褪黑素对创伤性脑损伤小鼠的抗氧化应激作用及机制研究

摘要

目的 探讨褪黑素对创伤性脑损伤(TBI)模型小鼠脑组织红系衍生的核因子2相关因子2(Nrf2)-抗氧化反应原件(ARE)信号通路的激活及抗氧化应激作用.方法 6～8周龄ICR雄性小鼠116只,按随机数字表法分为假手术组、模型组、溶剂组、褪黑素组.每组29只.后3组小鼠用经Flierl改良的自由落体撞击法制作TBI模型,褪黑素组小鼠于伤后0、1、2、3、4h腹腔注射褪黑素(10 mg/kg,3 mg/mL稀释于生理盐水),溶剂组小鼠于相同时间点注射等量生理盐水.伤后24 hFJC染色检测小鼠挫伤灶周围皮层变性神经元的数量;干湿重比法检测小鼠的脑组织含水量;比色法检测小鼠脑组织丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)的含量;Western blotting检测小鼠脑组织Nrf2核、浆蛋白以及磷酸酰胺腺嘌呤二核苷酸醌氧化还原酶1(NQO1)、血红素氧化酶1(HO-1)蛋白的表达;RT-PCR法检测小鼠脑组织HO-1和NQO-1 mRNA的表达;电泳迁移率实验(EMSA)观察小鼠脑组织中Nrf2和DNA的结合力.结果 与假手术组比较,模型组、溶剂组、褪黑素组小鼠挫伤灶周围皮层变性神经元数量、MDA含量、核Nrf2的表达、HO-1、NQO-1蛋白和mRNA的表达、脑组织含水量均较高,浆Nrf2的表达、GPx的含量较低;与溶剂组和模型组比较,褪黑素组小鼠挫伤灶周围皮层变性神经元数量、脑组织含水量、MDA含量、核Nrf2的表达均较低,浆Nrf2、HO-1、NQO-1蛋白和mRNA的表达、GPx的含量均较高,差异有统计学意义(P＜0.05).假手术组和褪黑素组小鼠脑组织SOD含量高于模型组、溶剂组,差异有统计学意义(P＜0.05).EMSA检测显示假手术组、模型组和溶剂组、褪黑素组小鼠脑组织Nrf2的DNA结合力依次增高.结论 褪黑素可以提高TBI后脑组织抗氧化酶的含量,其机制可能与激活Nrf2-ARE信号通路有关.%Objective To evaluate the potential involvement of melatonin in the activation of nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway and anti-oxidative stress in an experimental model of traumatic brain injury (TBI).Methods One hundred and sixteen ICR male mice,aged 6-8 weeks,were randomly divided into sham-operated group,model group,vehicle group and melatonin treatment group (n=29);mice in the later three groups were established TBI models by Flierl improved free-fall method;0,1,2,3 and 4 h after that,mice in the melatonin treatment group were performed intraperitoneal injection of melatonin (10 mg/kg,3 mg/mL diluted in normal saline),and those in the vehicle group were given the same volume of normal saline.Twenty-four h after TBI,the neuronal degeneration was investigated by Fluoro-Jade C (FJC) staining and brain water content was measured by wet-to-dry weight ratio method;colorimetric method was employed to detect the malondialdehyde (MDA),superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels;Western blotting was engaged to analyze the protein content of nuclear Nrf2,cytoplasmic Nrf2,NADPH quinine oxidoreductase-1 (NQO1) and heme oxidase-1 (HO-1);real-time quantitative-PCR was employed to evaluate the mRNA content of HO-1 and NQO-1;electrophoresis mobility shift assay (EMSA) was performed to measure the Nrf2 DNA binding activity.Results As compared with the sham-operated group,the other three groups had significantly increased number of cortical degenerative neurons,water content,MDA content,nuclear Nrf2 expression,and nuclear HO-1 and NQO-1 protein and mRNA expressions,and had significantly lower levels of cytoplasmic Nrf2 and GPx.As compared with the model group and vehicle group,the melatonin treatment group had significantly decreased number of cortical degenerative neurons,water content,MDA content and nuclear Nrf2 expression,and had significantly increased cytoplasmic Nrf2 level,GPx content and nuclear HO-1 and NQO-1 protein and mRNA expressions (P＜0.05).The SOD content in the sham-operated group and melatonin treatment group was significantly higher than that in the model group and vehicle group (P＜0.05).EMSA showed that the Nrf2 DNA binding activity increased accordingly in the order of sham-operated group,model group,vehicle group and melatonin treatment group.Conclusion The melatonin can improve the anti-oxidative stress neuroprotection following TBI,which may be associated with the activation of Nrf2-ARE signaling pathway.