| ObjectiveThe metabonomics technology was used to analyze the brain cortex,hypothalamus and stomach tissue samples of rats,finding the potential metabolic markers and key metabolic pathways of the pathology of liver depression and spleen deficiency in CAG,and exploring the correlation of different tissue metabolites.The purpose of this research was to explain the metabolic biological basis of brain and stomach related to TCM pathology of CAG,and it could provide objective basis for the aggravation of CAG caused by negative emotion.MethodsA total of 28 SPF Wistar male rats aged 5-6 weeks were divided into 2 groups by random number table method,including 8 normal group(N)and 20 model group(M).The N group rats were not given any treatment.Other rats were given 0.1% ammonia solution and20 mmol/L sodium deoxycholate solution alternately every day with the dietary control of2-day overfeeding and 1-day fasting.In the meantime,they were given chronic binding for 3h and tail clamping for 1h every day,continuous modeling for 16 weeks.And four rats were randomly selected for model evaluation during modeling.The standard for successful establishment of CAG rat model was determined by biological signs,behavioral experiments and pathological morphology of gastric mucosa of rats.Subsequently,CAG rats were randomly divided into model group(M,8 rats)and Chaishao Liujun Decoction group(C,8 rats,0.51 g/ml).Rats in N and M groups were gavaged with equal volume(10 ml/kg)of sterilized drinking water every day for 4 weeks.The histomorphology of gastric mucosa was observed with hematoxylin and eosin staining.Besides,the metabolites of rat cerebral cortex,hypothalamus and stomach were analyzed by non-targeted metabonomics.Combined with the results of univariate and multivariate statistical analysis,differential metabolites and metabolic pathways can be screened.At the same time,pearson correlation analysis can be performed on the expression of different metabolites in the cerebral cortex,hypothalamus and stomach samples of rats to find the metabolic relationship between different tissues.Results1.Model and intervention evaluation results: Compared with the N group rats,the M group rats gathered listlessly and quietly with hick and hard hair,light yellow and matte hair color,a strong smell of diarrhea and less body mass(P<0.01).The number of crossing small squares and standing in the open field experiment were decreased,and the motionless time of the forced swimming test was increased(P<0.01).In the M group,the glands in the lamina propria of gastric mucosa were atrophied and the number of chief cells and parietal cells decreased,accompanied by disordered arrangement of cells and infiltration of lymphocytes and plasma cells in the interstitium.Compared with the M group,the biological signs,behavioral results and histopathological morphology of gastric mucosa of rats in group C were improved.2.Metabonomic analysis of rat cerebral cortex samples: Twenty-three and four different metabolites were screened from the metabolic data of the cerebral cortex of rats in the M group and N group under the positive and negative ion mode respectively.The key metabolic pathways involved were mainly concentrated in pentose and glucuronate interconversions,thiamine metabolism,and histidine metabolism.And three common potential biomarkers were screened and identified among the N group,M group and C group.3.Metabonomic analysis of the rat hypothalamus: Seventy-two and thirty-four different metabolites were screened from the metabolic data of the hypothalamus of rats in the M group and N group under the positive and negative ion mode respectively.The key metabolic pathways involved were mainly concentrated in valine,leucine and isoleucine biosynthesis,glycerophospholipid metabolism,and aminoacyl-t RNA biosynthesis.And twenty-six common potential biomarkers were screened and identified among the N group,M group and C group.The key metabolic pathways involved were mainly concentrated in purine metabolism,glycerophospholipid metabolism,and D-glutamine and D-glutamate metabolism.4.Metabonomic analysis of the rat stomach: 63 and 23 different metabolites were screened from the metabolic data of the stomach of rats in the M group and N group under the positive and negative ion mode respectively.The key metabolic pathways involved were mainly concentrated in taurine and hypotaurine metabolism,thiamine metabolism,and valine,leucine and isoleucine biosynthesis.And 17 common potential biomarkers were screened and identified among the N group,M group and C group.The key metabolic pathways involved were mainly concentrated in thiamine metabolism,valine,leucine and isoleucine biosynthesis,and taurine and hypotaurine metabolism.5.Metabolite-metabolite correlation analysis: The correlation was obvious between different metabolites in the same tissue.And there were 1,41 and 15 pairs of metabolites in the cerebral cortex,hypothalamus and stomach of the M group rats,and there were 75 and 27 pairs of metabolites in the hypothalamus and stomach of the C group rats(|r|>0.7,P<0.05).The correlation was also observed between different metabolites in the cerebral cortex,hypothalamus and stomach.In the M group,10 pairs of metabolites were found between the cerebral cortex and hypothalamus,and 13 pairs of metabolites were found between the hypothalamus and stomach(|r|>0.7,P<0.05);One pair of metabolites was found between the cerebral cortex and hypothalamus of rats in the C group,3 pairs of metabolites were found between the cerebral cortex and stomach,and 15 pairs of metabolites were found between the hypothalamus and stomach(|r|>0.7,P<0.05).6.Analysis of characteristic metabolic pathways: The characteristic metabolic pathway of cerebral cortex in CAG rats with liver depression and spleen deficiency was cysteine and methionine metabolism;The characteristic metabolic pathways of hypothalamus were glycerophospholipid metabolism,alanine,aspartate and glutamate metabolism,and D-glutamine and D-glutamate metabolism;The characteristic metabolic pathway of stomach was steroid hormone biosynthesis.Conclusions1.The common characteristic of metabolites in cerebral cortex,hypothalamus and stomach of CAG rats with liver depression and spleen deficiency syndrome is that the body is mainly affected by the disorder of amino acid metabolism,and the disorder of amino acid metabolism may be the biological basis of brain and stomach related metabolism of CAG with liver depression and spleen deficiency syndrome.2.The metabolic characteristics in the cerebral cortex,hypothalamus and stomach of CAG rats with liver depression and spleen deficiency syndrome are respectively shown as changes in the contents of glycerol phospholipids,fatty acids and bile acids metabolites,and the above metabolite content changes may be the objective material basis of pathological changes in CAG with liver depression and spleen deficiency syndrome.3.Cysteine and methionine metabolism,glycerophospholipid metabolism,alanine,aspartate and glutamate metabolism,D-glutamine and D-glutamate metabolism,and steroid hormone biosynthesis may be used as a new direction to explore the essence of CAG with liver depression and spleen deficiency syndrome. |
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