Effect Of Huangqin Tang On Intestinal Flora In Mice With Ulcerative Colitis And Protective Mechanism Of Intestinal Mucosal Barrier

Objective:Ulcerative colitis(UC)is a chronic non-specific inflammatory disease of the rectum and colon and is a kind of inflammatory bowel disease(IBD).Which is often recurrent,refractory protracted illness,has been recognized as one of the three most import ant risk factors for colon cancer.Its etiology and pathogenesis are complicated.There is no technique to affect a radical cure at present.Therefore,UC-related mechanisms of pathogenesis and drag intervention research is very critical.Huangqin Tang(HQT)is a famous prescription from Shanghan Lun.It has been widely used to treat gastio intestinal diseases.A growing number of studies have confirmed that dysbacteriosis is one of the predisposing factors of ulcerative colitis.Through drug intervention,regulating the microecological balance of intestinal flora has become a new strategy for the treatment of UC.However,it has not been reported whether Huangqitang can regulate the microecological balance of intestinal microbiota and its relationship with protecting intestinal mucosal barrier and inhibiting inflammatory response.This study was based on 16SRNA high-throughput sequencing technology,intestinal bacterial interference and mouse model of ulcerative colitis,and a variety of molecular biological techniques to evaluate the pharmacodynamic effects and pharmacological molecular mechanisms of Huangqi Decoction on UC model mice.Investigate whether Huangqi Decoction plays a protective role in the UC by regulating intestinal flora.Method:Eighty 8-week-old SPF grade male Balb/c mice were randomly divided into 8 groups:Control group,Model group,High dose group of HQT(HQT-H),and Medium dose gropu of HQT(HQT-M),Low dose group of HQT(HQT-L),Antibiotic interference group(AB),Antibiotic interference model group(AB+DSS),Antibiotic interference High dose group of HQT group(AB+DSS+HQT).After one week of adaptive feeding,the experiment was started.First,prepare the intestinal flora interference model.AB,AB+DSS,AB+DSS+HQT three groups were given mixed antibiotics(bacilli peptide 200mg/kg,vancomycin 200mg/kg)Gavage for 7 consecutive days.Immediately after the collection of the bacterial colony AB fresh feces,the intestinal microbiota interference model was tested for success.Then,mouse ulcerative colitis model was induced by 3%sodium dextran sulfate(DSS)induction.Except for the normal control group,all other groups were given 3%DSS solution for 7 days of free drinking.After 4 days of free drinking,HQT-H,HQT-M,HQT-L,and bacterial interference groups AB+DSS+HQT.Daily dose of HQT at regular intervals,for 7 consecutive days.The body weight and disease activity index(DAI)index of each group were recorded during the experiment.After the last administration,feces of each group of mice were collected.After sacrifice,each group of mouse sera and colon tissue were collected.The levels of IL-4,IL-6,IL-10,and TNF-a in serum of mice were determined by enzyme-linked immunosorbent assay(ELISA);the gene of interest(IL-4)was detected by qPCR.The expression levels of IL-6,IL-10,TNF-?,Occludin,Claudinl,MUC1,ZO1)mRNA were detected by Western blotting and Western blot was used to detect the intestinal mucosal barrier proteins(Occludin,Claudinl,MUC1,ZO1).The expression of the expression was based on the design of primers for the 16S RNA V3-V4 region of bacteria,and the intestinal fecal microorganisms were sequenced on the Illumina Miseq platform.Result:1.After intragastric administration of mixed antibiotics,compared with the control group,the AB,AB+DSS,and the bacterial interference group AB+DSS+HQT.The mice in the three groups had reduced autonomic activity,reduced intake of water,and decreased body weight.Sequencing results of intestinal microbiota showed that the species diversity and abundance in fecal samples from the AB were significantly lower than those in the normal group.Principal component analysis(PCA),principal coordinate analysis(Pcoa),and diversity based on Beta Distance non-metric multidimensional scale analysis(NMDS)results show significant differences compared to the normal group.The above results indicate that the bacterial population disturbance model was successfully prepared.2.After drinking freely 3%dextran sodium sulfate(DSS),compared with the control group,model mice exhibited anorexia,weight loss,apathy,and diarrhea.The results of HE staining showed that compared with the control group,the model group(Model)had abnormal crypts,reduced goblet cells,disappearance of colon glands,marked thinning of the mucosal layer,severe epithelial damage,and infiltration of a large number of inflammatory cells in the muscular layer.The above results indicate that the ulcerative colitis model was successfully prepared.3.Compared with the control group,the serum levels of IL-4,IL-6 and TNF-a in the mouse of the model group(Model)increased significantly(P<0.01),and the IL-10 concentration decreased significantly.P<0.01),indicating that the mice in the model group had a severe inflammatory reaction.Compared with the model group,IL-4 in the serum of high-dose Huangqitang(HQT-H),middle-dose Huangqitang(HQT-M),and low-dose Huangqitang(HQT-L)mice.The concentrations of IL-6 and IL-10 were significantly decreased(P<0.01 or P<0.05),and the concentration of IL-10 was significantly increased(P<0.01),indicating that the administration of HQT can reduce the inflammatory response.4.Compared with the control group,the mRNA expression levels of IL-4,IL-6,and TNF-a in the model mice increased significantly(P<0.05 or P<0.01).After administration of HQT,the mRNA levels of pro-inflammatory cytokines IL-4,IL-6 and TNF-a were significantly decreased(P<0.05 or P<0.01).The expression levels of Occludin,Claudinl,MUC1,and ZO1 proteins in the model group(Model)were significantly reduced(P<0.01).In each administration group,high doses significantly up-regulated mRNA levels of MUC1 and Claudinl(P<0.01),mid-dose significantly up-regulated mRNA levels of Occludin and ZO1(P<0.01),and low doses significantly increased ZO1 protein levels.mRNA levels(P<0.01).It shows that HQT can significantly regulate the expression of inflammatory factors related genes and intestinal mucosal barrier protein gene expression to play a role in the treatament of UC.Compared with the control group,the Occludin,Claudin1,and ZO1 protein expressions of the model group(Model)decreased significantly(P<0.05 or P<0.01),and the expression of MUC1 protein was not significantly different.Compared with the model group,Occludin,Claudinl in the high dose group of Huangqitang(HQT-H),middle dose group of Huangqitang(HQT-M)and low dose group of Huangqitang(HQT-L)The expression of MUC1,ZO1 protein was increased(P<0.05 or P<0.01).Among them,the Occludin and claudin in the HQT-H group were significantly different(P<0.05 or P<0.01),and the ZO1 in the HQT-M group was significantly different(P<0.01).It was demonstrated that HQT exerts protective effect on intestinal mucosal barrier by increasing the expression of intestinal mucosal barrier proteins.6.Compared with the model group,the concentration of IL-4,IL-6,and TNF-a in the AB+DSS decreased significantly(P<0.05).IL-4,IL The mRNA level of-6 gene was significantly decreased(P<0.05 or P<0.01),indicating that the inflammatory response was reduced after the bacterial colony interference.Compared with the model group,the expression of each protein in the AB+DSS was reduced,but the difference was not significant,indicating that intestinal mucosal barrier injury exists after the bacterial population interferes.7,gut microbiota sequencing results:(1)The results of community composition analysis showed that,at the level of the phytoplankton community,the dominant phyla of the bacteria in the other groups were more than 80%of Firmicutes and Bacteroidetes.(2)The results of species test among the sample groups showed that,at the level of the gate,Bacteroidetes and Tenericutes of the model group(Model)significantly decreased compared with the control group(P<0.05).The Proteobacteria in the AB increased significantly(P<0.01),and the Firmicutes and Bacteroidetes significantly decreased(P<0.01).Compared with the model group,Bacteroidetes were significantly increased in the AB+DSS(P<0.05),and Firmicutes and Proteobacteria were significantly reduced(P<0.05 OR P<0.01).At the genus level,norank_f_Bacteroidales_S24-7_group(Bacteroides)was significantly reduced(P<0.01),and Escherichia-Shigella(Escherichia coli Shiga)was significantly increased(P<0.01)in the model group(Model)compared with the control group(Control).),Prevotellaceae_UCG-001(Prevotella)was significantly reduced(P<0.01),Staphylococcus(Staphylococcus)was significantly reduced(P<0.05),Odoribacter was significantly reduced(P<0.05),Alistipes(Scientia)Significantly reduced(P<0.05).This shows that the composition and abundance of intestinal flora.in the model group(Model)are significantly different from those in the control group.(3)The results of multiple comparisons showed that,at the level of the portal,Bacteroidetes and Tenericutes were significantly increased in the three groups of HQT(P<0.01 OR P<0.05).The number of Firmicutes decreased significantly(P<0.05).At the genus level,the abundance of Lactobacillus(Lactobacillus),Lachnospiraceae—NK4A136_group(Laccharomyces),Escherichia-Shigella(Escherichia coli Shiga),Helicobacter(Helicobacter)in the high,middle and low groups and the dose of HQT It is directly proportional to the fact that they are related to the role of Huangqitang in the treatment of UC.Conclusion:1.HQT has the effect of treating DSS-induced mouse UC model,and its mechanism may be to decrease the levels of serum inflammatory cytokines IL-4,IL-6,TNF-a and down-regulate the mRNA expression level,increase serum anti-inflammatory factors.IL-10 levels play an inhibitory role in inflammation.By increasing the expression of the proteins of the intestinal mucosal barrier proteins,such as Occludin,Claudinl,MUC1,and ZO1,intestinal mucosal barrier protection was exerted.2.The composition and abundance of gut microbiota in UC model mice are significantly different from those in normal mice.Bacteroidetes and Tenericutes are significantly reduced.Escherichia-Shigella(Escherichia coli Shiga)Significantly increased(P<0.01),Bacteroidales_S24-7_group(Bacteroides),Prevotellaceae_UCG-001(Prevotella),Staphylococcus(Staphylococcus),Alistipes(Rhizopus),Odoribacter were significantly reduced(P<0.05).Therefore,the disturbance of the microecological balance of the intestinal flora is a key factor in inducing UC.3.HQT may regulate Bacteroidales_S24-7_group(Bacteroides),Prevotellaceae—UCG-001(Prevotella),Erysipelato clostridium,Ruminococcaceae_UCG-014(Rumenia),Eubacterium_fissicatena_group(Euperia),Escherichia-Shigella((Escherichia coli Shiga),Abundance value of Lachnospiraceae_NK4A136_group(Loromyces)plays a role in the treatment of UC.4.Intestinal flora is a key factor in inducing inflammatory responses.Normally balanced intestinal flora has a protective effect on the intestinal mucosal barrier.In the disordered intestinal microflora,Akkermansia(E.mutans-Ekman)aggravate the inflammatory reaction by decreasing the thickness of the mucus layer.5.HQT can play a role in the treatment of UC by regulating the intestinal flora and protecting the intestinal mucosal barrier.