Mechanism of Traditional Tibetan Medicine Grubthobrildkr Alleviated Gastric Ulcer Induced by Acute Systemic Hypoxia in Rats

Objective This study was aimed at investigating the potential mechanism of Grubthobrildkr (GTB) on systemic hypoxia-induced gastric ulcers in rats and at detecting the chemical profile of GTB. Methods Male Sprague-Dawley rats were separated into control, hypoxia, hypoxia+omeprazole, and hypoxia+GTBs (0.25, 0.5, and 1.0 g·kg−1·d−1) groups. Systemic hypoxia was created in a hypobaric chamber to simulate 5000 m high altitude by adjusting the inner pressure and oxygen content for 6 days. After that, the ulcer index, pH, and volume of gastric juice were assessed. The levels of endothelin-1 (ET-1), gastrin (GAS), motilin (MTL), phospholipase A2 (PLA2), and prostaglandin E2 (PGE2) were detected by ELISA. The expression level of hydrogen potassium ATPase (H+-K+-ATPase), cyclooxygenase-1 (COX-1), and cyclooxygenase-2 (COX-2) was tested by western blotting. Chemical profile of GTB was revealed by UHPLC-Q-exactive hybrid quadrupole-orbitrap mass (UHPLC-Q-Orbitrap MS). Results GTB decreased the ulcer index in rats under hypoxia for six days, which was related to increased pH and volume of gastric juice, enhanced MTL and PGE2 levels, and decreased ET-1 and PLA2 levels of gastric mucosa. Furthermore, GTB decreased the level of H+-K+-ATPase and COX-2 while increased COX-1 levels in gastric mucosal tissue. 44 constituents were identified by UHPLC-Q-Orbitrap MS in GTB. Conclusion GTB exerted a gastroprotective effect to alleviate gastric ulceration induced by acute systemic hypoxia in rats. The effect of GTB increasing the volume and pH of gastric juice in rats under acute systemic hypoxia could be regulated by gastrointestinal hormones, including MTL and ET-1. Mechanically, gastrointestinal protection of GTB was based on inhibition of the protons pumping H+-K+-ATPase and regulation of prostaglandin family in rats.


Introduction
Some symptoms of digestive system such as peptic ulcer were frequently found in mountaineers and altitude people [1]. Both gastric acid and mucosal ischemia were involved in the etiology of stress ulcers [2]. In general, a physiological balance was maintained between gastric acid secretion and gastric mucosal defense. Mucosal lesions and subsequent gastric ulcers appeared when the balance was disrupted.
The decrease in gastric mucosal protective mechanism can be induced by many factors, including hypoxia [3]. A decrease in gastric mucosal blood flow led to gastric ischemia by destroying the lining of the mucosa, which is closely related to systemic hypoxia. The secretion of gastric acid is regulated by various gastrointestinal hormones, such as gastrin (GAS), motilin (MTL), and endothelin (ET) [4]. These gastrointestinal hormones also influenced the level of intercellular Ca 2+ and eventually activated H + -K + -ATPase. An inhibition of protons pumping H + -K + -ATPase as a means of preventing gastric ulcer has attracted considerable attention for several years [5].
Prostaglandins (PGs) were a family of lipid compounds derived from the arachidonic acid pathway and mediated several physiological functions, including the regulation of inflammation and gastrointestinal protection [6]. PGs were not only found to prevent the formation of ulcers but also improve the healing of the ulcer [7]. According to the reports, the secretion of gastric acid was regulated by PGs, which increased mucosal blood flow and promoted the healing of the mucosa. Enzymes involved in PG synthesis include PLA 2 , which influences the production of arachidonic acid, COX-1, and COX-2. The restoration of PGE 2 to normal levels can reduce gastric mucosa lesions [8,9].
In our previous study, we established a systemic hypoxia-induced gastric ulcer rat model by feeding rats in hypobaric chamber-stimulated altitude of 5000 m for 2, 4, 6, 8, and 10 days, respectively, and the severe gastric ulcer was found in the 6-day hypoxia group [13]. We also found the protective effect of GTB on systemic hypoxia-induced gastric ulcers in rat [14]. However, it remains to elucidate the mechanism of GTB on stress ulcer induced by systemic hypoxia. In this article, we focused on detecting the gastrointestinal protective mechanism of GTB in rats.

Experimental
2.1. Medicine Material and Preparation. GTB was purchased from Qinghai Provincial Tibetan Medical Hospital, the authority in the area on Tibetan medicine, with the batch number of Z20110562. According to the specification, the recommended dosage of GTB for adults was 3.0 g (total raw materials/day). In rat, equivalent dose was about 7 times the human dose. Based on clinical observation of the safety of this medicine, we chose 5, 10, and 20 times the human dose as lower (0.25 g·kg -1 ·d -1 ), middle (0.5 g·kg -1 ·d -1 ), and high dosage (1.0 g·kg -1 ·d -1 ), respectively. Three doses of GTB were suspended in distilled water and administrated by oral gavage for 6 days in this study. Omeprazole (Zhejiang Bohua Chemical Co., Ltd. Batch No. 1410021) at a dosage of 7 mg·kg -1 ·d -1 was used as a positive control medicine. Omeprazole was similarly suspended in distilled water and was mixed vigorously before oral gavage administration.

2.2.
Animal. The study was approved by the Institutional Animal Care and Use Committee of the Qinghai University in accordance with NIH guidelines for the care and use of laboratory animals. Male Sprague-Dawley rats (220-240 g) were obtained from Gansu Traditional Chinese Medicine College, China (certificate of quality: SYXK (甘) 2011-0001). The rats were housed with a 12 h light-dark cycle at 25°C ± 2°C and in a relative humidity of 50%-60%. The rats were fed ad libitum on a diet of standard pellets and water. All possible efforts were made to minimize suffering and reduce the number of rats used. No rat died during the experiment. Sprague-Dawley rats were randomly divided into control, hypoxia, hypoxia+omeprazole, and hypoxia+ GTBs (0.25, 0.5, and 1.0 g·kg -1 ·d -1 ) groups, with each group comprising of 12 rats. The hypoxic groups were exposed in hypobaric chamber (Guizhou Fenglei Aviation Ordnance Co., Ltd. DYC-3000), equal to the parameter in altitude of 5000 m. The rats were deprived of food for 24 h before research time point. Finally, the rats were sacrificed by bleeding from the abdominal aorta under urethane anesthesia (1.0 g·kg -1 ).
2.3. Measurement of pH, Volume of Gastric Juice, and Ulcer Index in Gastric Ulcer Tissue. The gastric secretion from sacrificed rat was gathered. The gastric content was centrifuged at 3000 rpm for 20 min (4°C), the volume of the gastric juice appearing in the supernatant was determined, and the total acidity was tested by pH 211 meter (Mettler Toledo Company). For ulcer index measurement, the stomach of the rat in each group was immediately filled with 5 mL of  Figure 1: Effect of GTB on mean gross lesion index and inhibition rate of gastric mucosal ulcer in rat under acute systemic hypoxia for 6 days. The five groups including hypoxia (H), H+omeprazole, H+GTB 0.25 g/kg, H+GTB 0.5 g/kg, and H+GTB 1 g/kg were induced by systemic hypoxia (rats exposed to hypoxia in hypobaric chamber, equal to the parameter in altitude 5000 m) for 6 days. Each value represents the mean ± S:D: value of eight animals. * P < 0:05 vs. hypoxia group.

BioMed Research International
10% phosphate-buffered formalin (pH 7.0) and submerged in the same solution for 30 min. To evaluate the extent of damage, the gastric sections were opened along the greater curvature and rinsed with normal saline to remove gastric content and blood clot. The degree of gastric mucosal damage was evaluated and rated for gross pathology according to the ulcer score scale described by Dekanski et al. [15] The criteria for assessing macroscopic damage were scored as follows: no ulcer (score = 0), ulcer < 1 mm (score = 1), 1 < ulcer < 2 mm (score = 2), 2 < ulcer < 3 mm (score = 3), and 3 < ulcer < 4 mm (score = 4). The sum of the total score was divided by the number of rats to obtain mean ulcer index for each group. The inhibition percentage was calculated using the following formula: ½ðUI untreated − UI treatedÞ/UI untreated × 100.

Determination of ET-1, GAS, and MTL Level in Blood.
Enzyme-linked immunosorbent assay (ELISA) kits were utilized to measure serumal ET-1, GAS, and plasmic MTL level. The test was performed in accordance with reagent instructions. The kits were obtained from R&D Systems, USA.  . Rats were exposed to hypoxia (in hypobaric chamber, equal to the parameter in altitude 5000 m), hypoxia (H)+omeprazole treatment (7 mg/kg/d), and hypoxia (H)+GTBs-treatment (0.25, 0.5, and 1.0 mg/kg/d) for 6 days. The volume and pH of the gastric juice were detected. Results are expressed as mean ± S:D: # P < 0:05 as compared with the control group; * P < 0:05 as compared with the hypoxia group. 3 BioMed Research International 2 min, 5-95% A for 2-42 min, 95% A for 42-46.9 min, and 5% A for 47-50 min (the equilibration time was 3 min). A Q-exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) included heat electrospray ionization (HESI) and was operated in both positive and negative ion modes to compete MS. The flow rate of sheath gas was 45 arbitrary units with the capillary temperature of 320°C. The auxiliary gas was set up to 15 arbitrary units at 350°C. In both positive and negative modes, the capillary voltage was set to +3.5 or -2.8 kV.
The resolution of the full MS scan was 70,000 with the range of 80-1200 m/z. Samples were analyzed under 20, 30, and 40 normalized collision energy (NCE) in MS2 mode and resolution (17,500). Thermo Xcalibur 3.0 software (Thermo Scientific, San Jose, CA, USA) was used for collection and analysis of data.

Statistical Analysis.
The results were expressed as means ± S:D: Differences between means were analyzed by one-way analysis of variance followed by Dunnett's or Student-Newman-Keuls test. Differences were considered statistically significant at P ≤ 0:05.

The Effect of GTB Treatment on Gastric Acidity, Ulcer
Index, and Volume of Gastric Juice. We found that the gastric mucosal ulcer induced by systemic hypoxia was alleviated by GTB administration. Meanwhile, ulcer index was significantly increased under systemic hypoxia. After administrated by middle and high dosage of GTB and omeprazole, the ulcer index was significantly reduced ( Figure 1). The volume of gastric juice was significantly reduced under systemic , and motilin (c) level in blood in rat under systemic hypoxia for 6 days ( X ± s, n = 12 ). Rats were exposed to hypoxia (in hypobaric chamber, equal to the parameter in altitude 5000 m), hypoxia (H)+omeprazole treatment (7 mg/kg/d), and hypoxia (H)+GTBs-treatment (0.25, 0.5, and 1.0 mg/kg/d) for 6 days. The level of gastrin, endothelin-1, and motilin in blood in rat was detected by enzyme-linked immunosorbent assay (ELISA). Results were expressed as mean ± S:D: # P < 0:05 as compared with the control group; * P < 0:05 as compared with the hypoxia group. hypoxia and was significantly increased after GTB and omeprazole treatment. Compared with hypoxia group, gastric acidity was significantly reduced after treatment with middle and high dosages of GTB and omeprazole ( Figure 2).

The
Effect of GTB Treatment on Level of GAS, ET-1, and MTL. GAS level was not obviously different among omeprazole and experimental groups. The level of ET-1 which was increased under hypoxia was significantly decreased after GTB and omeprazole treatment (P < 0:05). The MTL level had no significant difference between the hypoxia and control groups but was significantly increased after treatment with GTB ( Figure 3).   Figure 5: Effect of GTB on prostaglandin E 2 (PGE 2 ) level in blood (a) and gastric mucosa (b) in rat under systemic hypoxia for 6 days ( X ± s, n = 12). Rats were exposed to hypoxia (in hypobaric chamber, equal to the parameter in altitude 5000 m), hypoxia (H)+omeprazole treatment (7 mg/kg/d), and hypoxia (H)+GTB-treatment (0.25, 0.5, and 1.0 mg/kg/d) for 6 days. The level of PGE 2 in blood and gastric mucosa in rat was detected by enzyme-linked immunosorbent assay (ELISA). Results were expressed as mean ± S:D: # P < 0:05 as compared with the control group; * P < 0:05 as compared with the hypoxia group. 5 BioMed Research International decreased after GTB treatment (P < 0:05) (Figure 4). We found that the level of PGE 2 which was decreased in gastric mucosal tissue was increased in serum under systemic hypoxia. After treatment with GTB, the level of PGE 2 in serum and gastric mucosal tissue was both significantly increased compared with the hypoxia group (P < 0:05) ( Figure 5). 3.6. Identification of the Compounds in GTB Using UHPLC-Q-Exactive Hybrid Quadrupole-Orbitrap Mass. The total spectrum of chemical components in GTB aqueous extract was analyzed from both positive and negative ion models. 44 chemical components were identified by UHPLC-Q-Orbitrap MS analysis (Figure 9). It showed the characters of all 44 chemical constituents including chromatographic retention times, accurate molecular mass, and/or MS/MS data listed in Table 1. Among these, the peaks of 10, 11 12, 16, and 37 were identified as magnoflorine, boldine, phellodendrine, berberrubine, and dehydrocostus lactone, respectively, according to the data comparison with reference standards. Peak

Discussion
Acute gastric mucosal lesion was life-threatening at high altitude where gastric mucosal balance may be disrupted. It was found that blood flow to the gastric mucosa decreased because of systemic hypoxia affecting the physiological balance between gastric acid secretion and gastric mucosal defense. Provided changes in the gastrointestinal tissue during hypoxia are explored, which should be intervened by medicines, especially by traditional medicines. With a history going back approximately 2,500 years, Tibetan medicine is considered one of the world's oldest known traditional medicines [19]. Several traditional Tibetan medicines had been used to treat gastric diseases with obvious effect, and Grubthobrildkr is one of the classic Tibetan medicines to treat gastric problems. According to the reports, GTB attenuated acetic acid-induced gastric ulcer through reducing the expression of COX-2 and inflammatory reaction [20]. GTB also alleviated stress gastric ulcer induced by water immersion and pylorus ligature in rat [21]. Although GTB had been used for centuries as an effective and safe prescription for gastric disease treatment, its mechanism in the treatment of acute stress gastric ulcer under systemic hypoxia needs to be researched. In our previous study, we established systemic hypoxia-induced gastric ulcer rat model in 2, 4, 6, 8, and 10 days, respectively, and we observed that the severe gastric ulcer was in the 6-day hypoxia group [13]. The protective effect of GTB was also detected by hematoxylin and eosin staining and ultrastructural observation in systemic hypoxia-induced gastric ulcer rat model for 6 days [14]. In this article, we focused on detecting the gastrointestinal protective mechanism of GTB in rats.
The volume of gastric juice was significantly reduced, and total gastric acidity and ulcer index were significantly increased under systemic hypoxia in rat. The gastric ulcer index was reduced and pH of gastric juice and gastric secretion volume in rat were increased after GTB administration. GAS was from G cells of pyloric antrum for gastric acid secretion, and we found that GAS levels were not changed under systemic hypoxia for six days. MTL has been identified in the blood of dogs by means of radioimmunoassay [22], with function of stimulating pepsin output and enhancing activity of the stomach [23]. We found that MTL levels were not influenced by systemic hypoxia for six days in rats. ET-1 was one of the proinflammatory cytokines for the contraction of blood vessels, playing an important role in gastric ulcer formation. The increasing secretion of ET-1 results in the occurrence of hypoxia, acidosis, and ulcers. We found that ET-1 level was increased under hypoxia. GTB administration decreased ET-1 level and increased the level of MTL in the blood significantly compared with the hypoxia group but GAS level was not influenced. The results could be explained that the effects of GTB increasing pH of gastric juice and gastric secretion volume were mainly regulated by ET-1 and MTL levels. GAS, MTL, and ET-1 were all found to influence the level of intercellular Ca 2+ and eventually activated H + -K + -ATPase [24]. H + -K + -ATPase are responsible for secreting acid into the gastric lumen, which catalyzes the exchange of one H + for one K + at the expense of an ATP molecule [25]. We found that the H + -K + -ATPase Prostaglandins (PGs), targets for the prophylactic effect of probiotics in gastric ulcers [23], were participant in the ulcer healing process by decreasing acid secretion, stimulating the production of mucus, bicarbonate, and phospholipids [26]. Enzymes involved in PGs synthesis include PLA 2 , which influenced the production of arachidonic acid, COX-1, and COX-2. PGE 2 is a member of PGs, the restoration of which can reduce gastric mucosa lesions [8,9]. GTB treatment reduced PLA 2 level both in serum and in gastric tissue in rat under systemic hypoxia. Although the level of PGE 2 in serum was increased in the six-day hypoxia group, GTB treatment increased PGE 2 level both in the serum and in gastric tissue. Based on the dual contribution of PGs to inflammation and mucosal defense, the increased PGE 2 level after GTB administration could be deduced to play a protective role in ulcer lesions under systemic hypoxia.
COX-1 was a house-keeping enzyme that produces cytoprotective PGs, while COX-2 was an inducible form of the enzyme that produces inflammatory PGs. The protein expression of COX-1 was found to be reduced, but COX-2 was increased under acute systemic hypoxia. GTB treatment was found to increase the protein expression level of COX-1 and decrease that of COX-2 in gastric tissue in rat. 44 constituents in Grubthobrildkr were identified by UHPLC-Q-Orbitrap MS. To the best of our knowledge, we did not find articles which report the relationship between the 44 ingredients and the treatment of gastric ulcer.

Conclusion
Traditional Tibetan patent medicine Grubthobrildkr showed a protective effect and alleviated the ulceration in gastric mucosa under systemic hypoxia. The effect of GTB increasing volume and pH of gastric juice in rat under acute systemic hypoxia could be regulated by MTL and ET-1. The molecular mechanism of GTB might be related to reduction of H+-K+-ATPase protein expression and regulation of prostaglandin family by downregulating COX-2 expression and upregulating COX-1 protein expression in the gastric mucosa of rats under systemic hypoxia. 44 constituents in GTB were identified by UHPLC-Q-TOF-MS/MS. Furthermore, comprehensive studies are needed to elucidate the gastroprotective mechanism of GTB.

Data Availability
The data used to support the findings of this study are included within the article.

Ethical Approval
The study was approved by the Institutional Animal Care and Use Committee of Qinghai University in accordance with NIH guidelines for the care and use of laboratory animals.

Conflicts of Interest
We declare that we have no conflict of interest.

Authors' Contributions
Mei Yang and Zhanting Yang contributed equally to the work and should be considered co-first authors.