| Objective: To establish a diarrheal mouse model with intestinal dampness-heat syndrome(IDHS)based on the traditional Chinese medicine(TCM)pathogenesis and clinical features and treat the model with Ge-gen-qin-lian decoction(GD),investigate the effect of the model preparation on the intestinal microecology to disclose the microecological characteristics in the diarrheal mice with IDHS,investigate the influence of GD on the intestinal microecology in diarrheal mice with IDHS to disclose the relationship between the efficacy of GD and intestinal microecology.The results will provide references for the diagnosis and treatment research of different syndrome diarrhea,and the study of the efficacy mechanism of TCM.Method: The diarrheal mice with IDHS were established by the complicated pathogenic factors: the mice were fed with high sugar and fat diet first,then they were moved in a high temperature and humidity environment for 4 days with gastric infusion of alcohol in the morning and evening,and administration with freezing water at noon until all the model group mice occurred diarrhea,then GD was administrated to them.Then samples were gathered as soon as the model was successfully established and treatment,respectively.Mice eye blood was gathered for analysis of the substance P(SP)concentration,biochemistry index and blood routine index.Mouse organs were removed out to be weighted.Microbial culture technology was used to observe the changes of microbes in mice intestine.The intestinal mucosae and contents were collected under sterile condition following with the microbial activity analysis by Fluorescein diacetate(FDA),the activity determination of digestion enzyme by corresponding methods.Total bacterial genomic DNA samples in mice intestinal contents were extracted using CTAB,PCR amplication of the bacterial full length 16 S r RNA genes was performed to generate a library,and sequencing was performed using the Pac Bio platform based on Single molecule Real-Time sequencing following with the analysis of the bacterial characteristic.Results:(Ⅰ)Compared with the control group,the model mice activity decreased.Their weight gained slowly,their rectal temperature increased,their stool became tawny and thin causing the anus dirty.Compared with the control group,the SP concentration were increased obviously(P < 0.01)but TG was decreased(P < 0.05),the values of TC and GLU increased in the model mice blood.After treatment with GD,the diarrheal mice with IDHS regained its normal status,the concentration of SP recovered,TG and GLU level changed toward those of the control group.(Ⅱ)The amounts of WBC,GRA and EOS in the model mice blood increased and MCV decreased compared to the control group.The treatment group blood had less numbers of WBC and EOS than the untreated group,and the values of GRA and MCV returned to that of the control group(P > 0.05).The thymus index of the treatment group was significantly higher than that of the control group.(Ⅲ)Microbial culture results showed that numbers of Bacteria and Bifidobacterial in the model mice intestine were reduced significantly(P < 0.01 or P < 0.05).In the model mice intestine,the microbial activity increased significantly(P < 0.01).The activities of lactase,sucrase and amylase in the model mice intestinal contents increased,and protease activity decreased,however reversed changes appeared in these enzyme activities of the model mucosae.After treatment with GD,the treatment group exhibited more Lactobacillus number than the control group(P < 0.01),lower microbial activity than the untreated group,higher activities of lactase,sucrase,amylase and protease in the intestinal contents than the control group,and lower activities of lactase,sucrase,amylase and protease in the intestinal contents than the untreated mice.The lactase,sucrase,and amylase activities in the mucosae of the treatment mice surpassed that of the other two groups.(Ⅳ)The bacterial 16 S r RNA gene sequencing results showed that the alpha diversity index in the model group was increased,but difference between the model and control groups was not clear.PCo A and UPGMA clustering figures revealed that the model samples deviated from the control samples.At the phylum level,the model group had higher abundances of Bacteroidetes、Proteobacteria、Actinobacteria,and lower percentages of Cyanobacteria and Firmicutes,the abundance difference of Actinobacteria between the model group and control group was significant(P = 0.0325).The model group had lower proportion of Clostridium and Lactobacillus and higher proportion of Muribaculum、Streptococcus、Parasutterella、Prevotella、Enterorhabdus than the control group,and the proportion difference of Clostridium between the model and control groups was obvious distinction(P = 0.047).At the species level,the model group had lower abundances of Lactobacillus gasseri、Lactobacillus intestinalis、Lactobacillus reuteri、Lactobacillus vaginalis、Curvibacter lanceolatus and higher percentages of Muribaculum intestinale、Lactobacillus crispatus 、 Lactobacillus murinus 、 Staphylococcus Epeidermidis 、Porphyromonas gingivalis、Parasutterella excrementihominis、Enterorhabdus muris 、 Streptococcus acidominimus and Enterorhabdus mucosicola than the control group.LDA effect size(Lefse)exhibited that the relative abundance of Lactobacillus gasseri showed a significant difference in the control group.(Ⅴ)After treatment with GD,Alpha index of the treatment group returned to the level of the control group(P > 0.05).PCo A showed that the points representing the treatment group samples deviated from that of the control group samples.At the phylum level,the treatment group had a higher abundance of Bacteroidetes and lower percentages of Proteobacteria and Cyanobacteria than the control group,the treatment group and control group had almost equal percentage of Firmicutes.At the genus level,the treatment group had higher abundances of Muribaculum and Clostridium than the control group.The relative abundance difference of Lactobacillus between the control and treatment group was small.The relative abundance of Streptococcus and Neisseria in the treatment group and untreated group was almost equal.At the species level,the treatment group had a lower proportion of Lactobacillus reuteri and higher percentage of Muribaculum intestinale than the control group,but the difference was not clear.The abundance of Lactobacillus crispatus in the treatment group restored to that of the control group.Conclusion:(1)The diarrheal model with IDHS was successfully established.The efficacy of GD on the model confirmed it.(2)The model preparation induced the abnormal index of blood routine,GD had effect on WBC,GRA,EOS and MCV returning to that of the control group level,and benefited thymus to strengthen the host immune system.(3)The model preparation affected the numbers of the intestinal microbes and increased the intestinal microbial activity,changed the activities of digestion enzymes.GD could promote the proliferation of Lactobacillus,decrease the microbial activity,regulate the digestion enzyme activities in the diarrheal mice.(4)The bacterial community in the model mice intestine was changed inducing the imbalance of intestinal microecology,the decrease abundance of Lactobacillus gasseri may have correlation with diarrhea.GD could restore the bacterial community diversity,change the relative abundance of bacterial species in mouse intestine to regulate the balance of the intestinal microecology,which might reveal the therapeutic mechanism of GD on treating diarrhea with IDHS. |
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