| Background:Stress ulcer (SU) is one of the familiar stress diseases in clinic. It is a kind of acute disease specialized with gastrointestinal mucous and shallow ulcer, which will cause bleeding and perforation of gastrointestinal mucous with high mortality and difficult treatment. Stress ulcer does great harm to people's health. Many resources indicated that stomach acid is not the chief cause of stress ulcer, so prophylaxis and treatment should include both acid- suppression and integrated therapies. The mechanism and methods to prevent and treat has caused great attention of many scholars. However, current research are confined to nerve-fluid regulations in gastric mucous, deep mechanisms as nerve central regulation and nerve-endocrine-immune nets are neglected somehow.Treatment and prophylaxis by traditional Chinese medicine (TCM) has the predominance of whole-modulation. The study aims to illuminate molecular mechanisms of nerve central regulation to stress ulcer, and evaluate the prophylaxis by Zuojin pill. The curative are Zuojin pill with high, middle and low dose. We duplicated and improver stress ulcer model of water immersion restraint stress (WRS) . Gastric mucosal ulcer index (UI) and gastric PH level were observed commonly. Then we measure the expression of protein product of c-fos and CRHmRNA in hippothalamic paraventricular nucleus, and serum ACTH and cortisol. Methods:Male Wistar rats are divided into 6 groups: normal control group(A); stress ulcer model group(B); Zuojin pill with high dose(Cl); Zuojin pill with middle dose(C2);Zuojin pill with low dose(C3); Ranitidine treatment group(D). In each group,there are 6 rats. Rats of group A were not operated. Rats of group Cl, C2, C3 and D are treated with drugs, rats of group A and B are treated with saline for 3 times, one time each day. 3 days later, rats of group B, Cl, C2, C3 and D are going to be replicated model. The method of replicating model refers to stress ulcer model of water immersion restraint stress (WRS). Rats were confined in the mineral water bottles of 350 ml. After adjusting the volume, the bottom of the bottles are enclosed. The rats were immersed into warm water of 26 °C. 6 hours later,all rats were put to death. Gastric mucosal ulcer index (UI) and gastric PH level were observed commonly. The method of measuring UI refers to that of Guth. Their blood was taken from abdominal aorta and used to distill serum. Then their body were affused with saline and formalin of 10 percent. Their brain and stomach were fixed with formalin of 10 percent, embedded with olefin and sliced up. The tissue indexes were tested, normal pathology examination by HE dyeing; the expression of protein product of c-fos was examined by immunochemical technique in hippothalamic paraventricular nucleus; In situ hybridisation was used to detect the expression of CRHmRNA, serum ACTH and cortisol were analysed by RIA. Results: 1. effect of Zuojin pill to gastric mucosa of stress ulcer model rats1.1 gastric mucosal ulcer index ( UI) The study demonstrated that UI changed significantly under WRS compared with control group (P<0.01) . Zuojin pill could significantly alleviate ulcer ( P<0.01 ) , with obvious dose-effect relationship. UI in Ranitidine group was also decreased significantly compared with control group ( P<0.01) . Zuojin pill with high dose has no discrepancy compared with Ranitidine (P>0.05) .1.2 gastric PH level The study demonstrated that gastric fluid PH value increased significantly under WRS compared with control group (P<0.01) . Zuojin pill could significantly alleviate secretion of gastric acid (P<0.05) .without obvious dose-effectrelationship. Zuojin pill with high dose has no discrepancy compared with normal control group (P>0.05). PH level in Ranitidine group was also decreased significantly compared with control group ( P<0.01 ) . Zuojin pill with high dose has no discrepancy compared with Ranitidine (P>0.05) .1.3 observation of gastric mucosa Gastric mucosa of normal control group rats were normal and smooth, without congestion, edema and erosion. Gastric mucosa of stress ulcer model rats were congested and dropsical with many bleeding dots and ulcers; some material of light coffee color could be observed inside stomach. In groups of Zuojin pill with high and middle dose, rats' gastric mucosa were smooth with little gore and bleeding dots. In groups of Zuojin pill with low dose, rats' gastric mucosa were congested and dropsical lightly, with little gore and bleeding dots. In Ranitidine group, rats' gastric mucosa were smooth without obvious erosions.1.4 observation under microscope Gastric mucosa of normal control group rats were normal and smooth, with gland in order. Inflammatory cells and bleeding inclination could not be observed. Gastric mucosa of stress ulcer model rats were not intact, with many red cells on the surface of mucosa. The glands were broken through, and capillary vessel under ulcers were blocked and bleeding. Blots might be observed inside some little aorta. Some ulcers could destroy muscle layer, the muscle cells lined in disorder. In groups of Zuojin pill with high and middle dose, we found that epidermic cell of gastric mucosa were tumid and the clearance between glands were enlarged. Other tissue and the micro-circulation under mucosa were normal. In groups of Zuojin pill with low dose, under-mucosa layer of rats' gastric mucosa were destroyed with light pathology changes. Ranitidine group has no special discrepancy compared with group Zuojin pill with high dose. Gastric mucosa were smooth, with gland in order.2. effect of Zuojin pill to c-fos expression in brain of stress ulcer model rats2.1 observation under microscope In normal control group, c-fos could be found only in hypothalamic paraventricular nucleus (PVN) , ACd of origin system. Thedyeing colour is rather light. In stress ulcer model rats, we found more nerve cells with c-fos strong dyeing colour, especially in small cell region of PVN in hypothalamus. c-fos could also be seen at so. sot. of hypothalamus, pv of thalamencephalon, ce. shi. prcm. and cortex. Zuojin pill with high dose could significantly alleviate the expression of c-fos, but c-fos could also be seen at PVN in hypothalamus, so. hippocampus, cortex. In groups of Zuojin pill with middle and low dose and Ranitidine, we found that c-fos expression were rather comprehensive.2.2 counting nerve cells with c-fos dyeing under microscope In stress ulcer model rats, c-fos in hypothalamic paraventricular nucleus were greatly improved compared with control group ( P<0.01) . Zuojin pill with high dose could significantly alleviate the expression of c-fos, compared with stress ulcer model group (P<0.05) . Groups of Zuojin pill with middle and low dose and Ranitidine, has no discrepancy compared with control group (P>0.05) . 4. effect of Zuojin pill to ACTH and CORT in serum of stress ulcer model rats3.1 ACTH In stress ulcer model rats, ACTH in serum were greatly improved compared with control group (P<0.01) . Zuojin pill with high dose and middle dose could significantly alleviate the level of ACTH in serum, compared with stress ulcer model group ( P<0.05 ) . Zuojin pill with high dose has no discrepancy compared with normal control group ( P>0.05 ) . Groups of Zuojin pill with low dose and Ranitidine, has no discrepancy compared with control group (P>0.05) .3.2 CORT In stress ulcer model rats, CORT in serum were greatly improved compared with control group (P<0.01). Zuojin pill with high, middle and low dose could significantly alleviate the expression of CRHmRNA, compared with stress ulcer model group (P<0.05) . Zuojin pill with high dose has no discrepancy compared with normal control group (P>0.05) . In group Ranitidine, we found that CRHmRNA expression has no obvious discrepancy compared with model group (P>0.05) .Conclusion:1. The method of replicating model refers to stress ulcer model of water immersion restraint stress (WRS) and be meliorated. Avoided congestion and gore caused by binding limbs. The model successfully replicated the process of stress ulcer with 100 percent success rate and simple method.2. Zuojin pill could significantly improve the level of gastric fluid PH value,alleviate the damnification to gastric mucosa and decrease gastric mucosal ulcer index.3. Zuojin pill could significantly decrease the expression of c-fos in hypothalamic paraventricular nucleus, alleviate the intensity of stress reaction.4. Zuojin pill could significantly decrease the expression of CRHmRNA in hypothalamic paraventricular nucleus, restrain the activation of HPA axis.5. Zuojin pill could significantly decrease the level of ACTH and CORT in serum, restrain the activation of HPA axis and the intensity of stress reaction.In conclusion, Zuojin pill could prevent stress ulcer effectively through mulriple mechanisms. Zuojin pill could alleviate the expression of c-fos and CRHmRNA in hippothalamic paraventricular nucleus, restrain the activation of HPA axis, improve the level of gastric fluid PH value, decrease gastric mucosal ulcer index. Zuojin pill could directly intervene stress system, exert the action of nerve central regulation, alleviate secretion of gastric acid, protect gastric mucosa, prevent stress ulcer successfully. This is the advantage of traditional Chinese medicine about prophylaxis of stress ulcer. |
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