation response. This study was aimed to explore the changes and mechanism of GR expression in severely burned mouse and to clarify the function of diazepam-ketamine combined anesthesia in stress and inflammation response. Methods15-20% TBSA III0 burned mouse was used as experimental traumatic animal model, the study was carried out in two parts: 1. healthy male BALB/c mice were randomly divided into two groups: 10 for normal control group and 50 for experimental group, which was further distributed into 2h, 4h, 6h, 12h and 24h postburn subgroups and 10 for each. Western blot, reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assays (EMSA) were employed to examine serum corticosterone, hepatic GR and GR mRNA, JNK1 expression and phosphorylation status and its translocation from cytoplasm to nucleus as well as AP-1 DNA binding capacity; 2. healthy male BALB/c mice were randomly divided into normal control, burn injury, anesthesia control, diazepam treat, pretreatment (inflicted to burn 15min after anesthesia) and posttreatment(anesthesia 15min after burn) groups. 40 mice were used in normal control and burn injury group respectively, the rest for the other groups and 30 for each. The mice in burn injury group were given no anesthesia of any kind and mice in diazepam alone group were given diazepam 0.4mg/kg via muscle injection. An combination of 0.4mg/kg diazepam and 10mg/kg ketamine were used in mice of anesthesia control, pretreatment and posttreatment groups. Serum level of corticosterone, tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10) and GR expression in peritoneal macrophages and liver after burn or anesthesia were detected by radioactive immunoassay, ELISA and Western blot.Results1. Serum corticosterone level was markedly elevated and peaked at 4h postburn, it did not come back to the normal level at 24h. 2. Hepatic GR was markedly downregulated and reached its nadir at 4h postburn while the reduced GR mRNA expression was only observed at 2 h. 3. The expression of JNK1 in liver wasn't altered after burn injury. Phosphorylation status of JNK1 and the levels of JNK1 in nucleus obviously increased during the early stage, and peaked at 4h and gradually return to the normal level. 4. AP-1 DNA binding ability was markedly elevated and reached its maximum at 12h, and did not recover at 24h after burn.5. Serum TNF-α, IL-1β, IL-10 concentration were markedly elevated and diazepam-ketamine combined anesthesia before or after burn could significantly repressed the elevation.6. The increased serum corticosterone and reduced GR responses to burn injury were markedly suppressed in pretreat and even posttreat group. 7. The expression of GR in peritoneal macrophages was markedly decreased after burn, which could be inhibited by using diazepam-ketaminecombined anesthesia before or after burn. Further experiments demonstrated that there was no direct effect of diazepam-ketamine on GR of peritoneal macrophages in vitro. Conclusion 1. High glucocorticoids and low GR level occur in a stress status after severe trauma. The downregulation of GR is associated with high serum glucocorticoids during stress and may be regulated at mRNA level.2. The JNK1 phosphorylation and transfer of JNK1 from cytoplasm to nucleus are significantly up-regulated after severe trauma, indicating that the function of anti-inflammatory control of GR is weakened.3. The downregulation of the expression and function of GR are partly associated with the elevation of JNK1 and AP-1 activity.