| Objective: Establishing a mouse model of diarrhea with cold-dampness disturbing spleen syndrome(CDSS)and detecting the microbial basis and microecological mechanism,the intervention mechanism of Huoxiang Zhengqi powder(HXZQ)were studied.In order to provide experimental evidence for the clinical treatment of gastrointestinal diseases related to diarrhea with CDSS caused by exogenous cold dampness in Traditional Chinese Medicine(TCM),and provide ideas and references for the development and application of biological microbial resources of HXZQ.Methods:(Ⅰ)Simulating the stress intervention of cold dampness environment in an artificial climate box,a mouse model of diarrhea with CDSS was established.The syndrome diagnosis(such as loose stool rate,diarrhea index,defecation times,fecal water content,and abdominal temperature)were used to evaluate the establishment of an animal model of diarrhea with CDSS.Based on "Testing syndrome by Formula" the intervention of HXZQ was used to evaluate the animal model.(Ⅱ)The model mice were gavaged with "gentamicin sulfate +Cefradine" high-dose mixed antibiotics.The feces,intestinal contents,and mucosa were collected for bacterial culture and microbial activity detection.The mucus barrier of colonic tissue,such as HE and AB-PAS stained sections,mucus layer thickness,Muc2 by ELISA,etc.,were detected.The effect of intestinal microbiota homeostasis on intestinal mucus barrier in diarrhea with CDSS mice was evaluated.Moreover,the effect of intestinal microbiota homeostasis on diarrhea with CDSS mice was studied by using serum biochemical blood lipid and amino acid transferase indexes.(Ⅲ)To explore the microeco-logical mechanism of diarrhea with CDSS,the microbiota diversity,composition structure,and characteristic microbiota of intestinal mucosa of mice were studied by 16 S r RNA Ⅲ full-length highthroughput sequencing.The amino acid metabolism was detected by serum targeted amino acid LC-MS metabolome.(Ⅳ)After 7 d of modeling and 3 d of gavage with HXZQ,the morphology of the intestinal mucus layer,Muc2 by ELISA,serum biochemical blood lipid,and amino acid transferase were evaluated the effect of HXZQ on the intestinal mucus barrier in mice with CDSS diarrhea.The intestinal mucosa 16 S r RNA Ⅲ full-length high-throughput were used to study the effect of HXZQ on the microbiome of mice with CDSS diarrhea.The serum targeted amino acid LC-MS metabolome was used to study the amino acid metabolism function of "host-microorganism" interaction,so as to explore the microecological mechanism of HXZQ in the treatment of diarrhea with CDSS.(Ⅴ)The intestinal mucosa and contents of mice were collected in a sterile environment,and the bacteria in intestinal contents were cultured in vitro.Microbial activity was detected by the FDA method;amylase,xylanase,sucrase,and cellulase activities were detected by the DNS method;lactase activity was detected by the ONPG method,and protease activity was detected by the Folin-Ciocalteu method.Results:(Ⅰ)The model mice had a "heavy body,swollen chest and full abdomen",and the loose stool rate,defecation volume,and fecal water content increased significantly(P < 0.05);HXZQ significantly reduced the loose stool rate and diarrhea index(P < 0.05).(Ⅱ)After the intervention of high-dose mixed antibiotics: the microbial activity of feces and intestinal contents of mice with CDSS diarrhea decreased significantly(P < 0.05),and the microbial activity of intestinal mucosa increased significantly(P < 0.01).The goblet cells and thickness of the mucus layer and Muc2 in intestinal tissue decreased significantly(P < 0.01).(Ⅲ)Metastat and LEf Se biomarkers of intestinal mucosal microbiota in model mice showed that probiotics such as Lactobacillus reuteri decreased significantly and conditional pathogens such as Enterobacter cloacae increased significantly(P < 0.05).The targeted amino acid metabolome showed that Ala and Glu were the differential amino acid metabolites of model mice(P < 0.05).Correlation analysis between intestinal mucosal microbiota and amino acid metabolism showed that the differential bacteria of diarrhea with CDSS were closely related to Glu,Met,and Asp(P< 0.05).(Ⅳ)After the treatment of HXZQ,the thickness of the mucus layer and Muc2 returned to normal(P < 0.05).(Ⅳ)After the treatment of HXZQ,the thickness of the mucus layer and Muc2 basically returned to the normal level(P < 0.05).(Ⅴ)HXZQ significantly increased the abundance of Lactobacillus reuteri and Lactobacillus johnsonii in model mice,and significantly reduced the abundance of Clostridium sensu stricto 1,Clostridium sp.ND2 and Helicobacter(P< 0.05).Density curve analysis showed that HXZQ could restore the Asp and Glu in model mice.Z-score analysis showed that HXZQ could increase the GABA in model mice.(Ⅵ)In vitro microbial culture showed that HXZQ could promote the proliferation of lactic acid bacteria in the intestinal of mice with CDSS diarrhea(P < 0.05).The microbial activity of intestinal contents in model mice increased significantly(P < 0.01).The activities of lactase,amylase,and protease in intestinal contents and mucosa increased significantly(P < 0.01).HXZQ could significantly reduce the lactase activity of intestinal contents in model mice(P <0.05),and significantly increase the xylanase and amylase activities of the intestinal mucosa(P< 0.05).Conclusion:(Ⅰ)"Low temperature and high humidity environment simulation in artificial climate box method" can establish a mouse syndrome model basically in line with the clinical manifestations of diarrhea with CDSS.(Ⅱ)Intestinal microbiota homeostasis has an important effect on the intestinal mucus barrier in mice with CDSS diarrhea.The decrease of Lactobacillus reuteri and the increase of Enterobacter cloacae lead to the imbalance of intestinal mucosal microbiota homeostasis in mice with CDSS diarrhea.The imbalance of intestinal mucosal microbiota homeostasis is related to amino acid neurotransmitters such as Glu and its related metabolites such as Gln and GABA.(Ⅲ)HXZQ can increase the abundance of probiotics such as Lactobacillus reuteri and Lactobacillus johnsonii in intestinal mucosa of model mice,reduce the abundance of Clostridium sensu stricto 1,Clostridium sp.ND2 and Helicobacter,adjust the homeostasis of intestinal mucosal microbiota and the metabolism of amino acid neurotransmitters such as Asp and Glu and protect the intestinal mucus barrier.(Ⅳ)HXZQ has an important effect on the activities of lactase,xylanase and amylase in mice with CDSS diarrhea,which may be related to the increase of energy demand caused by cold dampness stress.(Ⅴ)The metabolism of neurotransmitter amino acids mediated by intestinal mucosal homeostasis and microbiota homeostasis may be one of the important microecological mechanisms for the formation of CDSS diarrhea and the intervention of HXZQ. |
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