| 摘要: |
| 目的:探讨H2 是否通过Nrf2 信号途径保护血脑屏障(BBB)减轻脓毒症相关脑病(SAE)。方法:将152 只雌性野生型(WT)和152 只Nrf2 基因敲除(Nrf2-/-)C57BL/6J 小鼠按随机数字表法分为假手术组、假手术+ 氢气组、脓毒症模型组和氢气治疗组。采用盲肠结扎穿刺法(CLP)制备小鼠脓毒症模型，假手术组和假手术+ 氢气组仅行开腹。假手术+ 氢气组和氢气治疗组分别在术后1 h和 6 h 开始吸入2% 氢气60min。术后24h，每组随机选取20 只小鼠观察并记录各组的7 天存活率；每组随机挑选4 只小鼠，采用EB 外渗法检测BBB 损伤程度；每组随机挑选4 只小鼠，切取其大脑皮质，采用Western blotting 法检测黏附连接蛋白(VE-cadherin)和紧密连接蛋白(ZO-1)的表达水平。于术后4 天将每组余下10 只小鼠进行连续7 天的Morris 水迷宫实验，以评价其认知功能。结果:在WT 小鼠中，氢气治疗组小鼠的7天存活率为60%，较脓毒症模型组的35% 显著提高(P<0.01)；其血管外EB 外渗量为[(3.68±0.18) μg/g]，较脓毒症模型组[(5.24±0.06) μg/g] 显著降低(P <0.01)；其第6~10 天的逃逸潜伏期分别为[(20.86±4.99) s、 (15.01±4.43) s、(15.45±4.95) s、(15.81±6.65) s、(12.37±4.15)s]，较脓毒症模型组各时点[(41.62±12.94) s、(39.37±12.74) s、(36.95±9.64) s、(34.55±13.57) s、(29.45±9.13) s] 显著延长(均P <0.01)；氢气治疗组穿越平台次数[(4.10±0.99) 次] 较脓毒症模型组[(1.20±0.79) 次] 显著增多(P <0.01)；氢气组治疗组小鼠大脑皮质中VE-cadherin 表达水平(0.37±0.02)较脓毒症模型组(0.18±0.01)显著增高(P <0.01)，ZO-1 水平(0.46±0.02)较脓毒症模型组(0.20±0.01)显著增高(P <0.01)。以上指标差异在Nrf2-/- 小鼠中均无显著差异(均P >0.05)。结论:吸入2% 氢气可通过Nrf2 依赖途径降低微血管内皮通透性来保护BBB，从而降低小鼠SAE 发生，改善认知功能。 |
| 关键词: 氢气 脓毒症相关脑病 血脑屏障 Nrf2 基因 盲肠结扎穿刺法 |
| DOI：10.3969/j.issn.1007-6948.2020.03.003 |
| 投稿时间：2020-02-11 |
| 基金项目:国家自然科学基金(81071533，81471842) |
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| Effects of Hydrogen Gas on Blood-brain Barrier Damage and Cognitive Dysfunction in Wild Type and Nrf2-knock out Mice with Sepsis |
| XU Ge,YU Yang,YANG Man |
| Department of Anesthesiology,Tianjin Medical University General Hospital, Tianjin Institute of Anesthesiology, Tianjin 300052, China |
| Abstract: |
| Objective To explore whether H2 can protect blood-brain barrier (BBB) and reduce sepsisassociated encephalopathy (SAE) through Nrf2 dependent pathway. Methods 152 female wild-type (WT) and 152 Nrf2 knockout (Nrf2-/-) C57BL/6J mice were randomly divided into Sham group, Sham+H2 group,CLP group and CLP+H2 group. In CLP and CLP+H2 groups, cecal ligation and puncture were performed to prepare sepsis model. Sham group and Sham+H2 group mice were only underwent open surgery. Sham group and CLP+H2 group mice began to inhale 2% H2 for 60 minutes at 1h and 6h after CLP or sham operation. 24 hours after sham operation or CLP, 20 mice in each group were randomly selected to observe and record the natural survival rate for 7days. 4 mice in each group were randomly selected to detect the blood-brain barrier injury by Evans blue extravasation method. 4 mice in each group were randomly selected to extract the cortex and the levels of VE-cadherin and ZO-1 were detected by Western blot. The rest 10 mice in each group were tested with Morris water maze for 7 days when 4days after sham operation or CLP operation to evaluate the cognitive function. Results In WT mice, the 7-day survival rate of CLP+H2 group (60%) was significantly higher than that of CLP group (35%) (P<0.01). The extravascular Evans blue extravasation in CLP+H2group [(3.68 ± 0.18) μg/g] was significantly lower than that in CLP group [(5.24 ± 0.06) μg/g] (P<0.05). The escape latency of CLP+H2 group[d6:(20.86±4.99)s;d7:(15.01±4.43)s;d8:(15.45±4.95)s;d9:(15.81±6.65) s;d 10 :( 12 . 37 ±4 . 15 )s] were significantly increased than those of CLP group [d 6 :( 41 . 62 ±12 . 94 )s; d7:(39.37±12.74)s; d8:(36.95±9.64)s; d9:(34.55±13.57)s; d10:(29.45 ±9.13)s] (all P<0.01).The time of crossing platform [(4.10 ± 0.99) times] in CLP+H2 group was significantly higher than that in CLP group [(1.20 ± 0.79) times] (P < 0.01);VE-cadherin/β-actin level (0.37 ± 0.02) was significantly higher than that in CLP group (0.18 ± 0.01) (P< 0.01), and ZO-1/β-actin level (0.46 ± 0.02) was significantly higher than that in CLP group (0.20 ± 0.01) (P < 0.01).There was no signi?cant difference in Nrf2 - / - mice (P> 0.05). Conclusion Inhalation of 2% H2 can protect BBB by reducing the permeability of BBB through Nrf2 dependent pathway, thus reducing SAE and improving cognitive function. |
| Key words: Molecular hydrogen sepsis-associatedencephalopathy blood brain barrier Nrf2 gene cecal ligation and puncture |
