Experimental Study On Energy Metabolism Of Spleen Insufficiency Of Spleen-yang Deficiency Syndrome And Regulation Mechanism Of Brain-gut Axis

Objective:Based on the spleen viscera theory of traditional Chinese medicine,this study used modern experimental techniques such as ELISA,Western Blot and UFLC-IT-TOF-MS to detect the mitochondrial respiratory chain enzyme complex I,IV,Na⁺-K⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATP enzyme,GSH-Px activity and MDA content in the ileum of rats with spleen yang deficiency syndrome.The content and the relative expression of protein of VIP,CCK,GIP,cAMP and PKA in the ileum tissue of rats were measured.The purpose of this study is to find the mechanism and interaction between pathological and relation and then to reveal the mechanism of spleen yang deficiency syndrome in the parlance of molecular biology,to pro-vide theoretical support for the quantitative index of spleen yang deficiency syndrome and provide the basis for clinical diagnosis and treatment.Materials and methods:44 SPF healthy SD rats were randomly divided into three groups,namely the normal control group,the spleen yang deficiency syndrome model group and the traditional Chinese Medicine counterevidence group.The model of spleen yang defi-ciency syndrome was modeled by the classical compound factor making method,saying,diet failure,overexertion unite drugs of bitter and cold nature were used.The model was divided into two stages.In the first stage,the model of spleen qi deficiency syndrome was induced,and the spleen yang deficiency syndrome model was established on the basis of spleen qi de-ficiency syndrome model in the second stage.Each stage lasted for 8 days in duration.In or-der to judge the success of the model,the overall situation of the rat model was evaluated by the method of symptom evaluation combined with the pattern recognition method of mathe-matics at two different time nodes at eighth,sixteenth days respectively.The pathological changes of tissues were observed by light microscopy.ELISA was used to detect the mito-chondrial respiratory chain enzyme complex I,IV,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,GSH-Px and MDA content in the ileum tissues of the rats.The changes of metabolic products were detected by non targeting metabolomics;ELISA and Western Blot were used to detect the content and the relative expression of protein of VIP,CCK,GIP,cAMP and PKA in the ileum tissue of rats.Result:1.Pathological examination results of ileum tissue of rats in each group:In the 100 X light microscope:the organizational structure of the ileum of the normal control group was intact,the texture was clear,and there was no inflammatory reaction and injury.The intestinal villa in the spleen yang deficiency model group appeared to have gone degen-erative changes,the glandular arrangement was disorderly,the structure was not clear.Locally inflammatory exudation were observed,gland atrophy,necrosis and other pathological changes were also found.Compared with the model group,the ileum tissue of the rats in the Chinese medicine counterevidence group returned to normal and the glandular arrangement was neat.2.The results of energy metabolism and biochemical indexes of lipid peroxidation in ileum tissues of rats in each group:The activity of mitochondrial respiratory chain complex I and IV,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase and GSH-Px in the ileum tissue of spleen yang deficiency syndrome mod-el group were all decreased,and were statistically significant?P<0.01?,but the counterevi-dence group showed a recovery trend?P<0.01,P<0.05?,still lower than normal level?P<0.05,P<0.01?.Compared with the normal control group,the content of MDA in the ileum tissue of the model group of spleen yang deficiency syndrome group was significantly in-creased?P<0.01?,and after the intervention treatment of Chinese medicine,there was a sig-nificant decrease?P<0.05?,but it did not reach the normal level?P<0.05?.3.Metabolomics test results of metabolites in plasma and ileum tissues of ratsNon target metabonomics test was performed on the serum and ileum tissues of rats with Spleen Yang deficiency syndrome and normal control rats.42 differential metabolites were screened in serum,such as proline,threonine 1,valine and oleic acid.Ileum tissue in rats of spleen yang deficiency syndrome model group and normal control group,and 24 different kinds of differential metabolites,such as mannose 1,methyl phosphate,tyrosine 2 and nuclear sperm,were screened in the serum.Including proteins,fatty acids and other abnormality of metabolites.4.The results of brain-gut axis related biochemical indexes?ELISA?in ileum of rats in each groupCompared with the normal control group,the content of VIP,CCK,GIP,cAMP and PKA in the ileum of spleen yang deficiency syndrome model group increased significantly?P<0.01?,after the prognosis of Chinese herbal,they showed a trend of decline,which was statistically significant at all conventional levels?P<0.01?.5.Results of Western Blot in ileum of rats in each group:Compared with the normal control group,the relative expression of VIP,CCK,GIP,cAMP and PKA in the ileum of spleen yang deficiency syndrome model group increased signifi-cantly?P<0.01?,after the prognosis of Chinese herbal,they showed a trend of decline,which was statistically significant at all conventional levels?P<0.01?.Conclusion:1.The spleen yang deficiency syndrome can affect the body mitochondria respiratory chain enzyme complex I,IV,Na⁺-K⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATP enzyme,GSH-Px activity and MDA content,as well as protein,lipid,sugar related metabolites,and then lead to material energy metabolism and lipid peroxidation.2.The changes of the brain gut peptide VIP,CCK,and GIP are regulated by the regulation of the brain-gut axis regulation,and the digestive and absorption function of the gastrointestinal tract is influenced by the cerebral intestinal axis.As the second messenger of the signal transduction pathway and the PKA as the signal transduction downstream kinase,cAMP is involved in the whole pathological process,and VIP-cAMP-PKA and GIP-cAMP-PKA are likely to be among them.The important transduction pathway is probably the biological mechanism of the spleen yang deficiency syndrome,which is caused by the abnormal func-tion of the cerebral intestinal axis,the main manifestation of the digestive tract symptoms.