海水浸泡对创伤性脑损伤小鼠转录组学图谱的影响及其机制

Effects and mechanism of seawater immersion on transcriptome profiles in mice with traumatic brain injury

目的观察海水浸泡后创伤性脑损伤(TBI)小鼠运动功能和脑组织转录组学图谱的变化,探讨海水浸泡对TBI的影响及其潜在机制。方法将51只雄性C57/BL成年小鼠随机分为假手术组、TBI组和TBI+海水组,每组17只。在损伤后1 d、3 d和7 d采用行为学测试(转棒和平衡木试验)检测小鼠耐力及运动协调能力的变化;于损伤后12 h和24 h进行血脑屏障通透性检测[伊文思蓝(EB)染色]、脑组织病理检测(HE染色);损伤后24 h采用Western blotting检测脑组织凋亡相关蛋白Bcl-2和Bax的表达水平,进行脑组织转录组学检测并分析差异表达基因及其相关的信号通路。结果损伤后1、3、7 d的行为学测试显示,与假手术组和TBI组比较,TBI+海水组小鼠在转棒仪上的停留时间均明显缩短(P<0.001),通过平衡木的时间均明显延长(P<0.001)。EB染色结果显示,TBI+海水组EB渗透面积明显大于TBI组(P<0.05);两组组内比较,损伤后24 h EB渗透面积均明显小于损伤后12 h(P<0.05)。HE染色结果显示,与TBI组比较,TBI+海水组病理损伤加重。Western blotting检测结果显示,与TBI组比较,损伤后24 h,TBI+海水组小鼠损伤区域脑组织Bax表达水平明显增高(P<0.05),而Bcl-2表达水平明显降低(P<0.05)。转录组学分析显示,与TBI组比较,TBI+海水组损伤区域脑组织有625个基因存在差异表达(P<0.05),且p53相关基因和自然杀伤细胞相关基因的表达量均明显增高(P<0.05);自然杀伤细胞免疫调节、淋巴细胞免疫调节和细胞因子-细胞因子受体结合等通路显著富集(P<0.05)。结论海水浸泡可促进TBI小鼠受损神经细胞凋亡,导致小鼠运动协调能力和耐力受损,其机制可能与p53介导的内源性凋亡和自然杀伤细胞介导的免疫调节有关。

Objective To observe the changes of motor function and brain tissue transcriptomic profiles in mice with traumatic brain inury(TBI)by seawater immersion,and to explore its potential mechanism.Methods A total of 51 male C57/BL adult mice were randomly divided into sham surgery group,TBI group and TBI+seawater group(17 mice in each group).Behavior tests(rotating bar and balance beam tests)were performed at 1 d,3 d and 7 d after injury to detect the changes of endurance and motor coordination ability in mice.Blood-brain barrier permeability(Evans blue staining)and brain tissue pathological changes(HE staining)were detected at 12 h and 24 h after injury.The expression levels of apoptosis-related proteins BCL-2 and Bax in brain tissues were detected with Western blotting 24 h after injury,and carry out brain tissue transcriptomics detection and analyze the related differentially expressed genes and signal pathways.Results Behavior tests showed that compared with the sham surgery group and TBI group,mice in TBI+seawater group had a significantly shortened time on the rotating bar(P<0.001)and spend a significantly prolonged time to pass through the balance beam(P<0.001)on 1 d,3 d,and 7 d after injury.Evans blue staining showed that the EB permeation area of TBI+seawater group was significantly larger than that of the TBI group(P<0.05),and the EB permeation area at 24 h after injury was significantly smaller than that at 12 h after injury in both groups(P<0.05).HE staining results showed that the pathological damage in TBI+seawater group was worsened compared with TBI group.Western blotting results showed that 24 h after injury,the expression of Bax in TBI+seawater group was significantly increased(P<0.05)while the expression of Bcl-2 was significantly decreased(P<0.05)in injured brain tissue compared with TBI group.Transcriptomic analysis showed that there were 625 differentially expressed genes in the injured brain tissue of TBI+seawater group compared with TBI group(P<0.05),and the expression levels of p53-related genes and natural killer cell-related genes were significantly increased(P<0.05).Pathway enrichment analysis showed that natural killer cell immune regulation,lymphocyte immune regulation,and cytokine-cytokine receptor binding pathways were significantly enriched(P<0.05).Conclusions Seawater immersion can promote apoptosis of damaged neural cells in TBI mice,leading to impaired motor coordination and endurance in mice.Endogenous apoptosis mediated by p53 and immune regulation mediated by natural killer cells may be associated with this phenomenon.