小鼠严重颅脑撞击伤早期咪唑安定-氯胺酮对肝脏GR与海马NR1变化的影响

Changes Of GR and hippocampal NR1 and effect of midazolam-Ketamine in mice after severe TBI

目的研究小鼠闭合性严重颅脑损伤（TBI）后肝脏糖皮质激素受体（GR）、海马N-甲基-D-天门冬氨酸受体（NR）蛋白功能亚单位1（NR1）蛋白水平变化及其与血清TNF-α、IL-1β变化的关系,以及应用咪唑安定-氯胺酮干预后的变化。方法利用BIM-Ⅲ型小型多功能动物撞击机对小鼠清醒致伤后将其随机分为5组,即假致伤组（J组）、致伤对照组（N组）、致伤后氯胺酮治疗组（K组）、致伤后咪唑安定治疗组（M组）、致伤后复合用药治疗组（F组）。于致伤后30min及2、8、24、48、72h采用West-ernblot免疫印迹法检测大脑皮质和肝脏GR蛋白水平变化,用酶联免疫吸附法（ELISA）检测各组外周血清中TNF-α、IL-1β含量。结果肝脏GR蛋白表达在致伤2h开始降低,8h呈现恢复趋势,72h仍未完全正常;海马NR1蛋白表达在致伤后2h开始明显降低,24h基本恢复,72h明显增加;应用咪唑安定-氯胺酮干预后可明显降低GR、NR1蛋白表达的这种变化趋势;外周血中TNF-α、IL-1β含量致伤后明显升高,均具有两个峰值特征,应用咪唑安定-氯胺酮干预后可明显降低二者的升高。结论严重TBI后存在糖皮质激素抵抗,NR激活可能是其中一个重要原因。咪唑安定、氯胺酮能明显抑制HPA轴的兴奋性,改善糖皮质激素抵抗,调控应激反应。其作用机制除直接抑制炎性细胞因子释放外,还可能有中枢性的作用机制,其中之一可能与调节NR1蛋白表达有关。

Objective To observe the changes of glucocorticoid receptors（GR）in hepatic tissue＇s,NR functional subunits 1（NR1）in hippocampus,and TNFα,IL-1βin serum,and after midazolam-ketamine administration following severe Traumatic Brain Injury（TBI）mice.Methods Severe TBI in mice was performed by a BIM-III biomechanical machine model.In all groups（J,N,K,M,F）,at these time points：30min,2,8,24,48,72 hafter TBI,total cytosolic GR in the liver and hippocampal NR1were detected with Western blot,Cytokine levels of TNFαand IL-1β were measured by ELISA technique.Results Serum TNFα,IL-1β and cortisol levels increased markedly,and there were two acme in the observe curve.After severe TBI,the hepatic GR was obviously downregulated at protein levels from 2-72h,the expression of hippocampal NR1 in protein levels was both markedly reduced at 2h,8h time point,and increased obviously in protein level from 48-72hafter injury.But after midazolam-ketamine administration,these changes was more significant retarded.Conclusion There were glucocorticoid resistant in the mice after TBI,These results indicate that it is more benefited to control the over excitation of HPA axis,glucocorticoid resistant and stress response by ketamine and midazolam management.Besides reduced directly inflammatory cytokine,one of mechanisms in nerve centre for this could be adjustment not only activation but expression of hippocampal NR1.