刘军彩1，谢立群1，陈金梅1，李俊美2，窦勇鹰1，海鸥1，李轩1
(1.武警医学院附属医院消化科，天津 300162：2.天津市第一中心医院，天津300192)
摘 要 【目的】探讨 PAR-2激动剂在大鼠应激性溃疡(stress ulcer, SU)中的作用。【方法】大鼠水浸束缚应激后, 肉眼计算胃黏膜溃疡指数(ulcer index, UI); 取动脉血和胃液做血气分析计算胃黏膜内pH值; 采用放射免疫方法检测内皮素（endothelin，ET），采用分光光度计检测一氧化氮（nitrogen monoxidem, NO）; 观察胃组织病理形态学变化.【结果】（1）PAR-2激动剂组与阴性对照组比较, pHi值、NO、氨基己糖水平显著升高(P<0.05), 而ET、UI显著降低(P<0.01)。病理形态学观察, 血栓形成减少。（2）PAR-2激动剂组与奥美拉唑组比较, 除ET显著降低外, 其他数据无统计学差异。（3）PAR-2激动剂组与辣椒素组比较，Phi、氨基己糖和NO显著升高，而ET则明显下降。【结论】PAR-2激动剂通过舒张血管, 增加胃黏膜血流量、促进粘液分泌起到保护胃黏膜的作用, 其机制可能与激活辣椒素敏感性神经元有关。
关键词：PAR-2受体；胃黏膜；辣椒素；血管收缩；血管扩张
The role of PAR-2 agonist in stress ulcer formation in Rats
LIU Jun-cai, XIE Li-qun, CHEN Jin-mei, LI Jun-mei, DOU Yong-ying, HAI Ou, LI-Xuan（Department of Gastroenterology, The Affiliated Hospital of Medical College of Chinese People's Armed Police Forces, Tianjin 300162,China）
Abstract ：【Objective】To investigate the protective effect of PAR-2 agonist in stress ulcer.【Methods】Ulcer index(UI) was observed after water immersion-restraint stressed (WRS); endothelin (ET) was measured by radioimmunoassay; NO（nitrogen monoxidu）and aminohexose were detected by spectrophotometer, and histopathology was analyzed.【Results】（1）Compared with the negative control group, PAR-2 agonist group revealed great increase in the level of pHi,
aminohexose and NO (P<0.01), but remarkable decrease in the level of ET and UI (P<0.01). Besides, the risk of thrombosis of PAR-2 agonist group was reduced. （2）There were no statistical differences between omeprazole group and PAR-2 agonist group except the obvious drop of ET
level in the latter group. （3）The level of pHi, aminohexose and NO were far higher while the ET level was quite lower in PAR-2 agonist group than that in capsaicin group.【Conclusion】Vasodilatation , gastric mucosal blood flow and gastric mucus secretion are stimulated by the
[作者简介] 刘军彩（1976-），女，河南省西平县人，本科主治医师，主要从事消化内科临床工作及应激性溃疡的发病机制研究。
[通讯作者] 谢立群（1966-），男，籍贯江西，副教授，博士，硕士研究生导师，主要从事应激性溃疡及消化道肿瘤早期诊断、治疗等方面研究。E-mail: xieliqun66@hotmail.com.
pretreatment with Protease-activated receptor-2 (PAR-2) agonist before WRS. And the protective effect of PAR-2 agonist in stress ulcer formation may be achieved by activating capsaicin sensitive neuron.
Key words: PAR-2 receptor; Gastric mucosa; Capsaicin; Vasodilatation; Vasoconstriction
1 Introduction
PAR-2 [1] is a G-protein-coupled, seven-trans-membrane-domain receptor activated by certain endogenous proteases including trypsin, tryptase and coagulation factors VIIa and Xa, and also possibly by some unknown proteases [2]. Trypsin and mast cell tryptase are considered as endogenous activators for PAR-2[3].Synthetic peptides based on the receptor-activating sequence of the tethered ligand are also capable of activating PAR-2 by direct binding to the receptor [3]. PAR-2 is unevenly distributed throughout the mammalian body, especially in the alimentary tract [3]. PAR-2 modulates gastrointestinal motility in vitro as well as in vivo [4]. It also participates in the regulation of alimentary exocrine secretion such as salivary and pancreatic secretion [5]. Recently, PAR-2 agonist, given in vivo, induced gastric mucus secretion accompanied by mucosal cytoprotection, via the release of calcitonin gene related peptide (CGRP) and tachykinins from sensory neurons [6]. We hereby reveal the difference of pretreating with PAR-2 agonist, Omeprazole and capsaicine when used to prevent stress ulcer.
2 Materials and methods
2.1 Animals and treatment
Male Wistar rats (200-250g) of both control and experimental groups kept separately in controlled condition were fasted for 24 h with water ad libitum. The negative control group received saline (1ml) only 30 minutes before WRS. While the experiment group received omeprazole or PAR-2 agonist (Calbiochem Company ,America) at 1.5μmol/Kg or Capsaicin (Fluka company, America) at 25, 50, 50mg/Kg intravenously.
2.2 Reagents
The following chemicals were used: PAR-2 agonist (2-Furoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2) was purchased from Calbiochem Company; capsaicin was provided by Fluka Company, and dissolved in a solution containing 10% ethanol, 10% Tween 80, and 80% saline; aminohexose was provided by the Third Hospital of Beijing Medical University.
2.3 Methods
2.3.1 Animals and treatment: 40 Wistar rats were divided randomly into 5 groups: ①negative control group (n=8) ②Omeprazole group (n=8) ③PAR-2 agonist group (n=8) ④capsaicin group (n=8) ⑤non WRS (water-immersion restraint stress) (n=8). Stress ulcer was induced by water-immersion restraint stress in the experimental groups. Rats were deprived of food for 24 hours and water for 2 hours before the experiments. Normal saline (1 ml) was administered ig 30 minutes before WRS to rats of negative control group; PAR-2 agonist (1.5 μmol/Kg, ig) to PAR-2 agonist group rats 30 minutes before; Capsaicin (25，50，50 mg/Kg, sc) to capsaicin group rats at 0,6,32 hours, and PAR-2 agonist (1.5 μmol/Kg, ig) 30 minutes before WRS after having been bred for 14 days .
2.3.2 Preparation of ulcer model: Before each experiment, rats were deprived of food but not water for 24 h. Then they were placed in a restraint cage contenting water and immersed up to a level of water the xiphoid process at 22℃ [7].
2.3.3 Preparation of gastric juice and blood sample: Immediately after that, all the rats used in this study were anesthetized in their abdominal cavities with 0.9 ml of 100 g/Kg sodium after treatment and there abdomens were opened for the observation on the changes of their gastric mucosa and exsanguinated via the abdominal aorta for blood sample. Blood samples were stored at -80℃ for detection.
2.3.4 Restraint Cold Stress-induced Gastric Ulceration
Before each experiment, rats were deprived of food but not water for 24 h. Then they were placed in a restraint cage contenting water and immersed up to the level of xiphoid process at 22℃ as described [7]. The severity of mucosal lesions was scored according to Guth′s method[8], the length of gastric mucosal damage area ＜1mm was scored 1 point; ≤2 mm 2 points;≤3mm 3 points; ≤4mm 4 points, ＞4mm scored 5 points. The sum of the total scores divided by the number of animals was expressed as the mean ulcer index.
2.3.5 Measurement of ET and NO
ET in the serum was analyzed by radioimmunology using the ET assay kit ( Nanjing Jiancheng Bioengineering Institute, China). NO was detected by colorimetric method using NO assay kit ( Nanjing Jiancheng Bioengineering Institute, China).
2.3.6 Measurement of pHi level
The PHi was calculated from the following equation: pHi= 6.1 +log (HCO3-/PCO2×
0.0307). In this calculation, the PHi are determined from the PCO2 in gastric cavity and HCO3- in blood of artery with the Henderson-Hasselbalch equation.
2.3.7 Measurement of aminohexose
The aminohexose in gastric mucosa was measured by Neuhaus method [9].
2.3.8 Histopathology of thrombus
The tissue blocks of rat stomach were fixed in 10% formalin for 48 h, HE stained after dehydration and embedding.
2.3.9 Statistical analysis: Values are summarized as means ± SE. Significant differences between values were determined with Student's t-test for paired or unpaired data. The P-value of differences between single subgroups was calculated with the Least Significance Difference post hoc test (LSD test). In all analyses, differences were considered statistically significant when P < 0.05.
3 Results
3.1 Macroscopic observation
Macroscopic observation showed lesions, most 2~3 mm in size, or petechial bleeding. The area of involvement was confined to the glandular part of the stomach. In contrast, PAR-2 agonist group and Omeprazole group rats had fewer lesions, as shown in Table 1.
3.2 Histopathology of thrombus
Endothelial cells of negative control group and capsaicin group rats were involved, which led to the discontinuance of gastric mucosa. The framework of glandular organ was deranged, and gaps were enlarged. There were thrombus in blood vessels. However, there were no thrombus in the blood vessels of both PAR-2 agonist group and Omeprazole group rats, but many akaryocyte aggregated, as shown in Figure 1~3.
Fig .1：Thrombus formation in negative control group rats（HE×40）
Arrow shows the thrombus formation.
Fig. 2：No thrombus formation in PAR-2 agonist group rats（HE×100）
Arrow shows the akaryocyte aggregation.
Fig .3：Thrombus formation in Omeprazole group rats （HE×100）
Arrow shows the akaryocyte aggregation.
Table 1 The content of UI, pHi, aminohexose, NO and ET (n=8, 
±S)
|
|
UI |
pHi |
aminohexose（mg/g） |
NO （mmol/g） |
ET（pg/ml） |
|
negative control group |
69.00±33.27 |
4.53±0.11 |
1.19±0.58 |
52.50±9.60 |
98.18±39.24 |
|
Omeprazole group |
26.88±14.13① |
4.72±0.47① |
5.20±2.69① |
54.06±10.65 |
77.46±17.11① |
|
PAR-2 agonist group |
37.50±24.42① |
4.71±0.23① |
5.26±1.48① |
62.20±6.91① |
48.53±8.26① |
|
capsaicin group |
59.00±12.46② |
4.43±0. 63② |
2.13±1.39② |
49.88±7.00② |
70.04±8.76② |
|
non WRS group |
|
4.94±0.22② |
6.21±2.67② |
39.44±8.97 |
32.35±4.11②② |
①：compared with negative control group，P<0.05; ②：compared with PAR-2 agonist group，P<0.05
4 Discussion
Protease-activated receptors (PARs) are a novel family of G-protein-coupled-seven-trans-
membrane-domain receptors, currently consisting of four family members [10]. Among them, PAR-1, PAR-3 and PAR-4 are thrombin receptors [1, 11-13], while PAR-2 is a receptor for trypsin, mast cell tryptase, coagulation factors VIIa and Xa, and possibly unknown proteinases [10]. Activation of PARs is achieved by proteolytic unmasking of the N-terminal cryptic receptor-activating sequence, which subsequently binds to the body of the receptor as a tethered ligand. The common signaling pathway following activation of distinct PARs is triggered by activation of phospholipase C via Gq/11 protein, although each PAR may also activate other distinct signal transduction pathways [2, 10]. The mRNAs of PAR-2 are abundantly expressed in the gastric mucosa [14]. Recent studies have suggested that the PAR-2 agonist, given systemically, exerts gastric mucosal protection by activating capsaicin-sensitive sensory neurons in distinct rat gastric mucosa injury models [15]. Few articles note whether there are differences between pretreatment with PAR-2 agonist and Omeprazole. Our experiment were to detect the agents of vasodilatation and vasoconstriction after pretreatment with PAR-2 agonist compared with Omeprazole and to observe the effect of PAR-2 agonist and capsaicin in precaution stress ulcer.
The mucus-bicarbonate barrier of stomach is one of the main factors of the mucosal defensive mechanism. The gastric mucosa is covered by mucus, which protects the mucosa away from mechanical catalyze of food and slows the diffusion speed of H+ in mucus. The 95% component of mucus is water, and other components are polyglycoprotein, galactose, N-kharophengalatose and fucose. Measurement of aminohexose can reflect the content of glycoprotein. Our results showed that the aminohexose level of PAR-2 agonist group rats was significantly higher than that of negative control group and capsaicin group rats. It indicates that PAR-2 agonist can acilitate gastric mucosa to secrete mucus. PAR-2 in mucosal sensory neurons, once activated appears to induce the release of CGRP and neurokinin A, which trigger mucus secretion via CGRP1 and NK2 receptors, respectively, resulting in mucosal cytoprotection[15].
Nitric oxide (NO) is a free radical constantly produced/released throughout the body by diverse tissues, i.e, endothelium [16]. Therefore, it presumably is part of numerous physiological processes and pathways. Yet, the role of NO in stress-related diseases represents an interesting field of investigation, since NO is involved in the stress physiology and apparently also takes part in stress-related disease processes. Hence, the role of NO in specific stress-related diseases will be discussed. NO is a potent visceral vessel expander. It can augment viscera blood flow, and protect gastric mucosa from lesion. Our experiment showed that NO level of PAR-2 agonist group was remarkably higher compared with that of negative control group and capsaicin group. It suggests that PAR-2 agonist can induce NO secretion, which can relax blood vessel and augment gastric mucosal blood flow.
ET is a short chain polypeptide constructed by 21 amino acid residue, which is secreted by blood vessel endothelial cells. ET can induce gastric mucosa lesion by constructing blood vessel and lessening circulating blood volume and gastric mucosal blood flow. Our results showed that ET level of PAR-2 agonist group rats was distinctly lower than that of negative control group rats. Our data indicate that PAR-2 agonist may be able to inhibit ET secretion and prevent blood vessel contraction.
pHi is the main index to detect the function of the gastrointestinal tract, and it can sensitively reflect stomach intestine hypoxia. The energy used to maintain internal environment and exercise functions comes from disassemble of ATP, namely ATP→ADP+Pi+H+ .After the organism suffered from wound, hemorrhagic and infection et al, the oxygen delivery to histiocyte is insufficient, and it can lead to the composition of ATP fewer than disassemble. Thus, it can produce the redundancy of H+ and the decrease of pHi. More severe the framework hypoxia is, more significantly the pHi decreases. Therefore, pHi provides the evidence whether apparatus organize is oxygenated or not. Our results showed that the pHi level was remarkably decreased in negative control group rats. The mechanism may be gastric mucosal artery contraction, which leads to mucosal ischemia and hypoxia and the composition of ATP less than disassembled. The pHi level of PAR-2 agonist group rats showed no statistical difference compared with Omeprazole group rats. These data indicate that both PAR-2 agonist and Omeprazole have the same effect in improving the organism hypoxia. PAR-2 agonist might have the ability in dilating blood vessel and augmenting framework blood and oxygen supply by inhibiting ET secretion and promoting NO secretion. According to the histopathology of the stomach, the blood vessels caliber of PAR-2 agonist group rats were found to be broadened, in which there were erythrocytic aggregation, so it also suggests that PAR-2 agonist may have the ability in dilating blood vessel. Omeprazole is the classic proton pump inhibitor, and it can protect mucus-bicarbonate barrier by inhibiting gastric acid secretion. In our experiment, Omeprazole could also reduce the depletion of gastric mucus, which may be relevant to slime layer thickness and H+ density. Gastric histopathology showed that there were erythrocytic aggregation in blood vessel of Omeprazole group rats, but no thrombus formation. However, thrombus formation appeared in negative control group rats. We deduce that PAR-2 agonist has the similar function to Omeprazole in prevention and treatment of stress ulcer.
In conclusion, Vasodilatation , gastric mucosal blood flow and gastric mucus secretion are
stimulated by the pretreatment with Protease-activated receptor-2 (PAR-2) agonist before WRS. And the protective effect of PAR-2 agonist in stress ulcer formation may be achieved by activating capsaicin sensitive neuron. It may be a potential drug for the precaution of stress ulcer.
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