| PurposeThe aim of this study was to screen the effective components of traditional Chinese medicine(TCM)for the treatment of intestinal microflora disorder induced by antibiotics in mice,16 S r DNA high-throughput sequencing and Non-targeted metabonomics were used to reveal the effect of traditional Chinese medicine and its active components on the imbalance of intestinal flora induced by antibiotics,to explore the mechanism of effective components of traditional Chinese medicine in regulating intestinal flora imbalance induced by antibiotics.Methods1.To establish an animal model of intestinal dysbacteriosis induced by antibiotics.Six-week-old(20±2 g)male Balb/c strain SPF mice were randomly divided into blank control group and antibiotic-associated diarrhea(AAD)model group.The AAD group were treated with 0.2 ml lincomycin hydrochloride(3g/kg)twice a day for one week,while the CON group were treated with the same volume of normal saline.The success of the model was evaluated by observing the general condition of mice,HE staining of colon tissue and 16 S r DNA high-throughput sequencing analysis.2.To screen the Chinese herbs and their active components which can effectively regulate the imbalance of intestinal flora caused by antibiotics.Six-week-old(20±2 g)male Balb/c strain SPF mice were used,they were randomly divided into control(Con)group,AAD Group,inulin(Inu)group,Cistanche deserticola(RCR)group,Prunus mume(WM)group,Cistanche deserticola Polysaccharide(RCRDT)group and Echinacoside(ECH)group with 8 rats in each group,the AAD model was induced by intragastric administration of Lincomycin hydrochloride(3 g/kg)for 7 days.Then INU(5 g/kg),RCR(5 g/kg),WM(5 g/kg),RCRDT(200 mg/kg)and ECH(60mg/kg)were given intragastrically,once a day,0.2 ml once a day for 7 days.By observing general signs of mice,colon HE staining and 16 S r DNA high-throughput sequencing analysis,to evaluate the effects of Polysaccharides and Echinacoside from Cistanche deserticola,Cistanche deserticola and their active components on intestinal dysbacteriosis induced by antibiotics in mice.3.To explore the mechanism of Echinacoside in regulating intestinal dysbacteriosis induced by antibiotics.Six-week-old(20±2 g)male Balb/c strain SPF mice were randomly divided into control(Con)group,AAD Group,inulin(INU)group,low,middle and high dose Echinacoside(ECH)group,eight mice in each group were treated with Lincomycin hydrochloride(3 g/kg)for 7 days to establish AAD model,while the mice in CON group were treated with the same volume of normal saline.Then Inu(5 g/kg)、 LECH(30mg/kg)、MECH(60 mg/kg)and HECH(90 mg/kg)were given intragastrically,once a day,0.2 ml once a day for 7 days.The intestinal mucosal histopathology were evaluated by observing diarrhea symptoms,colon HE staining,the serum levels of IL-6,TNF-α and Il-10 were detected by ELISA,the expression of ZO-1 and Muc2 proteins were detected by Western blot,the intestinal flora was detected by 16 S r DNA high-throughput sequencing,and the metabolic pathways were analyzed by non-targeted metabolomics,to explore the mechanism of echinacea glycoside on intestinal dysbacteriosis induced by antibiotics in mice.Results1.Establishment of an animal model of intestinal dysbacteriosis induced by antibiotics.Compared with the CON group,the weight of mice in the AAD group decreased,the number and amount of defecation increased,the water content of feces increased,the cecum became congested and swollen,and all the mice in the AAD group showed diarrhea symptoms,and the colon tissue showed obvious inflammatory changes,the abundance and diversity of intestinal flora decreased significantly,and the contents of probiotics such as Muribaculaceae,Bacteroides,Lachnospiraceae,Alistipes and Lactobacillus decreased,Escherichia coli-Shigella,Proteus,Enterococcus and other opportunistic pathogens increased(p < 0.05).2.Both Cistanche deserticola and its active components can effectively regulate the intestinal flora imbalance of AAD mice,improve the diarrhea symptoms of mice.Compared with AAD group,the weight of mice in Inu,RCR and WM groups recovered,diarrhea symptoms were improved,and the structure of intestinal flora was adjusted.The contents of Firmicutes and Desulphurites were significantly decreased in INU,RCR and WM groups(p < 0.01),the content of Parasutterella increased while that of Proteus decreased(p < 0.01).In addition,the colonic pathology of RCR group recovered to normal level.α diversity showed that the abundance and diversity of intestinal flora in RCR group were better than those in WM Group,lefse analysis showed that the structure of intestinal flora in RCR group was better than that in WM Group,and the contents of Parasutterella,Streptococcus,Bacillus,Bifidobacterium and Faecalibacterium in RCR group were higher than those in WM Group(p < 0.01).Compared with the AAD Group,the RCRDT Group and ECH group recovered their body weight and improved their diarrhea symptoms,and the pathological changes of the colon in the ECH group recovered to the normal level,and the content of the intestinal firmicutes in the RCRDT group and ECH group decreased significantly,Blautia and Lachnoclostridium contents increased and Clostridium contents decreased(p < 0.05).In addition,the abundance and diversity of intestinal flora in ECH group recovered to normal level compared with RCRDT group,and the structure of intestinal flora was well adjusted,the contents of Bacteroides,Flavonifractor,Agathobacter,Lachnoclostridium,Prevotella-9 increased(p < 0.01).3.Echinacoside can effectively regulate intestinal dysbacteriosis in AAD mice.Compared with AAD group,the weight and diarrhea of INU Group,LECH Group,MECH Group and HECH Group were significantly improved,and the colon pathology of HECH group was restored to normal level(p < 0.05),HECH could decrease the levels of serum inflammatory factors IL-6 and TNF-α,increase the levels of anti-inflammatory factors IL-10,Muc-2 protein and ZO-1 protein in the colon of AAD mice(p < 0.05),these results suggest that echinacea glycoside can modulate the serum inflammatory factors of AAD mice,alleviate the intestinal inflammatory reaction,protect the intestinal barrier function of AAD mice and decrease the intestinal permeability,the effect of high-dose echinacea glycoside was better.In addition,the abundance and diversity of intestinal microflora in AAD mice recovered after HECH treatment.The structure of intestinal flora was well adjusted,and the contents of probiotics such as Lactobacillus,Bifidobacterium,Parabacteroides and Prevotella were increased(p<0.01).Non-targeted metabonomics showed that Echinacoside increased SCFA(Propionic acid,Butyric acid,Isobutyric acid)levels after treatment,it is associated with Fatty acid metabolism,vitamin B6 metabolism,tyrosine metabolism,L-Phenylalanine metabolism,amino sugars and Nucleotide sugars metabolism pathways(p < 0.05).Conclusion1.Both Cistanche deserticola and Prunus mume could regulate the imbalance of intestinal flora caused by antibiotics and improve the symptoms of AAD.2.Both Cistanche deserticola and its active components Polysaccharides and Echinacoside can regulate the intestinal microflora of AAD mice,regulate the beneficial and pathogenic bacteria in the intestinal tract,and restore the imbalance of intestinal microflora,it can improve the diarrhea symptoms of mice,especially the effect of Echinacoside.3.Echinacoside can protect intestinal barrier,reduce intestinal permeability and improve intestinal inflammatory reaction by increasing the content of short-chain fatty acid metabolite in intestinal flora,so as to relieve diarrhea symptoms in mice The intestinal microecology can be regulated by benign regulation of the beneficial and pathogenic bacteria in the intestinal tract to prevent bacterial translocation and repair the intestinal mucosal barrier. |
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