分子氢对小鼠创伤性脑损伤神经转归的作用及机制研究

Effects and Mechanism of Molecular Hydrogen on Neurological Outcome after Traumatic Brain Injury in Mice

摘要: 目的：探索分子氢对小鼠创伤性脑损伤后脑水肿及神经转归的影响与细胞焦亡机制及全身炎症反应的作用。方法：雄性C57BL/6小鼠72只，依照随机数字法分为假手术组(S组)、创伤性脑损伤组(T组)和分子氢治疗组(H组)，每组24只。S组行气管插管后，暴露颅骨、钻孔，保留硬脑膜完整，2 h后拔除气管导管；T组采用液压冲击损伤(FPI)法构建小鼠创伤性脑损伤(TBI)模型；H组建立TBI模型后即日起每日吸入42% H₂-21% O₂-37% N₂混合气2 h，持续7 d。术后24 h处死小鼠，Western blot法检测细胞焦亡相关蛋白Caspase-1、ASC水平，ELISA法分别检测血清和创伤半暗带处脑组织IL-1β、IL-18水平；术后48 h处死小鼠，观察脑组织水肿情况并测量脑含水量，HE染色法观察创伤半暗带炎性细胞浸润情况，尼氏染色法观察创伤侧皮质尼氏小体数量及形态；同时，观察并记录小鼠创伤后7天神经功能评分、体重变化情况。结果：与S组比较，T组脑含水量显著增加，分子氢治疗后脑水肿显著改善(P < 0.05)；T组脑组织炎细胞浸润明显，异常尼氏小体数量增加，神经元排列松散，分子氢治疗后上述病理改变显著改善(P < 0.05)；T组脑组织Caspase-1、ASC蛋白表达显著升高，脑皮质及血清内IL-1β、IL-18水平明显增加，术后体重大幅降低，在96 h降至最低水平，后呈缓慢恢复趋势，mNSS评分结果显示，TBI后小鼠神经功能损伤明显加重。分子氢治疗后，上述病理改变均明显减轻(P < 0.05)。结论：分子氢显著改善了小鼠TBI后脑水肿及神经转归，其机制可能与抑制细胞焦亡与全身炎症相关。

Abstract: Objective: To explore the effects of molecular hydrogen on cerebral edema and neurological outcome after traumatic brain injury in mice and the mechanism of pyroptosis and systemic inflammation. Methods: 72 male C57BL/6 mice were randomly divided into sham operation group (group S), traumatic brain injury group (group T) and molecular hydrogen treatment group (group H), with 24 mice in each group. After endotracheal intubation, the skull of mice in group S was exposed, with the dura mater kept intact, and the endotracheal tube was removed 2 h later. Mice in group T were treated with fluid percussion injury (FPI) method to construct traumatic brain injury (TBI) model. The mice in group H inhaled 42% H₂-21% O₂-37% N₂ mixture 2 h daily for 7 days immediately after the establishment of TBI model. The mice were sacrificed 24 h after surgery; western blot was used to detect the levels of Caspase-1 and ASC. The levels of IL-1β and IL-18 in serum and brain tissue of traumatic penumbra were determined by ELISA. Other mice were sacrificed 48 h after surgery, and the edema of brain tissue was observed and the water content of brain was measured. HE staining was used to observe inflammatory cell infiltration in penumbra of trauma. The number and morphology of Nissl bodies in cortex were observed by Nissl staining. At the same time, neurological function scores and body weight changes of mice 7 days after injury were observed and recorded. Results: Compared with group S, brain water content in group T was significantly increased, while the brain edema was significantly improved after molecular hydrogen treatment (P < 0.05); in group T, inflammatory cell infiltration was obvious, the number of abnormal Nissl bodies was increased, and the arrangement of neurons was loose. The above pathological changes were significantly improved after molecular hydrogen treatment (P < 0.05). The expression of Caspase-1 and ASC protein in brain tissue was significantly increased in group T, and the levels of IL-1β and IL-18 in cerebral cortex and serum were also significantly increased. After surgery, the body weight was significantly reduced to the lowest level at 96 hours, and then showed a slow recovery trend. mNSS scores showed that the neurological impairment of the mice was significantly aggravated after TBI. After molecular hydrogen treatment, the above pathological changes were significantly alleviated (P < 0.05). Conclusions: Molecular hydrogen significantly improved brain edema and neurological outcome after TBI in mice, and the mechanism may be related to inhibiting pyroptosis and systemic inflammation.