OBJECTIVE To explain the metabolic characteristics of the stomach cold syndrome model rats, by constructing a rat model of stomach cold syndrome, analyzing the urine metabolites of the model rats.

METHODS  Twenty male SD rats were randomly divided into normal group and model group. The stomach cold syndrome model of rats was prepared by gavage with 5% acetic acid and refined lard, and the general behavioral observation of rats in each group was observed. After 14 days, the urine of rats in each group was collected, and the histopathological changes of gastric mucosa were observed by HE staining. The urine was preprocessed, and quality control(QC) samples were added to evaluate the stability of the experimental system and the repeatability of operation. Liquid chromatography-mass spectrometry was used for data collection, the molecular features of metabolites were extracted using Agilent Mass Hunter Profinder software. The obtained data set was imported into SIMCA-14.1 to perform PCA and OPLS-DA. According to the difference variables with variable importance projection greater than 1 in the OPLS-DA project, the screened difference variables were verified by t-test. The difference variables with P<0.05 were the potential markers screened. The potential biomarkers screened were analyzed by Metaboanalyst to screen the metabolic pathways related to stomach cold syndrome.

RESULTS  The establishment of stomach cold syndrome model was confirmed by general behavioral observation and gastric histopathological examination. The detection of QC samples in the PCA model proved that the data acquisition method was reliable and reproducible. The PCA model showed that there were differences in urine metabolites between the two groups of samples, and the OPLS-DA model showed that the separation between the samples in each group was obvious, and there were certain metabolic differences. There were a total of 12 biomarkers in the metabolic marker screening, which were 2-thiouracil, 6-(methylthio)purine, monoethanolamine, creatinine, δ-hexanolactone, N1-methylnicotinamine, 3-methylbenzyl alcohol, N1-acetylspermidine, scopoletin, N-acetyl-L-histidine, D-glucosamine-6-phosphate, estriol. Through metabolic pathway analysis, it was found that D-glucosamine-6-phosphate and estriol, D-glucosamine-6-phosphate were involved in alanine, aspartic acid and glutamic acid metabolic pathways, estriol was involved in steroid hormone biosynthesis pathway, ethanolamine was involved in glycerophospholipid metabolic pathway, D-glucosamine-6-phosphate was also involved in amino sugar and nucleotide sugar metabolic pathways.

CONCLUSION  Gastric perfusion with 5% acetic acid and refined lard can successfully build a rat model of stomach cold syndrome. In this experiment, through the metabolomic study of rat urine, found that the metabolic mechanism of rats with stomach cold syndrome includes four metabolic pathways, including alanine, aspartate and glutamate metabolism, glycerophospholipid metabolism, amino sugar and nucleoside sugar metabolism, steroid hormone biosynthesis, involving energy metabolism, inflammatory response regulation, etc.