3.1. NBP could improve hypoxia tolerance ability of rats and mice
To investigate whether NBP could benefit animals in hypobaric hypoxia, we firstly studied the effect of NBP on the survival time at 10,000 m exposure with 30 min as stopped time. The survival curve of experiments showed that 120 and 240 mg/kg NBP administration could significantly improve the survival time of rats instead of mouse (Figure 4A, B). Compared to control group (rat 100%, mouse 81.25%), the death percent at 30 min in 120 (90%) and 240 mg/kg group (80%) for rats and 180 mg/kg group (62.5%) for mice was declined (Figure 4C, D). Moreover, the standard tolerance time with closed hypoxia (control group, 12.18±2 min/100ml·g, 360 mg/kg group, 13.94±1.54 min/100ml·g, p=0.046) (Figure S1) of mice also markedly extended by 360 mg/kg NBP administration for day 5. The results indicated that NBP could improve the ability of hypoxia tolerance of rats and mice.
3.2. NBP improved physical ability under acute and chronic hypoxia
As hypobaric hypoxia is known to impair physical and cognitive functions in humans, we studied the role of NBP on the physical and cognitive behaviors of animals. The physical abilities of rats and mice were evaluated through treadmill running and motor-driven wheel-track treadmill experiments.
Under acute hypoxic conditions, NBP treatment at 120 and 240 mg/kg concentrations significantly increased the exhausted time of rats (control group, 38.28±17.85 min, 120 mg/kg group, 61.8±19.3 min, p=0.027, 240 mg/kg group, 73.26±26.89 min, p=0.001), and 90 mg/kg NBP dose significantly improved the exhausted time (control group, 6.79±8.72 min, 90 mg/kg group, 16.79±14 min, p=0.02) and distance (control group, 101.62±130.78 m, 90 mg/kg group, 251.49±210.07 m, p=0.02) for mice (Figure 5). Moreover, NBP at 60, 120, and 240 mg/kg concentrations could significantly increase the exhausted time for rats under chronic hypoxia (control group, 38.59±16.83 min, 60 mg/kg group, 66.83±28.1 min, p=0.024, 120 mg/kg group, 75±32.34 min, p=0.008, 240 mg/kg group, 79.1±26.33 min, p=0.001) (Figure 6). These results suggest that NBP may improve the physical ability of animals under acute and chronic hypoxia.
To clarify the mechanism underlying the NBP-mediated acceleration in physical activity under acute and chronic hypoxic conditions, we evaluated the levels of MDA, SOD, H2O2, lactate, and GSH-Px in the blood and ATP in the gastrocnemius muscle of rats. The levels of MDA (control group, 38.83±19.59 nmol/ml, 60 mg/kg group, 16.89±14.48 nmol/ml, p=0.008, 120 mg/kg group, 9.98±6.95 nmol/ml, p=0.0001, 240 mg/kg group, 11.11±9.17 nmol/ml, p=0.0003) and H2O2 (control group, 67.84±36.43 mmol/L, 120 mg/kg group, 42.44±12.26 mmol/L, p=0.042) decreased but that of SOD (control group, 148.47±24.8 U/ml, 60 mg/kg group, 169.35±20.92 U/ml, p=0.041, 120 mg/kg group, 173.78±29.33 U/ml, p=0.036) increased under acute hypoxia following treatment with various doses of NBP (Figure 7B–D). Under chronic hypoxia, levels of MDA (control group, 8.84±2.06 nmol/ml, 120 mg/kg group, 5.88±1.75 nmol/ml, p=0.002, 240 mg/kg group, 4.86±1.27 nmol/ml, p=0.00003) and H2O2 (control group, 351.13±111.57 mmol/L, 120 mg/kg group, 245.28±76.66 mmol/L, p=0.024) decreased, but GSH-Px expression was upregulated (control group, 457.01±326.03 U/mg protein, 120 mg/kg group, 976.35±462.23 U/mg protein, p=0.031, 240 mg/kg group, 1210.58±294.57 U/mg protein, p=0.001) (Figure 8B, D, F). Thus, NBP may increase the antioxidant capacity and exert opposite effects on oxidant capacity. The content of ATP (control group, 686.55±129.51 μmol/g protein, 60 mg/kg group, 1228.51±364.34 μmol/g protein, p=0.031) significantly increased under chronic hypoxia (Figure 8A) and lactate level significantly decreased under acute (control group, 32.79±8.63 mmol/L, 240 mg/kg group, 16.83±13.14 mmol/L, p=0.002) and chronic hypoxia (control group, 23.5±13.03 mmol/L, 60 mg/kg group, 13.38±5.15 mmol/L, p=0.028, 120 mg/kg group, 8.9±5.01 mmol/L, p=0.004, 240 mg/kg group, 12.48±8.65 mmol/L, p=0.047) (Figure 7E and 8E). These observations suggest NBP promoted ATP production via oxidative phosphorylation instead of glycolysis. NBP at 240 mg/kg dose significantly decreased red blood cell (p=0.01), hemoglobin (p=0.004), and platelet counts (p=0.002) as well as hematocrit level (p=0.028) under acute hypoxia (Table S1). NBP significantly increase white blood cell count at 60 mg/kg concentration (p=0.047) and decreased platelet count at 120 mg/kg concentration (p=0.042) under chronic hypoxia (Table S2).
3.3. NBP improved learning and memory ability under acute and chronic hypoxia
The cognition functions of rats were evaluated through the shuttle-box experiment. Under acute hypoxia, NBP at 240 mg/kg doses could significantly increase times of active escape (control group, 1.44±1.5 s, 240 mg/kg group, 3.13±2.17 s, p=0.035) (Figure 9). However, NBP had no effect on average time of active escape. The beneficial effect of NBP on learning and memory function was not as consistent as that on physical activity. Furthermore, MDA, SOD, H2O2, and GSH-Px levels were analyzed in animal blood samples. The level of H2O2 (control group, 155.7±89.57 mmol/L, 60 mg/kg group, 77.61±37.14 mmol/L, p=0.041, 120 mg/kg group, 75.59±33.71 mmol/L, p=0.035, 240 mg/kg group, 74.64±35.19 mmol/L, p=0.046) decreased and the expression of GSH-Px (control group, 5999.26±659.23 U/mg protein, 240 mg/kg group, 6704.55±451.45 U/mg protein, p=0.033) was upregulated under acute hypoxia (Figure 11C–D). Under chronic hypoxia, the levels of MDA (control group, 11.51±5.71 nmol/ml, 120 mg/kg group, 7.05±1.47 nmol/ml, p=0.028) and H2O2 (control group, 274.27±78.84 mmol/L, 120 mg/kg group, 200.67±47.33 mmol/L, p=0.029) decreased (Figure 12A, C). Thus, NBP could also increase the antioxidant capacity and decrease the oxidant capacity in animals.