创伤性脑损伤大鼠半暗带脑组织中Elk1的表达及作用

Expression and role of Elk1 in the contusion penumbra tissues of rats with traumatic brain injury

目的探讨创伤性脑损伤(TBI)大鼠半暗带脑组织中ETS结构域包含蛋白Elk1的表达及作用。方法将80只Sprague Dawley大鼠随机分为假手术组、TBI组、TBI-siRNA组和TBI-空病毒载体组,每组20只。TBI组、TBIsiRNA组和TBI-空病毒载体组大鼠建立中度液压脑损伤模型,假手术组大鼠手术步骤与TBI组、TBI-siRNA组和TBI-空病毒载体组相同,但未实施液压冲击。TBI-siRNA组及TBI-空病毒载体组大鼠于造模前24 h分别经侧脑室注射10μL Elk1-siRNA慢病毒表达载体和空病毒载体,假手术组和TBI组大鼠经侧脑室注射10μL磷酸盐缓冲液。各组大鼠分别于脑损伤后12、24、72 h采用Shapira和Wahl脑损伤神经功能评分法评估神经功能,神经功能评估后快速断头处死大鼠(每个时间点每组选取5只大鼠),取脑组织,采用实时荧光定量聚合酶链反应检测脑组织中Elk1 mRNA表达,Western blot法检测脑组织中Elk1蛋白表达,比色法检测脑组织中Caspase-3活性;各组大鼠脑损伤后24 h选取5只大鼠断头取脑组织,采用Fluoro-Jade染色法计数坏死神经元。结果大鼠脑损伤后12、24、72 h,TBI-空病毒载体组、TBI组大鼠神经功能评分显著低于假手术组(P<0.05),TBI-siRNA组大鼠神经功能评分高于TBI-空病毒载体组和TBI组(P<0.05),TBI-空病毒载体组与TBI组大鼠神经功能评分比较差异无统计学意义(P>0.05)。大鼠脑损伤后12、24、72 h,TBI组和TBI-空病毒载体组大鼠损伤半暗带脑组织中Elk1 mRNA及蛋白相对表达量显著高于假手术组(P<0.05),TBI-siRNA组大鼠损伤半暗带脑组织中Elk1 mRNA及蛋白相对表达量显著低于TBI组和TBI-空病毒载体组(P<0.05),TBI-空病毒载体组与TBI组大鼠损伤半暗带脑组织中Elk1 mRNA及蛋白相对表达量比较差异无统计学意义(P>0.05)。脑损伤后24 h,假手术组、TBI组、TBI-空病毒载体组和TBI-siRNA组大鼠大脑皮层坏死神经元数目分别为4.0±1.0、32.0±4.0、33.0±3.0、16.0±2.0,TBI组、TBI-空病毒载体组大鼠大脑皮层半暗带坏死神经元数目显著多于假手术组(P<0.05),TBI-siRNA组大鼠大脑皮层坏死神经元数目显著少于TBI组和TBI-空病毒载体组(P<0.01),TBI组与TBI-空病毒载体组大鼠大脑皮层半暗带坏死神经元数目比较差异无统计学意义(P>0.05)。脑损伤后12、24、72 h,TBI-空病毒载体组、TBI组大鼠大脑皮层半暗带脑组织中Caspase-3活性显著高于假手术组(P<0.05),TBI-siRNA组大鼠损伤半暗带脑组织中Caspase-3活性显著低于TBI组和TBI-空病毒载体组(P<0.05),TBI-空病毒载体组与TBI组大鼠损伤半暗带脑组织中Caspase-3活性比较差异无统计学意义(P>0.05)。结论Elk1在TBI大鼠损伤半暗带脑组织中表达上调,抑制Elk1表达可减轻损伤半暗带神经元坏死,改善神经功能。

Objective To investigate the expression and role of ETS-domain containing protein Elk1 in the contusion penumbra tissues of rats with traumatic brain injury(TBI).Methods Eighty Sprague Dawley rats were randomly divided into sham operated group,TBI group,TBI-siRNA group and TBI-empty virus vector group,with 20 rats in each group.The rats in the TBI group,TBI-siRNA group and TBI-empty virus vector group were established moderate hydraulic brain injury model.The operation procedure of rats in the sham operation group was the same as that in the TBI group,TBI-siRNA group and TBI-empty virus vector group,but no hydraulic shock was applied.The rats in the TBI-siRNA group and TBI-empty virus vector group were injected with 10μL El K1-siRNA lentivirus expression vector and empty avirus vector respectively in lateral ventricles at24 hours before modeling.The rats in the sham operation group and TBI group were injected with 10μL phosphate buffer.The neural function of rats in each group were assessed by Shapira and Wahl method at 12,24 and 72 hours after brain injury.The rats were sacrificed by rapid decapitation(5 rats in each group at each time point)after the nerve function score,and the brain tissues were taken.The expression of Elk1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction.The expression of Elk1 protein in brain tissues was detected by Western blot.The activity of Caspase-3 in brain tissues was detected by colorimetry.At 24 hours after brain injury,5 rats in each group were decapitated and their brain tissues were taken out,then the number of necrotic neurons was counted by Fluoro-Jade staining.Results At 12,24 and 72 hours after brain injury,the score of nerve function in the TBI group and TBI-empty virus vector group was significantly lower than that in the sham operation group(P<0.05),the score of nerve function in the TBI-siRNA group was higher than that in the TBIempty virus vector group and TBI group(P<0.05),but there was no significant difference in the score of nerve function between the TBI-empty virus vector group and TBI group(P>0.05).At 12,24 and 72 hours after brain injury,the relative expression of Elk1 mRNA and protein in contusion penumbra tissues of rats in the TBI group and TBI-empty virus vector group was significantly higher than that in the sham operation group(P<0.05),the relative expression of Elk1 mRNA and protein in contusion penumbra tissues of rats in the TBI-siRNA group was significantly lower than that in TBI group and TBI-empty virus vector group(P<0.05),but there was no significant difference in the relative expression of Elk1 mRNA and protein in contusion penumbra tissues of rats between the TBI-empty virus vector group and TBI group(P>0.05).At 24 hours after brain injury,the number of necrotic neurons in contusion penumbra tissues of rats in the sham operated group,TBI group,TBI-empty virus vector group and TBI-siRNA group was 4.0±1.0,32.0±4.0,33.0±3.0 and 16.0±2.0,respectively.The number of necrotic neurons in contusion penumbra of rats in the TBI group and TBI-empty virus vector group was significantly higher than that in the sham operation group(P<0.05),the number of necrotic neurons in contusion penumbra of rats in the TBI-siRNA group was significantly less than that in the TBI group and TBI-empty virus vector group(P<0.01),but there was no significant difference in the number of necrotic neurons in contusion penumbra of rats between the TBI-empty virus vector group and TBI group(P>0.05).At 12,24,72 hours after brain injury,the Caspase-3 activity in contusion penumbra of rats in the TBI-empty virus vector group and TBI group was significantly higher than that in the sham operation group(P<0.05),the Caspase-3 activity in contusion penumbra of rats in the TBI-siRNA group was significantly lower than that in the TBI group and TBI-empty virus vector group(P<0.01),but there was no significant difference in the Caspase-3 activity in contusion penumbra of rats between the TBI-empty virus vector group and TBI group(P>0.05).Conclusion The expression of Elk1 is up-regulated in contusion penumbra of TBI rats.Inhibiting Elk1 expression can reduce the necrosis of neurons in the contusion penumbra and improve the neural function.