| Objective:To establish and evaluate a mice model of kidney-yang deficiency diarrhea,examining the microecological mechanism of kidney-yang deficiency diarrhea and the role of Sishen Pill decoction from kidney pathology,energy metabolism,and gut mucosa microbiota,and providing an experimental foundation for the clinical treatment of kidney-yang deficiency diarrhea.Methods:The mouse model of diarrhea with kidney-yang deficiency syndrome by intragastric administration of adenine combined with Folium sennae,followed by the treatment of Sishen Pill decoction.The gut mucosa samples were collected after the modeling and treatment period,respectively:（1）Synthetically evaluate the syndrome attributes of model mice through the symptoms and signs corresponding to the clinical diagnostic criteria,and the efficacy of Sishen Pill"measured by formula".（2）The kidney structure was observed by hematoxylin eosin（HE）staining.Serum Na+-K+-ATP-ase and Ca2+-Mg2+-ATP-ase were detected by enzyme-linked immunosorbent assay（ELISA）,to evaluate the pathophysiological changes.（3）Full-length three-generation high-throughput sequencing of 16S r RNA gene for analysis of mouse gut mucosa microbiota.Results:（Ⅰ）The model mice showed symptoms of diarrhea with kidney-yang deficiency syndrome,（1）thin and soft feces that stick to the bedding,and feces adhering around the anus;（2）decreased food intake and increased water intake;（3）poor mental state,slow reaction,arching back and curling up,and preferring to gather and pile up;（4）cool body and cold tail,light paw nails,and sparse and lusterless fur.（Ⅱ）ELISA and HE staining revealed decreased Na+-K+-ATP-ase and Ca2+-Mg2+-ATP-ase activities and impaired kidney structures in model mice.（III）Analysis of gut mucosa microbiota revealed that the dominant and characteristic bacteria in the model group differed from the normal group at different taxonomic levels,and the gut mucosa microbiota was imbalanced.（Ⅳ）Following Sishen Pill therapy,the kidney structure recovered,the arrangement of cells was close,the level of energy metabolism was regulated,and gut mucosa microbiota was changed,which Muribaculum and Lactobacillus johnsonii were promoted,and Candidatus-Arthromitus,Lactobacillus murinus,and Lactobacillus intestinalis abundance were inhibited,and showed a tendency to the normal group.Conclusion:Adenine combined with Floumn senna could successfully construct a mouse model of diarrhea with kidney-yang deficiency syndrome,which caused kidney structural damage and affected energy metabolism in mice.Flora analysis revealed that Akkermansia muciniphila、 Clostridium cocleatum、Bacteroides vulgatus and Bacteroides sartorii might be the characteristic species of gut mucosal microbiota causing diarrhea with deficiency kidney-yang syndrome. Sishen Pill decotion can regulate energy metabolism,improve kidney structure and diversity of gut mucosa microbiota.In addition,Lactobacillus johnsonii may be a characteristic species of Sishen Pill decotion in the treatment of diarrhea mice with kidney-yang deficiency syndrome. |
