2.1. Experimental animals and NBP administration
Male pathogen-free Sprague-Dawley (SD) rats (6 to 8 weeks old, weighing 180–220 g) and male pathogen-free Kunming (KM) mice (6 weeks old, weighting 18–20 g) were used in this study. Rats and mice were obtained from the Laboratory Animal Center of Army Medical University, and the animal study protocol was approved by the Animal Care and Use Committee Guidelines of the Army Medical University. NBP (purity, 99.6%) was obtained from Shijiazhuang Pharma Group NBP Pharmaceutical Co., Ltd (Shijiazhuang, Hebei, China). Animals were randomly assigned to four experimental groups as follows: control group (n = 10), NBP low dose-treated group (60 mg/kg for rats and 90 mg/kg for mice, n = 10), NBP intermediate dose-treated group (120 mg/kg for rats and 180 mg/kg for mice, n = 10), and NBP high dose-treated group (240 mg/kg for rats and 360 mg/kg for mice, n = 10). NBP was intragastrically administered once every day. Animals had free access to food and water.
2.2. Survival analysis in 10,000 m hypobaric hypoxia chamber
NBP were given to SD rats and KM mice by intragastric administration at 1 ml/100 g body weight for 7 days. After 1.14 hours from last administration, animals were placed in hypobaric hypoxia chamber. The high altitude of chamber ascended to 10,000 m by around 1,000 m/min. The survival of animals were observed and recorded and the experiment stopped at 30 min in 10,000 m.
2.3. Standard hypoxia tolerance time of mice
NBP were given to KM mice by intragastric administration for 3, 5, or 7 days. After 1.14 hours from last administration, each mouse was put into a bottle with a nominal volume size of 125 ml, and 5 g of soda lime, respectively, was added to absorb the carbon dioxide and water vapor produced by breathing. Then, the lid was tightly sealed until the mouse’s breathing movement stopped. The ST, i.e., the time from when the mouse was sealed in the bottle to its death, was recorded. STT was calculated according to formulas .
STT = ST/(V-BW/0.94)
STT: standard hypoxia tolerance time (min/ml), ST: survival time (min), V: bottle volume (ml), BW: body weight of mice (g).
2.4. Gasping duration time of mice after decapitation
NBP were given to KM mice by intragastric administration for 7 days, twice per day. Mice were made to fast with free access to drinking water 12 h before the experiment. After 1.14 hours from last administration, the mice were decapitated, and the gasping duration of the isolated head was determined.
2.5. Treadmill running experiment for acute hypoxic rats
NBP was intragastrically administered to SD rats for 3 days at low altitude. Rats were then subjected to treadmill exercise everyday as follows: 5 min for adaptation, followed by 15 m/min for 10 min and 20 m/min for 20 min. On day 4, rats were placed in a 5,800 m hypobaric hypoxia chamber, administrated NBP and subjected to the above exercise regimen for 3 days. On day 7, exhaustive exercise was conducted by a treadmill running experiment. The experimental plan included 5 min for adaptation, followed by 15 m/min for 3 min, 20 m/min for 3 min, 25 m/min for 30 min, and then 30 m/min up to exhaustive status. Rats were anaesthetized and their arterial blood was collected for the analysis of MDA, SOD, H2O2, lactate, and GSH-Px levels as well as to perform routine blood tests. In addition, the gastrocnemius muscle tissues was excised and used for ATP detection. MDA, SOD, H2O2, lactate, and GSH-Px levels were detected by Nanjing Jiancheng assay agents (A003-1, A001-3, A064-1-1, A020-1-2, and A005, respectively). ATP level was analyzed by a Beyotime ATP assay kit (S0026). Blood routine tests were performed at the Xinqiao Hospital, Chongqing, China.
2.6. Forced exercise wheel-track treadmill experiments for mice
KM mice were intragastrically treated with NBP twice per day for 7 days at low altitude. On day 4, mice were exercised on a wheel-track treadmill (YLS-10B, Shandong Academy of Medical Sciences, Jinan, China) for 3 days under following conditions: 10 min/day, 0.8 mA for motor current limitation, 3 s for maximum electronic shock time, 30 s rest after electronic shock, and five times rest in 10 min as exhaustive standard. On the day 7, mice were placed in 5,000 m hypobaric hypoxia chamber and treated with NBP after 30 min. Following 1.14 h, exhaustive exercise was conducted through wheel-track treadmill running experiment and exhaustive time was recorded.
2.7. Treadmill running experiment for chronic hypoxic rats
Rats were placed in 5,800 m hypobaric hypoxia chamber and intragastrically administered with NBP for 14 days from day 8. Rats were exercised on treadmill as above for 3 days from day 18. On day 21, exhaustive exercise was conducted in treadmill running experiment. At exhaustive status, rats were anaesthetized and their arterial blood samples were obtained to analyze MDA, SOD, H2O2, lactate, and GSH-Px levels and perform routine blood test. Further, gastrocnemius muscle was excised for ATP detection.
2.8. Shuttle-box experiment for acute hypoxic rats
NBP was intragastrically administered to SD rats for 7 days in 5,800 m hypobaric hypoxia chamber. On day 4, rats were subjected to an exercise regimen in a shutter box for 3 days under following conditions: 2.2 mA of current, 10 s interval, 10 s rest for sound and light stimuli, and 10 s for electronic stimulus (50 times every day). On day 7, the cognition ability of rats was investigated through the shuttle-box experiment in 5,800 m hypobaric hypoxia chamber. Rats were then anaesthetized and their arterial blood was collected to measure MDA, SOD, H2O2, and GSH-Px levels.
2.9. Shuttle-box experiment for chronic hypoxic rats
Rats were placed in 5,800 m hypobaric hypoxia chamber and administered with NBP via intragastric route for 21 days from day 8. From day 25, rats were subjected to the exercise regimen mentioned above for 3 days. On day 28, cognition ability was studied through the shuttle-box experiment. At the end of the experiment, rats were anaesthetized and arterial blood was collected for the analysis of MDA, SOD, H2O2, and GSH-Px levels.
2.10. Statistical analysis
Statistical analysis was carried out using the SPSS 19.0 software. For survival analysis, a Kaplan-Meier curve was generated using the log-rank test. Other experiments were analyzed using an independent-samples t test. A value of p < 0.05 was considered statistically significant.