Saturated hydrogen saline protects the lung against oxygen toxicity
Juan Zheng1*, Kan Liu1*, Zhimin Kang1, Jianmei Cai1, WenWu Liu1, Weigang Xu1, Runping Li1, hengyi Tao1, John h Zhang2, XueJun Sun1
1Department of Diving Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, China; 2Department of Physiology and Pharmacology, Loma Linda University School
of Medicine, Loma Linda, California, USA
*The authors contributed equally to this work.
CoRReSponding auThoR: dr. Xuejun Sun –
dr. Juan Zheng –
AbStrAct
exposure to high oxygen concentrations leads to acute lung injury, including lung tissue and
alveolar edema formation, congestion, intra-alveolar hemorrhage, as well as endothelial and epithelial
cell apoptosis or necrosis. Several studies have reported that molecular hydrogen is an efficient anti-oxidant by gaous rapid diffusion into tissues and cells. moreover, consumption of water with
dissolved molecular hydrogen to a saturated level (hydrogen water) prevents stress-induced
cognitive decline in mice and superoxide formation in mice. The purpo of the prent study was
to investigate the effect of saturated hydrogen saline on pulmonary injury-induced exposure to >98% oxygen at 2.5 ATA for five hours. Adult male Sprague-Dawley (SD) rats were randomly divided into three groups: control group, saline group and saturated hydrogen saline group. hematoxylin and eosin (h&e) staining were ud to examine histological changes. The lung wet to dry (Wd) weight ratio was calculated. The concentration of protein and total cell counts in bronchoalveolar lavage fluid
(BaLF)were measured. Lactate dehydrogena (Ldh) in rum and BaLF were measured by spectro-photometer. The light microscope findings showed that saturated hydrogen saline reduced the impair-ment when compared with the saline group: Saturated hydrogen saline decread lung edema, reduced Ldh activity in BaLF and rum, and decread total cells and protein concentration in BaLF.
The results demonstrated that saturated hydrogen saline alleviated hyperoxia-induced pulmonary injury, which was partly responsible for the inhibition of oxidative damage.
jormungandIntroductIon
although esntial for survival, oxygen may be-come toxic at an elevated partial pressure; this puts limitations on the application of oxygen. hyperbaric oxygen therapy is administered for the treatment of tissue hypoxia, most commonly in an intensive care tting of respiratory-insufficient patients, though its potent toxicity is well-described (1). moreover, the relationship between pulmonary oxygen toxicity and acute lung injury (aLi) and its most vere form, the acute respiratory distress syndrome (aRdS), has attracted medical rearch attention. Studies have provided strong evidences for the similarities between pulmonary oxygen toxicity and aLi/aRdS. under-standing the similarities and the signaling path-ways is important for the mechanisms of aLi/aRdS.
The pathological manifestations of oxygen-induced lung injury include interstitial and alveolar edema, congestion and intra-alveolar hemorrhage, hyaline membrane formation, lysis of alveolar type i and capillary endothelial cells at 2-4 aTa oxygen pressure. The pathophysiology of oxygen injury is characterized by lung inflammation, including activation and recruitment of neutrophils and
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alveolar macrophages, tissue and alveolar edema, surfactant dysfunction, and excess production of free radicals and inflammatory cytokines (2-4). although the exact mechanisms of oxygen-induced lung injury are still unknown, compelling evidence suggests that reactive oxygen species such as super-oxide anions, hydroxyl radicals and hydrogen per-oxide are important mediators of lung injury (5-7).
The currently accepted primary mechanism for oxygen toxicity is the “free-radical” theory, which ascribes tissue damage to the incread generation of oxygen-derived radicals during exposure to elevated oxygen tensions (8, 9). it has been post-ulated that the source of oxygen-derived radicals associated with oxygen toxicity may be the poly-morphonuclear leukocyte (pmn) (10). models of lung injury have demonstrated that inflammation of the lung can interact with hyperoxic injury to potentiate lung damage (11). in addition, toxic products from pmn or non-cellular systems (e.g., xanthine plus xanthine oxida) in high concentration can damage lung cells (12,13). endothelial and epithelial injury and cell death are major features of pulmonary oxygen toxicity. There are two classical types of cell death in pulmonary oxygen toxicity: necrosis or apoptosis (14). apoptosis appears to be the major mode of cell death when cells experience lethal oxidative insult from exposu
re to oxidants, including h2o2 and superoxide (15,16).
Recently, it has been reported that molecular hydrogen is an efficient antioxidant by gaous rapid diffusion into tissues and cells (17-21). moreover, consumption of water with dissolved molecular hydrogen to a saturated level (hydrogen water) prevents stress-induced cognitive decline in mice and superoxide formation in mice (22). however, there is no direct evidence for the protective effect of hydrogen in pulmonary oxygen toxicity. Therefore, the purpo of the prent study was to investigate the effect of saturated hydrogen saline on pulmonary oxygen toxicity. 186MAterIAlS And MethodS
Animals
Thirty adult male Sprague-dawley rats weighing 270±10 g were ud for this study. all experimental procedures were conducted in accordance with the guiding principle in the Care and u of animals approved by the institutional animal Care and u Committee of Secondary military medical university, RoC.
experimental protocol
青春奉献演讲稿
For the saturated hydrogen saline preparation, purified H2was dissolved into normal saline for two hours under 0.6 mpa. The saturated hydrogen saline was stored under atmospheric pressure at 4°C in an aluminum bag with no dead volume. hydrogen-rich saline was freshly prepared every week, which maintained a continuous concentration of 0.6 mmol/L. all agents were intraperitoneally injected (6 ml/kg) 30 minutes before exposure. Rats were expod to >98% o2 at 2.5 ATA for five hours in a plexiglass chamber, described previously (23). during that time, the chamber was ventilated with pure oxygen, without interruption, to minimize Co2 accumulation. o2 levels were monitored hourly with an oxygen analyzer (hangtianpengcheng, Beijing, China) to maintain the level of 98%. Controls were kept in room-air temperatures. animals were killed by cervical dislocation, and lungs were fixed by perfusion of 10% buffered formalin at 20 cm h2O pressure and embedded in paraffin, as described previously (23).
Rats were randomly divided into three groups as follows:
• control group, n=10 – expod to room air; • saline group, n=10 – received saline and
expod to 2.5aTa oxygen;
• saturated hydrogen saline group, n=10 –
dragonboatfestival
received saturated hydrogen saline and
expod to 2.5aTa oxygen.
Quantification of lung wet/dry weight ratio immediately following exposure, lungs were excid en bloc and discted away from the heart and thymus. The middle lobe of right lung was imme-diately weighed and then placed in a drying oven at 60 Cº for 96 hours to stabilize dry weight. The ratio of wet/dry weight was ud to quantify lung water content.
Evaluation of lung injury
protein concentration and Ldh activity and total cells in bronchoalveolar lavage fluid (BALF) were measured for lung injury. after exposure, the low lobe of the right lung was inflated with paraform-aldehyde for histological studies. Bronchoalveolar lavage (BaL) was performed on the left lung with 4 ml of a phosphate-balanced saline solution in 2.5-ml aliquots after cannulation of the left trachea. The collected BaLF was centrifuged at 1000 g for 10 minutes, the supernatant was collected and stored at -20°C and -80°C for later protein assays and Ldh activity, and the total cell count was determined on a fresh fluid specimen using a hemocytometer. total protein in bAlFyielded
To asss the permeability of the broncho-alveolar–capillary barrier and cellular infiltration in the alveolar space, total protein content in BaLF was measured by the BCa protein assay reagents using BSa as a standard (pierce, Rockford, ill., uSa).
lactate dehydrogena activity in bAlF
The activity of Ldh, a cytosolic enzyme ud as an indicator for cellular oxidative damage, was measured at 490 nm using an Ldh determination kit according to the manufacturer’s instructions (Roche molecular Biochemicals, mannheim, germany). Ldh activity was expresd as u/L, using an Ldh standard.
lung histopathology
immediately following exposure, animals were exsanguinated by abdominal aorta. The right lungs were removed and then transferred to 4% formal-dehyde for 48 hours. The lungs were paraffin-embedded, and butterfly-shaped ctions of 5-mm thickness were cut and placed on glass microscope slides stained with hematoxylin and eosin (h&e). The h&e ctions were imaged at microscopic levels. tunel staining
The in Situ Cell death detection Kit, ap (1168-4809910) (Roche) was employed to demonstrate cell death and dna injury in lung tissue. The tissue ctions were dewaxed and rehydrated according to standard protocols. Then, coronal ctions were digested with proteina K (20 mg/ mL; Bio-Light) in 0.01 mol/L pBS at room temper-ature for 15 minutes. To interrupt digestion, the c-tions were dipped in 4% formaldehyde in 0.01 mol/L PBS for five minutes. After rinsing with PBS (five minutes, three times), the ctions were immerd in TuneL reaction mixture for one hour at 37 °C. in order to remove background staining caud by non-specific binding of antifluorescein-AP, the ctions were treated with 1% BSa in pBS for 30 minutes at 37 °C. After rinsing with PBS (five minutes, three times), the ctions were colorated in the dark with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Statistical analysis
all values are prented as mean ± Sem. differ-ences between groups were determined with one-way anoV a followed by Student-newman-Keuls test.
a level of p<0.05 was considered statistically significant.
reSultS
Morphology examination in lung学位班
Lung slices from animals subjected to chronic oxygen toxicity with or without saturated hydrogen saline are shown in Figure 1 (page 188). The lung tissues in saline group were injured, with vere edema, alveolar wall thickening, alveolar hemor-rhage, and infiltration of neutrophils into the lung interstitium and alveolar space. in the saturated hydrogen saline group, lung damage was alleviated, with results such as a reduction of infiltrated inflammatory cells and a marked improvement in lung architecture.
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欺诈猎人主题曲Copyright © 2010 Undera and Hyperbaric Medical Society, Inc.
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Figure 1 – Microphotographs of the lung tissue.
马来亚大学Lungs were obtained 24 hours after exposure; lung specimens were stained with hematoxylin and eosin. (A) control group; (B) saline group; (C) saturated hydrogen saline group. Original magnification: ×200.
Effects of saturated hydrogen saline on
cell apoptosis
陈如鉴The TuneL demonstrated apoptotic cell death in saline treatment animals, and saturated hydrogen saline reduced the number of apoptotic cells (Figure 2, this page).
Effect of saturated hydrogen saline on lung edema
The wet/dry ratio of the saline group is significantly higher than that of the control group (p <0.05). Compared to the saline group, saturated hydrogen saline signifi-cantly decread the wet/dry ratios (Figure 3, facing page) (p <0.05).
C
A
FIGURE 1
A
B
A
B
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B
A
FIGURE 2
Figure 2 – TUNEL staining.
(A) control group; (B) saline group; (C) saturated hydrogen saline group. Original magnification: ×200.
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Effects of saturated hydrogen saline on ldh in bAlF and rum
The concentration of Ldh in plasma was significantly higher in the saline group than that in the control group (Figure 4a , left). The saturated hydrogen saline group had significantly decread LDH level in plasma compared to the saline group (p <0.05). however, compared to the control group, the LDH level in the BAL fluid and plasma in the saturated hydrogen saline group were not significantly changed (p >0.05). The LDH level in the BAL fluid was significantly higher in the saline group than that in the control group (p <0.05). in contrast, a significant decrea of LDH in the BAL fluid was found in the saturated hydrogen saline group (Figure 4b , left)compared to that in the saline group. Effects of saturated hydrogen saline on total cells and protein concentration in bAlF
Total cells and protein concentration in the BAL fluid were determined as an indicator of lung hyperp
ermeability induced by hyperoxic exposure. Results demonstrate that total cells and protein in the BAL fluid in the saline group were significantly incread, compared to the control group (p <0.05) (Figure 5 and Figure 6, page 190). Converly, in the saturated hydrogen saline group, total cells and protein were much
appeton
Figure 3 – The wet/dry ratio of lung weight (W/D) determined after exposure. Saturated hydrogen saline attenuated the increa in W/D ratio in saline group. Numbers in the bars indicate the group size.
*p < 0.05 compared with the saline group; #p < 0.05 compared with the control group.
Figure 4 – Lactate dehydrogena activity in rum (a) and BALF (b) from each group of rats after five hours’ exposure to >98% O 2 at 2.5 ATA. Data are mean ± SEM. #p < 0.05 compared with the control group; *p < 0.05 compared with the saline group.
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lower than tho of the saline group (p <0.05). In addition, there was not a significant difference between the control group and the saturated hydrogen saline group (p >0.05).
伊尔维萨克dIScuSSIon
in the prent study, we have demonstrated for the first time that saturated hydrogen saline alleviated the lung injury induced by hyperbaric oxygen. Treatment with saturated hydrogen saline, a potent free-radical scavenger, decread the wet/dry ratio of lung weight and protein concentration and Ldh activity in the BAL fluid, as well as LDH in rum. in addition, saturated hydrogen saline decread total cells in the BAL fluid and inhibited apoptosis.
Figure 5 – Protein concentration in BAL from each group. Data are mean ± SEM.
#p < 0.05 compared with the control group; *p < 0.05 compared with the saturated hydrogen saline group.
– Total cell counts in BAL from < 0.05 compared with the control group; < 0.05 compared with the saturated
FIGURE 6
The currently accepted primary mechanism for oxygen-induced cellular injury is enhanced oxygen-d
erived free radical generation, with subquent oxidative attack upon basic cell constituents. Free radicals, such as superoxide and hydroxyl radicals, are important mediators of lung injury, whether they are produced inside lung parenchyma cells or by immigrant neutrophils (14). ikuroh ohsawa et al. have found that molecular hydrogen lectively reduces hydroxyl radicals (17). hydrogen has already been ud in diving for humans to help prevent decompression sickness in divers at the level of 2 mpa partial pressure of hydrogen, suggesting that 16 mm hydrogen in blood could be safe (25).
