将氢气用于脓毒症治疗研究最早是第四军医大学谢克亮等的工作，连续在Shock上发表论文两篇，他们的系列研究是最早证明氢气可以促进身体内抗氧化酶活性的研究。尽管这些发现目前无法有合理的解释，但仍对该领域有比较好的影响，此后有不少关于氢气的研究都使用抗氧化酶作为解释氢气效应的指标，可以说起源都是他们的工作。

现在这个工作也是氢气治疗脓毒症的研究，和以前工作的不同点是，采用注射氢气盐水的方法和过去呼吸氢气的方法不同。研究的器官是脑，这不同于过去外周器官的研究。不过从整体上考虑，即使研究脑功能，其他一些相关器官的功能也应该研究，例如肝脏和肺功能，因为这些器官的功能也会影响到脑功能，不仅仅是炎症一个方面的因素。这个论文是来自兰州大学附属医院麻醉科的，过去没有听说过他们的研究，不过通讯作者是华西大学，他们是开展氢气研究最早的单位之一。也曾经和我们有合作关系，去年发表一篇关于肺缺血再灌注损伤的论文。

Hydrogen-rich saline reverses oxidative stress, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and puncture

• Jun Zhou, MDa,Ye Chen, MDb,Guo-Qing Huang, MDc,Jun Li, MDb,Gang-Ming Wu, MDa,Li Liu, MDa,
• Yi-Ping Bai, MDa,Jian Wang, MDd, ,
  
• a Department of Anesthesiology, Affiliated Hospital of Luzhou Medical College, Luzhou, P.R. China
• b Department of Traditional Chinese Medicine, Affiliated Hospital of Luzhou Medical College, Luzhou, P.R. China
• c Department of Emergency Medicine, Xiangya Hospital of Central South University, Changsha, P.R. China
• d Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, P.R. China
  
• Received 31 August 2011. Revised 12 January 2012. Accepted 25 January 2012. Available online 1 April 2012.

Abstract

Sepsis is associated with high morbidity and mortality, and survivors can present with cognitive dysfunction. The present study was performed to investigate the effects of hydrogen-rich saline (HRS) on oxidative stress in the brain, cognitive dysfunction, and mortality in a rat model of sepsis.

Methods

A rat model of sepsis was induced by cecal ligation and puncture. Physiologic saline or HRS was administered intraperitoneally (2.5 mL/kg or 10 mL/kg) 10 min before the operation. The survival rate was recorded, and cognitive function was tested using the Morris water maze. The reactive oxygen species and malondialdehyde levels and superoxide dismutase activity in the hippocampus were observed to evaluate the oxidative stress levels. The caspase 3 levels were measured to detect apoptosis. The histopathologic changes in the hippocampus were evaluated by hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay.

Results

Cecal ligation and puncture resulted in a poor survival rate, evidence of brain injury, and cognitive dysfunction. The hippocampal reactive oxygen species and malondialdehyde levels increased significantly, and superoxide dismutase activity decreased significantly. HRS reversed these changes in a dose-dependent manner.

Conclusions

These findings indicate that HRS could attenuate the consequences of sepsis induced by cecal ligation and puncture in rats, at least in part, by the inhibition of oxidative stress.

Keywords

• Sepsis;
• Hydrogen-rich saline;
• Cognitive function;
• Oxidative stress
  

Figures and tables from this article:

Fig. 1. Changes in blood hydrogen concentrations among four experimental groups (n = 16; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 2. Survival rates among four experimental groups ≤10 d. Data expressed as percentages (n = 20; *P < 0.05 versus sham; #P < 0.05 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 3. H&E staining in hippocampal CA1 region. Representative sections from hippocampal CA1 region observed 48 h after CLP (magnification ×400). Arrows indicate injured neurons. (A) Regular morphology of hippocampal CA1 region were seen in sham group. (B) Many injured neurons, characterized by shrunken nuclei and stained dark, were observed in CLP group. (C) Normal neurons were significantly increased in CLP+H21 group. (D) Neurons with eumorphism were significantly preserved in CLP+H22 group. (E) Normal neuronal cell counting of hippocampal CA1 field among different groups. Data expressed as mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 4. Immunostaining for cleaved caspase 3 in hippocampal CA1 region. Cleaved caspase 3 immunopositive cells were darkly stained (arrows) in representative micrographs 48 h after CLP. Magnification ×400. (A) Sham, (B) CLP, (C) CLP+H21, and (D) CLP+H22 groups. (E) Quantitative analysis of positive cells among three groups. Bars represent mean ± standard error mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 5. Change in cleaved caspase 3 protein expression in hippocampus after CLP. Western blot analyses demonstrated coordinated increase in cleaved caspase 3 protein within CA1 region of hippocampus at 48 h. Quantitation of Western blots confirmed significant increases in immunoreactive band densities. Data are presented as mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 6. TUNEL staining in hippocampal CA1 region at end of 48 h of CLP. TUNEL-positive cells (arrows) and contain nuclei stained dark brown. Representative micrographs were taken 48 h after CLP event (magnification ×400). (A) Sham, (B) CLP, (C) CLP+H21, and (D) CLP+H22 groups. (E) Quantitative analysis of TUNEL-positive cells of different groups. Bars represent mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 7. ROS generation in hippocampus by 48 h after CLP. ROS levels were assessed using dichlorofluorescein (DCF) assay. Bars represent mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 8. MDA level and SOD activity in hippocampi at 48 h after CLP. (A) MDA level. (B) SOD activity. Bars represent mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21).

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Fig. 9. Cognitive impairment assessed by hidden platform trial. (A) Latency to platform, (B) swimming distance to platform, and (C) swimming speed throughout experiments. Data presented as mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21 at corresponding measurement points).

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Fig. 10. Probe trial test. Cognitive impairment occurred at days 4 and 7 after CLP and HRS improved cognitive impairment. (A) Number of crossings in the former platform region. (B) Percentage of target quadrant. (C) Time spent in quadrant of former platform position. Bars represent mean ± standard error of mean (n = 8; *P < 0.05, **P < 0.01 versus sham; #P < 0.05, ##P < 0.01 versus CLP; †P < 0.05 versus CLP+H21 at corresponding measurement points).