| BackgroundGastric ulcer (GU) is a common disease of digestive system, referring to ulcerative lesion of gastric mucosa because of self-digestion by gastric juice. It mainly relates to role of gastric acid and pepsin, H.pylori(Hp) infection, use of nonsteroidal anti-inflammatory drugs (NSAIDs), abnormal gastroduodenal motility, influence of stress and heredity, etc. In summary, it is caused by disbalance between damage and protection factors to gastric mucosa. Recently, histamine-2 receptor antagonists (H2RA), proton pump inhibitors (PPI) and protecting medicament of gastric mucosa have been widely used in GU treatment, thus this disease can be cured shortly. However, its recurrence rate is still very high after medication stop, because of low quality of ulcer healing(QOUH). Treating GU with traditional Chinese medicine (TCM) based on differentiation of symptoms could increase QOUH significantly and decrease recurrence rate of ulcer, through a series of machanisms.Acetic acid-induced gastric ulcer model in rats is widely used as the standard model for anti-ulcer drugs screening. Its spontaneous healing and relapse highly resemble human chronic ulcers, but its mechanisms are still not so clear. Frequently, single or several indexes are choosed to evaluate Chinese medicine of anti-ulcer, but they can't reflect the muti-targets and comprehensive action of TCM in the whole.Weiweifang (WWF), a Chinese herbal preparation, has been used to cure gastric ulcer clinically. Present studies have demonstrated that WWF can regulate levels of gastrin (GAS), interleukin-2 (IL-2), nitric oxide (NO), epidermal growth factor(EGF) and inducible nitric oxide synthase(iNOS) in serum, and increase hexosamine level in gastric mucosa, thus to decrease the damage and enhence the protection to mucosa, so as to promote gastric ulcer healing effectively.Metabonomics, a new "omics" after genomics, transcriptomics, and proteomics, is referring to a systematic approach to analyzing the dynamic changes of low molecular mass organic endogenous metabolites of living organisms in response to pathophysiological stimuli or genetic medication or pharmacological effects, et al. Pattern recognition is utilized to findout the biomarker that characterize specified state and to analyze the metabolic network defect of different state or intervention effect on metabolic network, so as to reveal the biological essence of specified state or the mechanisms of intervention. Nowaday, metabonomics has been gradually used to the researches of pathology, diagnosis of disease, mechanisms of medicine, essence of TCM syndrome, pharmacology and toxicology of Chinese herbal medicine.Objective1. To observe the chages of mucosa ulcer area and pathological features in acetic acid-induced gastric ulcer rats during the lesion occurring and spontaneous healing; to observe the effect of WWF on gastric ulcer area and pathological features.2. To find out the biomarkers characterizing the process of gastric ulcer occurring and spontaneous healing in rats using metabonomics; to explain their mechanisms in the whole according to the chages of biomarkers levels, so as to provide an experimental basis for evaluating anti-ulcer drugs comprehensively.3. To evaluate the therapeutic effect of WWF on acetic acid gastric ulcer in rats viewing from metabonomics, and to explore the mechanisms of it according to the changes of biomarkers levels after treated with WWF.MethodsThe male Wistar rats were randomly divided into normal group, model-1d group, model-3d group, model-7d group, model-10d group (spontaneous healing group), ranitidine group and 3 WWF groups (high-dose, middle-dose and low-dose), with 8 rats in each group. Gastric ulcer models were made by applying acetic acid to the anterior serosal surface of gastric antrum. Ulcer area of each group was measured and histological specimens with HE staining were made. Gastric mucosa and 12h urine were collected, then the former was derivatized after homogenizing, while the latter was derivatized directly. Subsequently, metabolites spectra of these samples were acquired using gas chromatography-mass spectrometry (GC-MS). Identification After processed, the data was subjected to SIMCA-P+12.0 software(Umetrics AB, Umea, Sweden) for principal component analysis (PCA) and least squares discriminant analysis (PLS-DA). The modeling and therapeutic effects were judged by PCA visually; distinguishing effect among groups were further inspected by PLS-DA, and the correlation with classification of metabolites were evaluated according to the "variable importance in the projection (VIP) "value. When a certain metabolite's VIP>1, it would be taken as potential metabolic marker. The differences of potential biomarkers levels in different groups were conducted with univariate statistical analysis using software of SPSS 13.0, and those with significant difference were chosen as final biomarkers.The machanisms of developing and spontaneously healing of gastric ulcer in rats were interpreted according to the changes of biomarkers levels in different model groups and combining known biochemical theory. The therapeutic machanisms of WWF on gastric ulcer were investigated according to the effect of this preparation on biomarkers levels.The measurement data was presented as(x±s). Two groups means were compared with independent-samples T test; multiple groups means were compared with One-way ANOVA and the pairwise comparison was performed with LSD test if the variance of the data was homogeneous, otherwise with Games-Howell method. All significant levels were 0.05 (two tailed).Results1. Gross appearence of gastric mucosa:In normal group, the gastric mucosa was smooth, integrated and reddish, without hyperemia, edema, erosion and bleeding. In model-1d group, there were round or oval defects in gastric antrum mucosa with size ranged from 2 to 5 mm in diameter, hyperemia and edema around. In model-3d group, there were obvious deep and clear-margin ulcers with size ranged from 5 to 6 mm in diameter, covered with gray-white or yellowish-white mosslike substance, and there was hyperemia surrounding. In model-7d and model-10d groups, the areas of the ulcer were less than model-3d group, with pale hyperplastic scar forming plica around. In WWF middle-dose group, the ulcers had healed well, with smooth plica centralizing, without moss. In WWF high-dose and low-dose groups, the ulcers had decreased significantly, covered with white thin moss, with plica centralizing, without inflammation. In the ranitidine group, the situation was similar to the WWF high-dose and low-dose groups.2. Comparision of ulcer areas (S):One-way ANOVA result of ulcer areas in different model groups and treatment groups showed that there was significant difference in population mean (P<0.01); the S in model-3d group were significantly larger than in model-1d group (P<0.05); compared to model-3d group, the S decreased significantly in model-7d and model-10d groups(P<0.05 or P<0.01). When compared with spontaneous healing group, the S in treatment groups were significantly smaller (P<0.01); compared to ranitidine group, the S in WWF middle-dose group decreased significantly (P<0.01); while there was no significant difference in WWF high-dose, low-dose groups and ranitidine group (P>0.05).3. Pathological changes:Normal group:the mucosa was smooth and intact; the glandular cells were in order, and the submucosa, muscularis and serosa were intact. Model-1d group:mucosal defects occurred in ulcer side, some glands were destroyed; the blood circulation in capillaries was stagnating, and the submucosa and serosa were infiltrated by a large collection of inflammatory cells. Model-3d group: large mucosal defects occoured in ulcer side, the glands were totally destroyed and even dispeared; the blood circulation in capillaries was still stagnating, even some capillaries had rupture and were bleeding; the submucosa and serosa were still infiltrated by a large number of inflammatory cells. Model-7d group: the defects were extending to muscularis, with some regenerated glandular cells arranging disorderly; some inflammatory cells were infliltrating in submucosa with thrombosis in capillaries; spontaneously healing group:some mucosa defects extended to muscularis and regenerated glands arranged disorderl, still with inflammatory cells infiltrating and thrombosis formed. Ranitidine group:the mucosal defects had narrowed to a certain degree and there were regenerated epithelium cells and glandular cells arranged compactly but irregularly on them; there were still some inflammatory cells infiltrating in submucosa and the scar tissure formed in muscularis. WWF high-dose and low-dose group:the defects diminished and the density of regenerated glandular cells was uneven, with inflammatory cells infiltrating; there were no pathogenesis changes in submucosal vasculars; WWF middle-dose group: the mucosa was almost intact, and the regenerated glands were even more, with only few inflammatory cells in submucosa.4. Gastric mucosal biomarkers and their changes in different model groups: results of one sample t test showed that, compared to normal group, model-1d group displayed significantly higher levels of cholest, octadecanoic acid, arachidonic acid, oleic acid, linoleic acid, hexadecanoic acid, lactate, malate and nicotinamide (P<0.01), and lower levels of lysine, pyroglutamate, tyrosine, leucine, glutamic acid, alanine, histidine, glycine, methionine and valine (P＜0.01); model-3d group displayed significantly higher levels of arachidonic acid, cholest, oleic acid, hexadecanoic acid, octadecanoic acid, malate and lactate, linoleic acid (P＜0.01), and lower levels of lysine, tyrosine, pyroglutamic acid, glutamic acid, histidine, leucine, proline, phenylalanine and alanine (P<0.01); model-7d group displayed significantly higher levels of arachidonic acid, cholest, hexadecanoic acid, linoleic acid, octadecanoic acid, nicotinamide, malate, oleic acid, lactate, glutamine, tryptophan and alpha-hydroxy-benzeneacetic acid (P<0.01 or P＜0.05), and lower levels of tyrosine, glutamate, proline, lysine, leusine, glysine, pyroglutamic acid, histidine and phenylalanine (P＜0.01 or P<0.05); moedel-10d group displayed significantly higher levels of arachidonic acid, isoleusine, aspartic acid, malate, cysteine, cholest, lactate, 3-methylcytosine and valine (P＜0.01 or P＜0.05), and lower levels of proline, octadecanoic acid, oleic acid, glutamic acid, hexadecanoic acid, dimethylglycine, lysine, linoleic acid and tyrosine (P＜0.01 or P＜0.05).5. Gastric mucosal biomarkers characterizing the dynamic process of gastric ulcer occouring and spontaneously-healing included arachidonic acid, malate, isoleusine, aspartic acid, proline, cysteine, valine, lactate, methionine, glutamic acid, tyrosine, cholest, lysine, oleic acid, pyroglutamic acid, octadecanoic acid, histidine and hexadecanoic acid. The gastric mucosal biomarkers changed dynamically in different phases of gastric ulcers.6. Urinary biomarkers and their changes in different model groups: compared to normal group, model-1d group display significantly higher levels of phenaceturic acid, acetoacetic acid, p-hydroxyphenylacetic acid,3-hydroxyhexanoic acid,3-hydroxy-butanoic acid and proline(P<0.01), and lower levels of citrate, hippurate, aconitate, succinate,5-acetamidovaleric acid,3-indoleacetic acid and isocitrate (P<0.01); model-3d group displayed significantly higher levels of Phenaceturic acid, p-hydroxyphenylacetic acid,5-acetamidovaleric acid, phenylacetic acid, tyramine and malonate (P<0.01), lower levels of citrate, hippurate, aconitate, succinate, indoleacetate, isocitrate and quinaldate (P＜0.01); model-7d group displayed significantly higher levels of phenaceturic acid,5-acetamidovaleric acid, 3-hydroxybutanoic acid, p-hydroxyphenylacetic acid, phenylacetic acid and benzoic acid (P＜0.01 or P＜0.05), and lower levels of hippurate, citrate, succinate, quinaldate, glycine,3-methoxybenzenepropanoic acid, methylpropanedioic acid,3-indoleacetic acid and isocitrate (P<0.01 or P＜0.05); model-10d group displayed significantly higher levels of phenaceturic acid,5-acetamidovaleric acid, p-hydroxyphenylacetic acid and 3-indoleacetic acid (P＜0.01 or P＜0.05), and lower levels of citrate, aconitate, quinaldate,3-methoxybenzenepropanoic acid, succinate and glycine(P＜0.01 or P＜0.05).7. Urinary biomarkers characterizing the dynamic process of gastric ulcer occouring and spontaneously-healing included phenaceturic acid,5-acetamidovaleric acid, citrate, hippurate, aconitate,3-hydroxyhexanoate, acetoacetic acid, glysine, quinaldate, succinate, p-hydroxyphenylacetic acid, pimelate, proline,3-indoleacetic acid and phenylacetic acid. The urinary biomarkers changed dynamically in different phases of gastric ulcers.8. Gastric mucosal biomarkers and their changes in treatment groups: compared to normal group, ranitidine group displayed significantly higher levels of arachidonic acid, phenylalanine, glutamine, valine, lactate, tryptophan, isoleusine, malate and aspartic acid (P＜0.01), and lower levels of cholest, tyrosine, glutamic acid, glycine, leusine, oleic acid, pyroglutamic acid, lysine, hexadecanoic acid and octadecanoic acid (P＜0.01 or P＜0.05); WWF high-dose group displayed significantly higher levels of pyrimidinone, leusine,3-acetamidopropanal and lysine (P＜0.01 or P＜0.05), and lower levels of tyrosine, hydroxyproline, oleic acid, glycine, alanine and arachidonic acid (P＜0.01 or P＜0.05). WWF middle-dose group displayed significantly higher levels of glutamate, histamine, leusine and lysine (P＜0.05), tyrosine, proline (P＜0.01 or P＜0.05). WWF low-dose group displayed significantly higher levels of 3-acetamidopropanal, lysine, leusine, pyrimidinone and nicotinamide (P＜0.01 or P＜0.05), and lower levels of tyrosine, proline, alanine, glysine, pyroglutamic acid and valine (P＜0.01 or P＜0.05).9. Urinary biomarkers and their changes in treatment groups:compared to normal group, ranitidine group displayed significantly higher levels of:phenaceturic acid, tyramine, p-hydroxyphenylacetic acid and phenylacetic acid (P＜0.01), and lower levels of citrate,5-acetamidovaleric acid, quinaldate, succinate,3-indoleacetic acid, ethylmalonic acid and malate (P＜0.01). WWF high-dose group displayed significantly higher levels of: phenaceturic acid and proline (P＜0.05), and lower levels of citrate, aconitate, 3-methoxybenzene -propanoic acid,3-indoleacetic acid, succinate, homocitrate, quinaldate and 2-oxo -glutarate(P＜0.01 or P＜0.05). WWF middle-dose group displayed significantly higher levels of: proline and 3-hydroxyphenylacetate (P＜0.01), and lower levels of 3-methoxybenzene-propanoic acid,3-indoleacetic acid and homocitrate (P＜0.01 or P＜0.05). WWF low-dose group displayed significantly higher levels of phenaceturic acid, ethylmalonic acid, tyramine and proline (P＜0.01 or P＜0.05), and lower levels of citrate, aconitate, 3-indoleacetic acid,3-methoxybenzene -propanoic acid, succinate, quinaldate, aspirate and homocitrate (P＜0.01 or P＜0.05).Conclusion1. The gross obsveration and comparison of ulcer areas in different model groups demonstrate that gastric ulcers are developing in model-1d group, have formed in model-3d group, and are spontaneously healing in model-7d and 10d groups; but the healing effect is not satisfied. Pathological examinations demonstrate that the acetic acid gastric ulcers are closely related to inflammation injury and microcirculatory disturbance of gastric mucosa; the regenerative tissues of gastric mucosa are abnormal under spontaneous healing mechanisms.2. The gross obsveration and comparison of ulcer areas in treatment groups demonstrate that the therapeutic effect of middle-dose WWF is better than those of high-dose and low-dose WWF; the effect of ranitdine is similar to the latter two, but better than spontaneous healing. Pathological examinations demonstrate that the regenerative tissues in WWF middle-dose group approximate normal.3. The dynamic changes of metabolites spetras from gastric mucosa and urine are revealed using GC-MS based metabonomics approach, and the biomarkers that characterize the process of gastric ulcer developing and spontaneously healing are acquired, including lipoid, organic acids, fatty acids and amino acids. The abnormal marks levels in different model groups demonstrate that the energy and substance metabolisms are disorder during the ulcers developing and spontaneously healing.4. The biomarkers from gastric mucosa and urine in treatment groups are acquired using GC-MS based metabonomics approach. The levels of gastric ulcer biomarkers in different treatment groups are tending toward normal, expecially in WWF middle-dose group, showing that the therapeutic effect of middle-dose WWF is the best and WWF cures gastric ulcer effectively by way of regulating the energy and substance metabolisms.5. The biological characteristics and their machanisms of acetic acid gastric ulcer in rats are interpreted integrally by metabonomics. The therapeutic mechanisms of WWF on the ulcerative focus and the whole body are revealed systematically by metabonomics, displaying prominently the characteristics of Chinese medicine multi-targets comprehensive therapy. Metabonomics would be widely used in TCM, because the general characteristc of integrality between them.
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