Character Istics Of Intestinal Flora In Lingnan Damp-heat Model Mice And Effects Of Traditional Chinese Medicine

ObjectiveLingnan region is characterized by high temperature,humidity and rainy climate.Lingnan people are weak in spleen and stomach and prefer seafood liquor.The dampness-heat syndrome of traditional Chinese medicine is mostly caused by external dampness and internal dampness.Lingnan dampness-heat syndrome just accords with the etiology and pathogenesis of dampness-heat syndrome of traditional Chinese medicine.The changes of intestinal microecology have similar symptoms with Damp-heat Syndrome of TCM.The mechanism of pathological state caused by the changes of intestinal microenvironment of damp-heat syndrome of TCM is not clear.The aim of this study was to establish Lingnan damp-heat model and to explore the changes of intestinal microflora and intestinal mucosal immune barrier under damp-heat pathological conditions and the effects of probiotics,Yinchen and Forsythia suspensa on Damp-heat syndrome.MethodsThe model of Lingnan damp-heat syndrome mice was established by the compound factor method of "external damp-heat+internal damp-heat+strain injury".The artificial climate chamber with full-closed automatic temperature and humidity regulation increased temperature and humidity,[T:(30+0.15)C,HR:80%-85%]12 hours a day,simulated the unique damp-heat external environment of Lingnan;"high-protein feed+liquor infusion every other day 0.3ml" simulated the internal damp-heat factors;and running 30 minutes daily simulated labor.The factor of spleen deficiency.After the completion of the model,probiotics,Yinchen and Forsythia suspensa were administered for 7 days respectively,while the control group was administered with ultra-pure water for 7 days.The daily symptoms and signs of mice in normal group,model group and drug group were observed,and the weekly weight of mice was measured.Pathological sections and HE staining were performed on the root of tongue and colon tissues of mice.The expressions of LPS,Muc2 and AQP1 in colon and intestine tissues of mice were detected by immunohistochemistry;goblet cells were stained by PAS and Pan cells were stained by fluorescent peach red lemon yellow;and antimicrobial peptides in colon and intestine tissues were stained by Global-def,Crypt,CRS4C,IFN-?,TLR2,TLR4 real-time fluorescence relative quantitative PCR detection;serum TNF-?,IFN-?,IL-6,LPS concentration enzyme-linked immunosorbent assay(ELISA);16S rRNA detection of intestinal flora in mice.ResultsThe typical damp-heat syndrome symptoms of model group mice were fatigue,sleepiness,reduced diet,sticky hair,short yellow urine and sticky stool.Histopathological HE staining of tongue showed increased keratosis of mucosa with hyperkeratosis,inflammation of lamina propria with connective tissue injury,and histopathological HE staining of colon showed gland injury.The results of goblet cell staining and Muc2 immunohistochemistry showed that the concentration and increase of goblet cells and mucus in the intestinal;the results of Pan' s cell staining and PCR of antimicrobial peptide indicated that the intestinal microflora disordered and the increase of intestinal harmful bacteria;the expression of AQP1 in the colonic tissue increased significantly,indicating the retention of intestinal water.The contents of TNF-?,IFN-? and LPS increased significantly(P<0.001),suggesting that the intestinal mucosal immune barrier was destroyed and the intestinal flora shifted.The OTU number of enteric bacteria increased,and the intestinal flora of PCA diagram changed greatly,suggesting that the number of intestinal flora increased,the species of bacteria increased,and the intestinal microbial environment became disordered in mice with Damp-heat model;the proportion of Bacilli and Ruminococcus decreased,Clostridia and Erysipelotrichi increased,and the proportion of Betaproteobacteria and Deltaproteobacteria increased in Proteus.The proportion of Prevotella in Bacteroides decreased,and the proportion of TM7-3 increased,suggesting that mice with damp-heat syndrome were in an unhealthy state,and the factors of damp-heat syndrome could have a negative effect on the body.Compared with the model group,(1)The pathological condition of mice in probiotic group was improved significantly,the structure of the lamina propria of tongue tissue was intact,but the keratinization of mucosa was not improved;the pathological changes of colon tissue were improved significantly;the number of Pan' s cells and the expression of antimicrobial peptides in colon tissue decreased;the content of IFN-y decreased(P<0.001),and the content of IL-6 increased(P(0.05),LPS content decreased,intestinal mucosal immune barrier function and intestinal flora improved significantly.(2)In Yinchen group,the pathological condition of mice was improved,the lamina propria and muscularis of tongue tissue have a tendency to improve,and the histopathological section of colon showed that the histological structure was improved;the expression of AQP1 in colon tissue was significantly reduced;the content of IFN-? in serum was decreased(P<0.01);the contents of intestinal flora TM7-3,Desulfobrio and Escheria were increased.(3)The pathological symptoms and signs of mice in Forsythia suspensa group were improved;the histological sections of tongue and colon showed that the histological structure improved,but the keratosis of tongue mucosa did not decrease;the expression of AQP1 in colon tissue did not decrease significantly;the levels of serum inflammatory factors TNF-? and IFN-? decreased significantly(TNF-?,P<0.05;IFN-?,P<0.001),and the levels of IL-6 increased(P<0.05);Dow flora did not improve significantly.ConclusionAccording to the modeling method of damp-heat syndrome model in traditional Chinese medicine,the typical clinical symptoms of damp-heat syndrome appeared in Lingnan damp-heat syndrome model mice,suggesting that the model was successful.The model mice showed slow weight gain,tongue tissue damage and mucosal keratinization increase,colon tissue damage,intestinal microflora disorder,intestinal mucosal immune barrier damage and intestinal flora displacement.Probiotics can improve the symptoms,signs and colon tissue damage of model mice,especially the intestinal microflora,improve the intestinal disorder flora environment,and repair the intestinal mucosal immune barrier,but it can not reduce the keratosis of the tongue mucosal layer.This may be due to the role of probiotics in intestinal flora,for example,for intestinal microflora.Oral flora had no significant regulatory effect.The effect of Yinchen and Forsythia suspensa on dampness-heat syndrome of TCM is mainly in the aspects of water metabolism and immune inflammation,but it has no obvious improvement on intestinal microflora,but it has significant effect on the overall improvement of the body of model mice.The pathological results of tongue and colon tissue also show that the tissue damage has been well recovered.This shows that Yinchen and Forsythia suspensa,as natural medicines,can achieve a good fit with natural organisms.Their role is not limited to a certain point,but to the overall regulation of organisms,which is different from the combined probiotics.