文章摘要
瘦素减轻高浓度氧气所致幼鼠脑损伤
Leptin alleviated hyperoxia-induced brain injury in neonatal rats
  
DOI：10.12089/jca.2021.05.016
中文关键词: 瘦素  高浓度氧气  脑损伤  炎症  凋亡  幼鼠
英文关键词: Leptin  Hyperoxia  Brain injury  Inflammation  Apoptosis  Neonatal rats
基金项目:河南省科技攻关计划项目（172102310022）
作者单位E-mail
朱永锋 450003,河南大学人民医院,河南省人民医院麻醉与围术期医学科  
王倩楠 450003,河南大学人民医院,河南省人民医院麻醉与围术期医学科  
王建平 450003,河南大学人民医院,河南省人民医院麻醉与围术期医学科  
王静瑞 450003,河南大学人民医院,河南省人民医院麻醉与围术期医学科  
张加强 450003,河南大学人民医院,河南省人民医院麻醉与围术期医学科 hnmzxh@163.com 
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中文摘要:
      
目的 探讨瘦素在高浓度氧气所致幼鼠脑损伤中的作用及相关分子机制。
方法 健康清洁级SD雄性幼鼠60只，14日龄，体重40~50 g，采用随机数字表法分为三组：对照组（C组）、高浓度氧气组（H组）和高浓度氧气+瘦素组（L组），每组20只。C组呼吸正常空气，于每日同一时点腹腔注射生理盐水0.5 ml，连续7 d。H组放置于有机玻璃氧箱中呼吸高浓度氧气（氧气浓度78%~80%），持续吸氧7 d，于吸氧前30 min腹腔注射生理盐水0.5 ml，后每日同一时点腹腔注射生理盐水0.5 ml，连续7 d。L组放置于有机玻璃氧箱中呼吸高浓度氧气（氧气浓度78%~80%），持续吸氧7 d，于吸氧前30 min腹腔注射瘦素50 μg/kg+生理盐水稀释至0.5 ml，后每日同一时点腹腔注射瘦素50 μg/kg+生理盐水稀释至0.5 ml，连续7 d。药物干预结束后次日采用神经功能缺损评分（NDS）评估神经功能，采用水迷宫实验评估认知功能。水迷宫实验结束后采用安乐死法处死幼鼠，取海马组织测定湿/干重比，采用TUNEL法测定海马组织CA1区细胞凋亡情况并计算凋亡细胞百分比，实时荧光定量PCR法检测海马组织CIRP、TNF-α和IL-1β mRNA表达量，Western Blot法检测海马组织冷诱导RNA结合蛋白（CIRP）、TNF-α和IL-1β蛋白含量，采用HE染色法于光镜下观察海马CA1区正常锥形细胞数及病理学形态。
结果 与C组比较，H组和L组NDS明显升高（P<0.05），逃逸潜伏期明显延长（P<0.05），平均游泳速度明显减慢（P<0.05），平台所在象限滞留时间明显缩短（P<0.05），海马组织湿/干重比、凋亡细胞百分比、CIRP、TNF-α和IL-1β mRNA表达量及相应蛋白含量均明显升高（P<0.05），海马组织CA1区正常锥体细胞计数明显减少（P<0.05），海马组织病理学形态学结构发生损伤。与H组比较，L组NDS明显降低（P<0.05），逃逸潜伏期明显缩短（P<0.05），平均游泳速度明显加快（P<0.05），平台所在象限滞留时间明显延长（P<0.05），海马组织湿/干重比、凋亡细胞百分比、CIRP、TNF-α和IL-1β mRNA表达量及相应蛋白含量均明显降低（P<0.05），海马组织CA1区正常锥体细胞计数明显增多（P<0.05），海马组织病理学形态学结构损伤减轻。
结论 瘦素可能通过下调海马组织CIRP、TNF-α和IL-1β的表达、减轻脑组织炎症反应、抑制脑细胞凋亡，从而减轻高浓度氧气所致幼鼠脑损伤和脑损伤后的认知功能障碍。
英文摘要:
      
Objective To investigate the effect of leptin on brain injury caused by hyperoxia and related molecular mechanism in neonatal rats.
Methods Sixty healthy and clean neonatal Sprague-Dawley male rats, aged 14 days, weighing 40-50 g, were randomly divided into 3 groups：control group (group C), hyperoxia group (group H) and hyperoxia combined with leptin group (group L), 20 rats in each group. Group C breathed normal air and intraperitoneally injected normal saline 0.5 ml at the same time every day for 7 days. Group H was placed in plexiglass oxygen box with hyperoxia for 7 days, the concentration of oxygen was 78% - 80%, normal saline 0.5 ml was first injected intraperitoneally 30 minutes before oxygen inhalation, and then normal saline 0.5 ml was injected intraperitoneally at the same time every day for 7 days. Group L was placed in plexiglass oxygen box with hyperoxia for 7 days, the concentration of oxygen was 78% - 80%, leptin 50 μg/kg diluted to 0.5 ml with normal saline was first injected intraperitoneally 30 minutes before oxygen inhalation, and then leptin 50 μg/kg diluted to 0.5 ml with normal saline was injected intraperitoneally at the same time every day for 7 days. The neurological deficit score (NDS) was assessed the day after drug intervention. Morris water maze test was used to evaluate cognitive function. After Morris water maze test, the neonatal rats were killed by euthanasia, and the hippocampi were used. The ratio of wet weight / dry weight (W/D) was determined. The apoptosis of CA1 area of the hippocampus and the percentage of apoptotic cells was calculate by TUNEL. Real-time fluorescent quantitative PCR was used to detect the expression of hippocampal CIRP, TNF-α and IL-1β mRNA, and Western blot was used to detect the expression of hippocampal CIRP, TNF-α and IL-1β protein. The counts of normal pyramidal cell and pathological morphology of hippocampal CA1 area were observed under light microscope with HE staining.
Results Compared with group C, NDS was significantly increased (P < 0.05), the escape latency was significantly prolonged (P < 0.05), the average swimming speed significantly were slowed (P < 0.05), the residence time in the quadrant of the platform was significantly shortened (P < 0.05), W/D of the hippocampus, the percentage of apoptotic cells, the expression of hippocampal CIRP, TNF-α and IL-1β mRNA and the expression of CIRP, TNF-α and IL-1β protein were all significantly increased (P < 0.05), the normal pyramidal cell count in hippocampal CA1 area significantly decreased (P < 0.05), and the hippocampal morphological structure damaged in groups H and L. Compared with group H, NDS was significantly decreased (P < 0.05), the escape latency was significantly shortened (P < 0.05), the average swimming speed significantly were accelerated (P < 0.05), the residence time in the quadrant of the platform was significantly prolonged (P < 0.05), W/D of the hippocampus, the percentage of apoptotic cells, the expression of hippocampal CIRP, TNF-α and IL-1β mRNA and the expression of CIRP, TNF-α and IL-1β protein were all significantly decreased （P < 0.05），the normal pyramidal cell count in hippocampal CA1 area significantly increased (P < 0.05), and the damage of hippocampal morphological structure alleviated in group L.
Conclusion Leptin may down-regulate the expression of CIRP, TNF-α and IL-1β in hippocampus, reduce inflammation in brain tissue, and inhibit brain cell apoptosis, thereby reducing brain injury and cognitive function in neonatal rats after brain injury caused by hyperoxia.
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