The Antioxidative Effect of a Novel Free Radical Scavenger 4′-Hydroxyl-2-substituted Phenylnitronyl Nitroxide in Acute High-Altitude Hypoxia Mice

Abstract

Acute mountain sickness is caused by sub-acute hypoxia in healthy individuals going rapidly to altitude. Both tissue hypoxia in vitro and whole-body hypoxia in vivo have been found to promote the release of reactive oxygen species. Nitronyl nitroxide can trap free radicals such as ·NO or ·OH, and may therefore be efficient protective agents. This study assessed the ability of nitronyl nitroxide to against acute mountain sickness as a free radical scavenger in acute high-altitude hypoxia mice model. Normobaric hypoxia and hypobaric hypoxia model were used to estimate the protect effects of nitronyl nitroxide against acute mountain sickness. Low pressure oxygen compartment system was used to stimulate high-altitude hypobaric hypoxia environment. Mice in nitronyl nitroxide groups survived longer than acetazolamide group in normobaric hypoxia test. Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) increased in both cerebrum and myocardium in vehicle group. The results indicated more radicals were generated during high-altitude hypobaric hypoxia environment. In therapeutic groups H₂O₂ and MDA were significantly reduced while the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were similar to normal group. These results demonstrated that nitronyl nitroxide was an efficient tissue radical scavenger and a potential protective agent for acute mountain sickness.

More than 140 million people worldwide live >2500 m above sea level. Eighty million live in Asia, and 35 million live in the Andean mountains. The latter region has its major population density living above 3500 m.¹⁾ Barometric pressure falls with increasing altitude and consequently in the partial pressure of oxygen reduced. This leads to a hypoxic challenge to any individual ascending to altitude. A spectrum of high-altitude illnesses can occur when the hypoxic stress outstrips the subject’s ability to acclimatize.²⁾ Acute mountain sickness (AMS) is a condition affecting otherwise healthy individuals going rapidly to altitude. It is caused by sub-acute hypoxia in susceptible subjects.³⁾ Acute hypoxia induces pulmonary vascular permeability and contributes to forms of noncardiogenic pulmonary edema such as high-altitude pulmonary edema and acute respiratory distress syndrome.^4,5) AMS can sharply limit people’s recreation and working at high altitude, especially in the first few days following arrival at a new, higher altitude.⁶⁾ More and more studies were concerned on the prophylaxis and therapy of AMS.^7–9)

In vitro hypoxia and in vivo whole-body hypoxia can cause tissue release reactive oxygen species (ROS) which are potentially damaging to the cardiovascular system.¹⁰⁾ Hypoxia rapidly increases the levels of free radicals in organism, which is thought to overwhelm the reserves of scavengers. The radicals then damage cell walls, reduce the flexibility of blood vessels, destroy enzymes, and cause other molecular damage in the neurological pathways. Free radical reactions can result in deleterious modifications in membranes, proteins, enzymes and DNA.¹¹⁾ Therefore, it is important to discover effective free radical scavengers for prevention and treatment of related disorders. Previous study reported that antioxidant supplementation exhibited a trend toward lower severity, although it did not diminish AMS incidence.¹²⁾ There were conflicting evidences of free radical scavenger’s effectiveness in prevention of AMS with some studies showing a benefit while others not.¹³⁾ So the doubt about whether antioxidants have some effects on AMS still remains unclear.²⁾

Nitronyl nitroxides are chemically remarkable because of their capability to trap ·NO, ·OH, hydrogen peroxide (H₂O₂), and O₂^•−, protecting endothelial cells from the attack of free radicals.^14–19) Among nitronyl nitroxides, 4′-hydroxyl-2-substituted phenylnitronyl nitroxide (HPN), chemical name 2-(4′-hydroxyl)-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl, is a novel antioxidant and free radical scavenger (Fig. 1). It could trap free radicals such as ·NO, H₂O₂ and ·OH.¹⁹⁾ The aim of current work was to study the effect of HPN to against AMS as a free radical scavenger in acute high-altitude hypoxia mice model in vivo. Acetazolamide (ACZ) is one of the unequivocally effective drugs.^7,20–22) It is concerned that antioxidants may interfere with the action of ACZ on the normocapnic hypoxic ventilatory response.²³⁾ In our work we chose ACZ as a positive drug to compare the effects of HPN with ACZ. The first step of the present study was to prepare the free radical scavenger by literature reported procedures.¹⁸⁾

Fig. 1. Chemical Structure and General Synthesis Process of HPN

MATERIALS AND METHODS

Drugs and Chemicals

HPN represented by the formulae in Fig. 1 was synthesized according to the literature method.¹⁸⁾ Its general synthesis process was listed in Fig. 1. Generally, 2,3-dimethyl-2,3-bis(hydroxylamino)butane (1.48 g, 10.0 mmol) and 4-hydroxylbenzaldehyde (1.22 g, 10.0 mmol) were dissolved in methanol (30.0 mL). The reaction was filtered after stirring for 24 h at room temperature. The resulting white powder was washed by cool methanol and suspended in the solution of dichloromethane (30.0 mL). Then the reaction mixture was added to an aqueous solution of NaIO₄ (30 mL) and stirred for 15 min in an ice bath to give a dark blue solution. The aqueous phase was extracted with CH₂Cl₂ and the organic layer was combined and dried over Na₂SO₄. Then the solvent was removed to give a dark blue residue which was purified by flash column chromatography with the elution of n-hexane–ethyl acetate (1 : 2) to yield 1.24 g (50%) of the title compound as a dark blue powder. The structure of HPN was determined by IR, MS, and ESR. The total yields of the compound were comparable to previous literature. mp 135–136°C. IR (KBr) 3252, 1510, 1495, 1360, 843 cm⁻¹; electrospray ionization (ESI)-MS (m/z)=249 [M]⁺; Anal. Calcd for C₁₃H₁₇N₂O₃^•: C, 62.63; H, 6.87; N, 11.24; O, 19.25; Found C, 62.41; H, 6.53; O, 19.53. ESR: a five-line pattern with intensity ratios of 1 : 2 : 3 : 2 : 1, a_N=7.69 G, g=2.0068. HPN was prepared with physiological saline before use. ACZ was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) as positive control. The measurement kits for lactate dehydrogenase (LDH), lactic acid (LD), bicinchoninic acid (BCA) protein assay, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and ATPase activities assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Animals

All BALB/c mice (22±2 g) and Wistar rats (200±20 g) used in this experiment were SPF animals and obtained from the Center for Experimental Animals, Lanzhou Institute of Biological Products (Lanzhou, China). The mice were housed in the Laboratory Animal Care Center of Lanzhou Command General Hospital (elevation 1520 m). Animals were allowed access to food and water ad libitum. The animals were kept on a 12-h day–night cycle. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Lanzhou Command General Hospital. Sixty BALB/c mice and sixty Wistar rats were randomly divided into six groups: normal control group, decompression hypoxia model group, ACZ group (200 mg/kg) and HPN group (50, 75, 100 mg/kg).

Effect of HPN on Survival Time of Mice under Normobaric Hypoxia

In the normobaric hypoxia test, groups of overnight fasted mice were treated by vena caudalis administration with HPN (50, 75, 100 mg/kg), vehicle (10 mL/kg) or ACZ (200 mg/kg). Twenty minutes after administration, each mouse was put into a 250 mL airtight container with 5 g medical soda lime inside. The bottle neck was treated with petroleum jelly for a hermetic condition. Bottle cap was sealed after mouse was put into the bottle. Time from bottle cap sealed to mouse stopped breathing was recorded as survival time. The survival time of oxygen deprivation and prolongation rate (prolongation rate=(survival time of treatment group−survival time of model group)/survival time of model group) were used to compare the anti-hypoxic activity. The thorax was opened as soon as the mouse stop breathing. About 0.5 mL blood sample was withdrawn from mice hearts and 0.4 mL was added to the centrifuge tube citrate-stabilized with 3% natrium citricum. The mixture was centrifuged at 3500 rpm for 5 min. The plasma was collected and used to determine the concentration of lactic acid and lactate dehydrogenase.

The Lactice Acid (LD), LD Accumulation Rate and LDH Assessment

Blood samples were collected, centrifuged and kept at −20°C until analyses to assess the LD, LD accumulation rate and LDH.²⁴⁾ Standard techniques using commercialized assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China) were performed for analysis. LD accumulation rate were calculated as LD/survival time. LD values were expressed as mmol/L. LD accumulation rates were expressed as µmol/L·min. LDH values were expressed as U/L.

Hypobaric Hypoxia Test

The method reported by Ma et al. was adjusted and used in this test.²²⁾ Low pressure oxygen chamber FLYDWC50-IIC (Guizhou Fenglei, China) was used to stimulate high-altitude condition. HPN and ACZ were administrated as mentioned above. Twenty minutes after being veinly administrated with HPN or vehicle, mice except normal control group were put into a hypobaric hypoxia chamber and decompressed at a speed of 100 m/min. At last the simulated altitude of 8000 m was obtained. Mice were adapted to this hypobaric hypoxia environment (8% oxygen and 92% nitrogen, 0.035 MPa) for 6 h as previous described,²⁵⁾ and then slowly recovered to normal altitude in half an hour. Opened the chamber door, sacrificed the animals by cervical dislocation. Mice hearts and brains were collected and stored at −80°C which were used for morphological analysis, hydrogen peroxide, malonaldehyde and enzymatic activity assays.

Morphological Analysis

After treatment with hypobaric hypoxia, the mice hearts and brains were subjected to morphological analyses. The ultrastructure pathology changes were examined by electron microscopy (EM).

Effect of HPN on Heart Rate and Blood Pressure of Rats under Hypobaric Hypoxia Test

Large low pressure oxygen compartment (Guizhou Fenglei, China) was used to stimulate high-altitude condition. HPN and ACZ were administrated as mentioned above. Twenty minutes after being veinly administrated with HPN or vehicle, Rats except normal control group were put into a the hypobaric hypoxia compartment and decompressed at a speed of 100 m/min. At last the simulated altitude of 8000 m was obtained. Rats were adapted to this hypobaric hypoxia environment (8% oxygen and 92% nitrogen, 0.035 MPa) for 12 h and then recovered to altitude of 4500 m (100 m/min, 0.06 MPa). Meanwhile the experimenters entered the large low pressure oxygen compartment through a transfer chamber (4500 m) and test the blood pressure and heart rate (HR) of rats by BP-2010A Series Blood Pressure Meter (Softron, Japan).

Malondialdehyde (MDA) Assessment

The extent of lipid peroxidation in the mouse blood was estimated by MDA level, which was measured by using the spectrophotometric diagnostic kits (Nanjing Jiancheng Biotechnology Institute, China) as described by Uchiyama and Mihara.^26,27)

H₂O₂ Measurement Assay

H₂O₂ production in tissue homogenate was measured as quantitative index of ROS generation (indirect indicator of the free radical O₂^•−) by H₂O₂ assay kit (Nanjing Jiancheng Institute, China). H₂O₂ was performed on monitoring at the absorbance at 405 nm of the molybdenic acid-peroxide complex. The absorbance values were calibrated to a standard graph generated with known content of H₂O₂. The unit was defined as 1 mmol of H₂O₂ per gram fresh protein. Vehicle and HPN groups were treated under hypobaric hypoxia mentioned above for 6 h. After 6 h of exposure, brains and hearts were grasped and washed with cold physiological saline once then homogenated and centrifugated in refrigerated centrifuge. The supernatant (100 µL) was added with H₂O₂ assay solution (100 µL).

Antioxidant Enzyme Activities

To prepare homogenates, the mice cerebral cortex, heart and liver were homogenized with a homogenizer (400 rev./min, 60 s) at 4°C in cold buffer (1/9, tissue/buffer, w/v) containing 0.01 mol/L Tris–HCl, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.01 mol/L saccharose, 0.8% saline.²⁸⁾ The tubes with homogenate were kept in ice water for 30 min and centrifuged at 4°C (1000 g, 10 min), as recommended in the assay kits. The supernatant was separated and stored at −80°C for assay of various enzymatic activities. Measurement of protein concentration was estimated using commercial BCA assay kits (Nanjing Jiancheng Institute, China). The activities of SOD, GSH-Px, CAT and ATPase were measured using commercial assay kits (Nanjing Jiancheng Institute, China) according to the manufacturer’s instructions. Briefly, SOD activities were measured following the reduction of nitrite by a xanthine–xanthine oxidase system which was a superoxide anion generator. The activities were expressed as U/mg protein. GSH-Px activities were assayed by the decrease of the GSH, which can be reflected by the alteration of the absorbance at 412 nm. CAT activities were determined by decrease of H₂O₂ absorption at 405 nm. The activities of SOD, GSH-Px, CAT and ATPase were expressed as U/mg protein, U/g protein, U/mg protein and µmolPi/mgprot/h, respectively.

Statistical Analysis

All data were expressed as the mean±S.D. Data was subjected to ANOVA followed by Student–Newman–Keuls tests. p≤0.05 was considered significant.

RESULTS

The defining characteristic of an antioxidant is that it should protect an organism against oxidative stress.²⁹⁾ To determine the antioxidant capability of HPN, we administrated mice with three concentrations of HPN and subjected them to oxidative challenges via normobaric hypoxia and hypobaric hypoxia. Our initial aim was to discover whether free radical scavenger could protect mice against oxidative stress caused by high-altitude hypoxia. Our study was to estimate the protective effects of HPN in vivo compared with ACZ, and also to identify an appropriate dose that offering beneficial effect with no toxicity. To test whether HPN could protect mice through the hypobaric hypoxia progress, we carried out both histology and biochemistry assay to determine the morphology and physiology changes during the test.

Alterations in Cellular and Mitochondrial Morphology in Hypoxia-Treated Mice Brains and Hearts

The morphology of cardiac myocytes and neurons taken from hypoxia-treated mice brains and hearts were examined (Fig. 2). We observed cardiac and nerve cell mitochondrial morphology by EM at ×6000 to ×10000 magnification. Figures 2A–D showed the cell and organelle morphology of normal control, vehicle, positive control and HPN 75 mg/kg group in hearts (1, 2) and brains (3, 4) under normal and hypobaric hypoxia conditions, respectively.

Fig. 2. Typical Electron Micrograph of Mice Cerebral Cortex and Heart

A normal group, B hypobaric hypoxia model group, C positive control group, D HPN 75 mg/kg group. A1, B1, C1 and D1 showed morphology of nucleus, ×6 k. A2, B2, C2 and D2 showed mitochondria and cardiac muscle fibers, ×12 k. A3, B3, C3 and D3 showed the neurons in parietal cortex, A3×8 k, B3×4 k, C3×6 k, D3×5 k. A4, B4, C4 and D4 showed the cell organelle in neurons, ×20 k.

The mitochondria were precisely positioned between the myofibrils in normal control group healthy aerobic hearts (Figs. 2A1, 2). With respect to sarcomere structure, the mitochondria were almost always positioned between the Z lines at the level of the A band. The precisely fixed position of mitochondria in cardiac muscle cells has been found to be important for facilitating energy exchanges with myofibrils and sarcoplasmic reticulum, with which mitochondria seem to form functional complexes.³⁰⁾ Figure 2A2 showed the mitochondrial structure in control group at higher magnification. Both the outer and inner membranes were intact and the mitochondria were firmly attached to the myofibrils. Dramatic changes in the mitochondrial structure were seen in model group (Fig. 2). Hypobaric hypoxia treated hearts showed a marked increase in mitochondrial size (Fig. 2B1) with loss of membrane integrity and a reduction in mitochondrial number. Following hypobaric hypoxia treatment, mitochondria were swollen and fragmented with clear membrane damage, ruptured cristae, and missing matrix. Figure 2B2 showed the ultrastructure reversible changes in mitochondira, such as augmented volume, lower electronic density in matrixes, damage of vacuolar degeneration after the hypobaric hypoxia treatment. Mitochondria were always and homogeneously detached from the myofibrils, clustered (Fig. 2B1) and often swollen with a clearly broken outer membrane (Fig. 2B2). The morphological changes, observed microscopically, were closely related to alteration of the respiratory function of mitochondria. In heart of the positive and HPN 75 mg/kg group, the mitochondria were still firmly attached to the myofibrils (Figs. 2C1, 2 and D1, 2). Mitochondrial morphology in HPN 75 mg/kg group was not significantly changed compared with vehicle group.

The EM results showed normal primary cortical neurons contained large oval nuclei with homogeneously distributed euchromatin, clear intracytoplasmic mitochondria, and well-defined rough endoplasmic reticulum with abundant ribosomes (Figs. 2A3, A4). In vehicle model group, nucleus in edema neurons was irregular in shape, accompanied with obvious nuclear atypia, wellen and vacuolized mitochondria, declined electronic density in perinuclear cytoplasm. The number of organelle decreased as some organelles dissolved (Figs. 2B3, B4). By contrast, in positive control group the nuclear chromatin was homogeneously distributed and the ultramicrostructure was largely comparable to that of untreated control neurons, although there were some evidences of mitochondrial swelling (Figs. 2C3, C4). In HPN groups the injuries were lessened compared with model group and positive control group (Figs. 2D3, D4). Mitochondria, endoplasmic reticulum and other organelle were in good state.

HPN Prolonged the Survival Time of Mice in Normobaric Hypoxia Test

In normobaric hypoxia test, the treatment with HPN significantly prolonged the survival time of oxygen deprivation mice (Table 1). Data showed that HPN had a dose-dependent effect of increasing the mice survival time under hypoxia condition. The prolongation rates were 38% (low dose), 32% (middle dose), 54% (high dose) and 16% (positive control) compared with model, respectively.

Table 1. Effects of HPN on the Survival Time of Mice under Normobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	Survival time (min)	Prolongation rate (%)
Vehicle	—	32.33±2.26	—
Acetazolamide	200	37.40±5.70*	16
HPN	50	44.70±10.90*	38
	75	42.60±2.30**	32
	100	49.70±8.70**	54

Each group represents the mean±S.D. * p<0.05 vs. vehicle. ** p<0.01 vs. vehicle.

LD

We tested LD level of different groups in normobaric hypoxia test. There were not significant changes during each group, but the treatment with HPN significantly decreased the lactic acid accumulation rate comparing with vehicle³¹⁾ (Table 2). The decrease rates of LD accumulation rate were 11% (low dose), 24% (middle dose), 44% (high dose) and 19% (positive control) compared with model, respectively.

Table 2. Effects of HPN on LD Content, LD Accumulation Rate and LDH Activity in Mice Plasma under Normobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	LD content (mmol/L)	LD accumulation rate (µmol/L·min)	LDH activity (U/L)
Control	—	4.85±0.47	—	2275.6±226.7
Vehicle	—	9.11±0.53##	282.85±27.10	1608.8±91.2#
Acetazolamide	200	8.59±0.70	229.67±16.43*	1997.4±226.0*
HPN	50	10.75±0.43	251.91±63.53	1423.2±77.2
	75	9.18±0.80	215.29±11.57**	1911.2±245.3*
	100	8.80±0.61**	159.61±11.35**	2593.4±175.2**

Each group represents the mean±S.D. ^# p<0.05 vs. control. ^## p<0.01 vs. control. * p<0.05 vs. vehicle. ** p<0.01 vs. vehicle.

Activity of LDH

The activity of LDH was coincidence with the trend of lactic acid accumulation rate. Treatment with HPN significantly decreased the LDH activity compared with model (Table 2).

Heart Rate and Blood Pressure

Heart rate could vary as the body’s need to absorb oxygen and excrete carbon dioxide changes and was expressed as beats per minute (bpm). The heart rate increased significantly in hypobaric hypoxia model group while the blood pressure including systolic blood pressure (SBP), mean artery pressure (MAP) and diastolic blood pressure (DBP) decreased on the contrary. Treatment with HPN significantly attenuated these changes compared with model (Table 3).

Table 3. Effects of HPN on Heart Rate and Blood Pressure of Rats under Hypobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	HR (bpm)	SBP (mmHg)	MAP (mmHg)	DBP (mmHg)
Control	—	402.4±29.1	112.1±14.8	95.2±13.3	86.7±13.9
Vehicle	—	456.7±22.7##	90.7±10.9#	74.6±14.4#	68.1±7.4#
Acetazolamide	200	429.6±13.2*	107.3±10.0*	92.8±7.3	84.8±7.5
HPN	50	450.7±28.1	85.6±7.5	76.9±9.4	62.5±9.5
	75	427.9±21.9*	100.8±13.6*	87.7±8.3*	78.7±6.3*
	100	420.3±22.4*	131.3±18.0*	102.2±17.8*	91.5±12.5*

Each group represents the mean±S.D. ^# p<0.05 vs. control. ^## p<0.01 vs. control. * p<0.05 vs. vehicle.

Malondialdehyde

The level of malondialdehyde was coincidence with the trend of lactic acid accumulation rate. Treatment with HPN significantly decreased the malondialdehyde level compared with model group (Table 4).

Table 4. Effects of HPN on MDA and H₂O₂ in Mice Cerebrum and Myocardium under Hypobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	MDA (nmol/mg prot)		H2O2 (mmol/g prot)
		Cerebrum	Myocardium	Cerebrum	Myocardium
Control	—	2.42±0.33	2.45±0.19	7.15±0.85	24.41±3.24
Vehicle	—	3.75±0.08##	3.43±0.35##	10.16±1.52##	43.41±8.46##
Acetazolamide	200	3.23±0.34*	3.15±0.39	9.21±1.91	55.21±12.74*
HPN	50	3.27±0.29*	3.58±0.09	8.82±0.68	45.15±7.66
	75	2.99±0.41*	3.18±0.19	8.52±1.85	47.30±10.05
	100	2.59±0.43**	2.73±0.13**	7.28±0.69*	32.69±10.94*

Each group represents the mean±S.D. ^# p<0.05 vs. control. ^## p<0.01 vs. control. * p<0.05 vs. vehicle. ** p<0.01 vs. vehicle.

HPN Directly Degrade Hypoxia-Induced H₂O₂ Production in Hypobaric Hypoxia Mice

We determined production of H₂O₂ as an indication of ROS formation in mice brains and hearts after 6 h hypobaric hypoxia test. Hypoxia induced oxidative stress stimulated cerebrum and myocardium to increase H₂O₂ production compared to normal control. Treatment with HPN significantly decreased H₂O₂ production in both cerebrum and myocardium in hypobaric hypoxia mice model (Table 4). The decrease rate of H₂O₂ in HPN 100 mg/kg group was 28% in mice cerebrum and 25% in myocardium compare with model group.

Effect of HPN on the Activities of SOD, GSH-Px, CAT and ATP in Hypoxic Mice

As the biomarker of the antioxidant defenses, the activities of SOD in cerebrum and myocardium, GSH and CAT in liver were measured (Table 5). The activity of SOD conspicuous decreased in cerebrum and myocardium in vehicle. Activity of GSH-Px was also descent significantly. There were no reduced GSH and SOD deficiency observed throughout the trial period in high doses of HPN and ACZ compared with the vehicle group. The activity of CAT was different from SOD and GSH-Px. It was not descent but ascent, which probablely associated with the positive feedback regulation of increased H₂O₂. HPN (especially the high does group) but not ACZ maintained the activities of CAT to normal level. These results indicated that the mice in HPN group remained in normal status even after 6 h hypobaric hypoxia test.

Table 5. Effects of HPN on SOD in Mice Cerebrum and Myocardium, GSH-Px, CAT in Liver, under Hypobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	SOD (U/mg prot)		GSH-Px (U/g prot)	CAT (U/mg prot)
		Cerebrum	Myocardium
Control	—	137.57±22.07	75.54±3.39	431.56±47.40	30.88±5.32
Vehicle	—	20.84±4.05##	52.08±3.97##	368.86±27.41#	38.58±4.14#
Acetazolamide	200	86.72±19.70**	56.31±4.53	495.42±69.83**	41.01±6.85
HPN	50	46.57±8.44**	71.25±11.32*	389.50±58.21	24.53±3.03**
	75	45.75±4.40**	65.83±6.62*	395.48±70.35	27.02±5.73**
	100	78.08±1.10**	73.15±8.11**	421.79±54.76*	30.09±7.53*

Each group represents the mean±S.D. ^# p<0.05 vs. control. ^## p<0.01 vs. control. * p<0.05 vs. vehicle. ** p<0.01 vs. vehicle.

Reduction of Cerebrum and Myocardium ATP Activities in Hypoxic Mice

Mice ATP activities in cerebrum and myocardium were examined using a luciferase assay kit. The biochemical activities of Na⁺–K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase in mice cerebrum and myocardium were significantly lower in vehicle group after hypoxia treatment compared with normal group. On the contrary HPN lessened the decrement of ATPase activities in hypoxic mice cerebrum and myocardium compare to vehicle group. The data showed that HPN had a dose-dependent effect (Table 6).

Table 6. Effects of HPN on ATPase Activities in Mice Cerebrum and Myocardium under Hypobaric Hypoxia Condition (n=10)

Group	Dose (mg/kg)	K+Na+-ATPase (µmol Pi/mg prot/h)		Mg2+-ATPase (µmol Pi/mg prot/h)		Ca2+-ATPase (µmol Pi/mg prot/h)
		Cerebrum	Myocardium	Cerebrum	Myocardium	Cerebrum	Myocardium
Control	—	6.11±0.17	2.07±0.33	5.51±0.23	1.94±0.29	2.96±0.28	2.03±0.24
Vehicle	—	3.41±0.39##	1.30±0.16##	3.75±0.41##	0.69±0.39##	1.69±0.07##	0.98±0.13##
Acetazolamide	200	6.16±0.33**	2.11±0.23**	4.87±0.46*	1.82±0.25**	2.20±0.36*	1.69±0.22**
HPN	50	5.03±0.64**	1.76±0.28*	3.67±0.51	1.43±0.23*	1.59±0.62	0.92±0.13
	75	5.93±0.16**	1.91±0.21**	4.56±0.23*	1.69±0.15**	2.11±0.29*	1.68±0.19**
	100	5.86±0.40**	2.13±0.15**	4.76±0.40*	2.22±0.28**	2.36±0.38*	2.26±0.09**

Each group represents the mean±S.D. ^# p<0.05 vs. control. ^## p<0.01 vs. control. * p<0.05 vs. vehicle. ** p<0.01 vs. vehicle.

DISCUSSION

The novel 2-substituted nitronyl nitroxides with enhanced radical scavenging capacities may be potential drug leads against the deleterious action of ROS or reactive nitrogen species.¹⁸⁾ HPN has been previously shown to confer improved resistance against oxidative insults in vitro. We wished to verify whether it also functioned as an antioxidant in mice. Normobaric hypoxia and hypobaric hypoxia model were used as antioxidant screening model in developing drug against acute high-altitude hypoxia. The present study showed HPN prolonged the mice survive time during normobaric hypoxia test. It also decreased tissue MDA and H₂O₂ under hypobaric hypoxia test. The functional changes in hypobaric hypoxia mice hearts described above were related to intracellular alterations. The mitochondrial structural deformations were most obvious, as revealed by electron microscopic observations of cardiac and brain tissue. After altitude of 8000 m 12 h the heart rate of rats was increased for model group but the SBP, MAP and DBP were decreased on the contrary. These results indicated that the cardiac function impairment occurred after hypobaric hypoxia treatment. Administration of HPN or ACZ significantly decreased these changes compared with model. HPN alleviated brain edema, protetcted mitochodria, mitigated the ultrastructural changes in both “neurovascular unit” and cadiocyte in mice under hypobaric hypoxia treatment. HPN also sustained the activities of SOD, GSH-Px and CAT within a normal range.

ACZ was judged ineffective as a prophylactic for people with increased susceptibility to high-altitude illness at daily doses lower than 750 mg,³²⁾ but the ideal dose is undecided.⁸⁾ So we first determined the ideal effective administration does and route for ACZ as a positive control in hypoxia mouse model. We chose three route of administration including oral administration, intraperitoneal injection and intravenous injection to determine the best effective does with fewer side effects. We found that intraperitoneal injection at 300 mg/kg and intravenous injection at 200 mg/kg for BALB/C mouse had similar effect to prolong the survival time in normobaric hypoxia model. Considering first pass effect in intraperitoneal injection and decomposition in oral administration we finally chose intravenous injection for ACZ at 200 mg/kg and for HPN at 50, 75, 100 mg/kg. We observed an increase in the surviving time resulting from normobaric hypoxia model. We found that prior treatment of HPN had remarkable effect on the survival prolongation rates of mice in normal hypoxia (Table 1).

HPN’s protection against hypoxia-induced H₂O₂ could result from the direct decomposition of H₂O₂ or from a maintenance on the activities of H₂O₂-degrading enzymes. Positive control ACZ exhibited no appreciable degradation of H₂O₂ in cerebrum but upgradation in myocardium (Table 4). MDA, a major oxidation product of membrane peroxidized polyunsaturated fatty acids, is another indicator for oxidative stress.³³⁾ MDA level indirectly reflects the severity degree of the body cells attacked by free radicals. It is one of the indicators to evaluate organism ischemic injury.³⁴⁾ MDA level in hypobaric hypoxia mice had changed significantly, which indicated that lipid peroxidation occurred in early hypobaric hypoxia stage.

Human antioxidant defense system is equipped with enzymatic scavengers, such as SOD, GSH-Px, CAT, hydrophilic scavengers and lipophilic radical-scavengers.³⁵⁾ SOD plays a critical role in protecting mitochondria from superoxide anions generated during respiration. It accelerates the dismutation of superoxide anions into hydrogen peroxide, which can be regarded as a primary defense in preventing the generation of free radicals.³⁶⁾ GSH-Px is another important enzymatic antioxidant enzyme in antioxidant systems. It catalyzes monomeric glutathione and H₂O₂ into glutathione disulfide and water. So the enzymatic activities of these antioxidases are critical for the clearance of free radical. It is beneficial if antioxidase activities could maintain or increase. Preventing mitochondrial ROS generation and scavenging ROS are essential in limiting hypoxia tissue damage. We hypothesized that HPN may have protective effect in mice brains and hearts under hypobaric hypoxia condition. To study the protective role of this antioxidant free radical scavenger in vivo, we used hypobaric hypoxia mice model.²⁵⁾ This model stimulated the hypobaric hypoxia condition in high altitude. In the present study, severe morphological alterations in mitochondrial were first detected as a marker of the deleterious effects of hypobaric hypoxia on mitochondrial function and energy metabolism (Fig. 2). We found that the activities of SOD and GSH-Px were down-regulated in vehicle group after hypobaric hypoxia treatment, which led to superoxide anion-scavenging activity decreasing and oxidative damage to organism. These results partially explained the increase of H₂O₂ and MDA in vehicle group as there were not sufficient cellular antioxidants. Interestingly the activities of SOD, GSH-Px and CAT showed different trends during the hypoxia progress in current study. Unlike SOD and GSH-Px, the activity of CAT was up-regulated. This may attributed to the back donation of increased H₂O₂ in organism.

Cai et al. found the activity of ATP enzyme in hypoxia model group in myocardial cell membrane was decreased and lead to transport disorders of Na⁺, K⁺ and Ca²⁺, intracellular calcium overload and seriously cardiac cells damaged.³⁷⁾ Enhancement of Na/K pump activity by chronic intermittent hypobaric hypoxia protected against reperfusion injury.³⁸⁾ The recovery of Na⁺, K⁺-ATPase activity in the hippocampus was responsible for neuroprotection induced by brain ischemic preconditioning.³⁹⁾ Previous finding also indicated the important role of Na⁺, K⁺-ATPase activity to in cellular neuroprotection.⁴⁰⁾ Under hypoxic conditions, increased release of ROS from the inner mitochondrial membrane to the intermembrane space leads to internalization of the membrane Na⁺, K⁺-ATPase from the basolateral membrane of alveolar epithelial cells.⁴¹⁾ So the mitochondrial Na⁺, K⁺-ATPase activity could be an indication of mitochondrial disfunction. In our work we found the activities of ATPase were down regulated as signs of mitochondrial dysfunction.

We found that hypobaric hypoxia markedly increased myocardium ROS generation thus it also intensified lipid peroxidation. ROS directly lead to mitochondrial dysfunction under oxidative stress. The morphological changed in both cerebrum and myocardium mitochondria showed that alteration of the respiratory function happened during early hypoxia period. HPN protected mitochondria in hypobaric hypoxia treatment by scavenging free radical. These results indicated that in the early stage of AMS the body may already in decompensation accompanied with cellular organelle changes in brain and heart. So it is necessary for the residents to receive preventive treatment when they go to high altitude areas from low altitude areas.

We also tested the antioxidative activities of other 2-substituted phenylnitronyl nitroxides in high-altitude hypoxia mice model. Phenylnitronyl nitroxides such as 4′-ethoxyl-3′-methoxyl-2-substituted phenylnitronyl nitroxide and 4′-hydroxyl-3′-methoxyl-2-substituted phenylnitronyl nitroxide showed toxicity while 4′-hydroxyethyl-2-substituted phenylnitronyl nitroxide showed little effect for the model. So we chose 4-hydroxyl-2-substituted phenylnitronyl nitroxide which showed the best safety and activity as our investigate object. Thus, we can conclude that in our hypoxia mice model HPN was comparable in protect effect to the commonly used antioxidant ACZ or perhaps more effective and less toxic at the same dosage.

In the end, we found that mice administrated with HPN could survive longer than ACZ group in normobaric hypoxia test. We also found that HPN was more effective to clear ROS than ACZ in hypobaric hypoxia mice model. This may partially answer the doubt whether free radical scavenger was effect to AMS. Our results suggested that free radical scavenger administration may be a potential therapeutic way for prophylaxis and therapy of acute high-altitude sickness.

Acknowledgment

We are thankful to the support of Grant 2008ZXJ09014-010 and the support of National Natural Science Youth Foundation 81202458 from National Sci. & Tech. Department, P.R. China. Thanks for the help from professor Wei Zhang (Research Center for High Altitude Medicine, Qinghai University, Xining, China).

REFERENCES

• 1)  Penaloza D, Arias-Stella J. The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic mountain sickness. Circulation, 115, 1132–1146 (2007).
• 2)  Imray C, Wright A, Subudhi A, Roach R. Acute mountain sickness: pathophysiology, prevention, and treatment. Prog. Cardiovasc. Dis., 52, 467–484 (2010).
• 3)  Milledge JS. Acute mountain sickness: pulmonary and cerebral oedema of high altitude. Intensive Care Med., 11, 110–114 (1985).
• 4)  Bärtsch P, Mairbäurl H, Maggiorini M, Swenson ER. Physiological aspects of high-altitude pulmonary edema. J. Appl. Physiol., 98, 1101–1110 (2005).
• 5)  Fagan KA, McMurtry IF, Rodman DM. Role of endothelin-1 in lung disease. Respir. Res., 2, 90–101 (2001).
• 6)  Hackett PH, Roach RC. High altitude cerebral edema. High Alt. Med. Biol., 5, 136–146 (2004).
• 7)  Hackett PH, Roach RC. High-altitude illness. N. Engl. J. Med., 345, 107–114 (2001).
• 8)  Basnyat B, Murdoch DR. High-altitude illness. Lancet, 361, 1967–1974 (2003).
• 9)  Davis PR, Pattinson KT, Mason NP, Richards P, Hillebrandt D. High altitude illness. J. R. Army Med. Corps, 151, 243–249 (2005).
• 10)  Baillie JK, Bates MG, Thompson AA, Waring WS, Partridge RW, Schnopp MF, Simpson A, Gulliver-Sloan F, Maxwell SR, Webb DJ. Endogenous urate production augments plasma antioxidant capacity in healthy lowland subjects exposed to high altitude. Chest, 131, 1473–1478 (2007).
• 11)  Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact., 160, 1–40 (2006).
• 12)  Bailey DM, Davies B. Acute mountain sickness; prophylactic benefits of antioxidant vitamin supplementation at high altitude. High Alt. Med. Biol., 2, 21–29 (2001).
• 13)  Dubey AK, Shankar PR, Upadhyaya D, Deshpande VY. Ginkgo biloba—An appraisal. Kathmandu Univ. Med. J., 2, 225–229 (2004).
• 14)  Akaike T, Yoshida M, Miyamoto Y, Sato K, Kohno M, Sasamoto K, Miyazaki K, Ueda S, Maeda H. Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor. NO through a radical reaction. Biochemistry, 32, 827–832 (1993).
• 15)  Koshland DE Jr. The molecule of the year. Science, 258, 1861 (1992).
• 16)  Haseloff RF, Zöllner S, Kirilyuk IA, Grigor’ev IA, Reszka R, Bernhardt R, Mertsch K, Roloff B, Blasig IE. Superoxide-mediated reduction of the nitroxide group can prevent detection of nitric oxide by nitronyl nitroxides. Free Radic. Res., 26, 7–17 (1997).
• 17)  Blasig IE, Mertsch K, Haseloff RF. Nitronyl nitroxides, a novel group of protective agents against oxidative stress in endothelial cells forming the blood-brain barrier. Neuropharmacology, 43, 1006–1014 (2002).
• 18)  Wu Y, Bi L, Bi W, Li Z, Zhao M, Wang C, Ju J, Peng S. Novel 2-substituted nitronyl nitroxides as free radical scavengers: synthesis, biological evaluation and structure–activity relationship. Bioorg. Med. Chem., 14, 5711–5720 (2006).
• 19)  Zhao M, Li Z, Wu Y, Tang Y-R, Wang C, Zhang Z, Peng S. Studies on logP, retention time and QSAR of 2-substituted phenylnitronyl nitroxides as free radical scavengers. Eur. J. Med. Chem., 42, 955–965 (2007).
• 20)  Kronenberg RS., Cain SM. Effects of acetazolamide on physiologic and subjective responses of men to 14000 feet. SAM-TR-67-81, Tech. Rep. SAM-TR, 1–10 (1967).
• 21)  Forwand SA, Landowne M, Follansbee JN, Hansen JE. Effect of acetazolamide on acute mountain sickness. N. Engl. J. Med., 279, 839–845 (1968).
• 22)  Ma HP, Fan PC, Jing LL, Yao J, He XR, Yang Y, Chen KM, Jia ZP. Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata. J. Ethnopharmacol., 137, 1510–1515 (2011).
• 23)  Teppema LJ, Bijl H, Romberg RR, Dahan A. Antioxidants reverse depression of the hypoxic ventilatory response by acetazolamide in man. J. Physiol., 572, 849–856 (2006).
• 24)  Hu X, Zhou X, He B, Xu C, Wu L, Cui B, Wen H, Lu Z, Jiang H. Minocycline protects against myocardial ischemia and reperfusion injury by inhibiting high mobility group box 1 protein in rats. Eur. J. Pharmacol., 638, 84–89 (2010).
• 25)  Ma HP, Fan PC, Jing LL, Yao J, He XR, Yang Y, Chen KM, Jia ZP. Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata. J. Ethnopharmacol., 137, 1510–1515 (2011).
• 26)  Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem., 86, 271–278 (1978).
• 27)  Deng X, Wu F, Liu Z, Luo M, Li L, Ni Q, Jiao Z, Wu M, Liu Y. The splenic toxicity of water soluble multi-walled carbon nanotubes in mice. Carbon, 47, 1421–1428 (2009).
• 28)  Wang QS, Zhang CL, Zhao XL, Yu SF, Xie KQ. Malondialdehyde and catalase as the serum biomarkers of allyl chloride-induced toxic neuropathy. Toxicology, 227, 36–44 (2006).
• 29)  Schriner SE, Avanesian A, Liu Y, Luesch H, Jafari M. Protection of human cultured cells against oxidative stress by Rhodiola rosea without activation of antioxidant defenses. Free Radic. Biol. Med., 47, 577–584 (2009).
• 30)  Boudina S, Laclau MN, Tariosse L, Daret D, Gouverneur G, Bonoron-Adèle S, Saks VA, Dos Santos P. Alteration of mitochondrial function in a model of chronic ischemia in vivo in rat heart. Am. J. Physiol. Heart Circ. Physiol., 282, H821–H831 (2002).
• 31)  Young RS, Petroff OA, Aquila WJ, Cheung A, Gore JC. Hyperglycemia and the rate of lactic acid accumulation during cerebral ischemia in developing animals: in vivo proton MRS study. Biol. Neonate, 61, 235–242 (1992).
• 32)  Dumont L, Mardirosoff C, Tramèr MR. Efficacy and harm of pharmacological prevention of acute mountain sickness: quantitative systematic review. BMJ, 321, 267–272 (2000).
• 33)  Freeman BA, Crapo JD. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem., 256, 10986–10992 (1981).
• 34)  Cai Y, Lu Y, Chen R, Wei Q, Lu X. Anti-hypoxia activity and related components of Rhodobryum giganteum par. Phytomedicine, 18, 224–229 (2011).
• 35)  Ratnam DV, Ankola DD, Bhardwaj V, Sahana DK, Kumar MN. Role of antioxidants in prophylaxis and therapy: A pharmaceutical perspective. J. Control. Release, 113, 189–207 (2006).
• 36)  Van Remmen H, Williams MD, Guo Z, Estlack L, Yang H, Carlson EJ, Epstein CJ, Huang TT, Richardson A. Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis. Am. J. Physiol. Heart Circ. Physiol., 281, H1422–H1432 (2001).
• 37)  Cai Y, Lu Y, Chen R, Wei Q, Lu X. Anti-hypoxia activity and related components of Rhodobryum giganteum par. Phytomedicine, 18, 224–229 (2011).
• 38)  Guo HC, Guo F, Zhang LN, Zhang R, Chen Q, Li JX, Yin J, Wang YL. Enhancement of Na/K pump activity by chronic intermittent hypobaric hypoxia protected against reperfusion injury. Am. J. Physiol. Heart Circ. Physiol., 300, H2280–H2287 (2011).
• 39)  de Souza Wyse AT, Streck EL, Worm P, Wajner A, Ritter F, Netto CA. Preconditioning prevents the inhibition of Na⁺,K⁺-ATPase activity after brain ischemia. Neurochem. Res., 25, 971–975 (2000).
• 40)  Carletti JV, Deniz BF, Miguel PM, Rojas JJ, Kolling J, Scherer EB, de Souza Wyse AT, Netto CA, Pereira LO. Folic acid prevents behavioral impairment and Na(+), K(+)-ATPase inhibition caused by neonatal hypoxia-ischemia. Neurochem. Res., 37, 1624–1630 (2012).
• 41)  Schumacker PT. Lung cell hypoxia: role of mitochondrial reactive oxygen species signaling in triggering responses. Proc. Am. Thorac. Soc., 8, 477–484 (2011).