Objective

2

Taking the rat model of spleen-stomach damp-heat syndrome（SSDHS） as the research object， this study aimed to investigate the potential biomarkers of SSDHS and the related metabolic pathways based on urine metabolomics， and tried to reveal the essence of SSDHS at the level of endogenous small molecular metabolites.

Method

2

Sixteen SD rats were randomly divided into normal and model groups. The normal group was fed normal chow and the model group was fed with 200 g·L

-1

honey water daily， and lard and Chinese Baijiu alternately on alternate days for 17 days. The SSDHS model rats were exposed to external dampness-heat environment with temperature at 30-34 ℃， relative humidity of 95% for 2 h at the same time every day from the 10

th

day for 7 d. Then， the model was evaluated by observing the general conditions of the rats， measuring the contents of motilin（MTL） and gastrin（GT） in plasma by enzyme-linked immunosorbent assay（ELISA）， and examining the histopathology of gastronitestinal tissues. In additon， the urine metabolomics analysis was performed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， and the detection conditions was as follows：ACQUITY™ UPLC BEH C

18

column（2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） for gradient elution （0-3 min， 1%-18%B； 3-8 min， 18%-40%B； 8-10 min， 40%-100%B）， the flow rate of 0.4 mL·min

-1

， electrospray ionization（ESI） in positive and negative ion modes， scanning range of

m

/

z

50-1 000. The univariate and multivariate statistical analysis were constructed for screening inter-group differential ions， the element composition was calculated according to the precise relative molecular weight， and ion information was matched with databases such as Human Metabolome Database（HMDB） to identify biomarkers. Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to obtain the biological information of metabolites， and their associated metabolic pathways were analyzed by MetaboAnalyst 5.0.

Result

2

Compared with the normal group， the rectal temperature of the model group increased significantly（

P

<

0.01）， the levels of plasma MTL and GT decreased significantly（

P

<

0.05，

P

<

0.01）， and pathological changes such as bleeding， congestion and inflammatory infiltration in the gastric and colonic tissues. A total of 25 differential metabolites such as

L

-histidine， citric acid and isocitric acid were found to be the potential biomarker of SSDHS by urine metabolomics， 13 of which were phase Ⅱ metabolites of endogenous substances（glucuronic acid conjugates， sulfuric acid conjugates and acetyl conjugates）， involving the metabolic pathways of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism.

Conclusion

2

SSDHS primarily causes disorders of histidine metabolism， tricarboxylic acid cycle， glyoxylate and dicarboxylate metabolism， as well as the imbalance of the activation/inactivation of endogenous metabolites， which may involve the immune response， material and energy metabolism， inflammatory response and intestinal flora， and may provide a basis for the establishment and application of SSDHS model.