| Background and Aim: Gastric cancer is the fifth most common malignant tumor in the world.Its occurrence is a multi-factor and multi-step process,which is affected by genetic factors,environmental factors and Helicobacter pylori(Hp)infection.Hp,a common gastric pathogen,infects more than 50% of the global population.Hp infection can cause several diseases including chronic gastritis,peptic ulcer,gastric mucosa-associated lymphoid tissue(MALT)lymphoma and gastric cancer.Almost all Hp-infected patients have chronic active gastritis.However,only 1-3% of Hp-infected patients eventually develop into gastric cancer,indicating that there are potential factors other than Hp that play an important role in gastric carcinogenesis.Recently,many studies have demonstrated that the microbiota is closely associated with the occurrence and development of diseases.The perturbation of gastric microbiota in gastric carcinogenesis has gradually attracted attention.Current studies mainly focus on association between gastric cancer and gastric mucosal microbiota.However,there is a lack of research on gastric fluid microbiota.In addition,further study is required to investigate the relationship between alterations of gastric microenvironment(gastric acid,microbiota and metabolites)and gastric cancer risk.It has been reported that Hp infection can affect the structure of gastrointestinal microbiota,which may be involved in Hp-induced gastric inflammation and gastric carcinogenesis.Previous studies have shown that probiotics could inhibit inflammation,modulate immunity and microbiota.Further,it has been demonstrated that probiotics could inhibit Hp-induced inflammation.However,the effect of probiotics on Hp-induced gastric carcinogenesis has not been deciphered.Eradicating Hp can significantly reduce gastric cancer risk.The PPI and antibiotics included in the eradication regimen can influence the gastrointestinal microbiota.Disturbance of the gastrointestinal microbiota is closely associated with diseases.Hence,the large-scale eradication of Hp should consider its impact on the gastrointestinal microbiota.Therefore,we studied the alterations of gastric microenvironment(gastric acid,gastric mucosal microbiota,gastric fluid microbiota,metabolites)in gastric carcinogenesis,as well as the short-and long-term effects of Hp eradication on gastrointestinal microbiota at the human level;In addition,this study also explored the influence and possible mechanism of probiotics on Hp-induced gastric carcinogenesis,as well as the modulatory effects of probiotics on the gastrointestinal microbiota,gut barrier function and the contents of short-chain fatty acids after Hp eradication at animal level.Materials and Methods: 1.We have collected gastric mucosa and fluid samples from patients with gastritis,intestinal metaplasia,and gastric cancer patients.Extract sample DNA and amplify the target 16 S r RNA V4 hypervariable region segment.After sample mixture,purification and library construction,it is ready for subsequent sequencing.Paired-end sequencing was performed using the Illumina Mi Seq high-throughput sequencing platform.When the sequencing has finished,bioinformatics methods are used to analyze the structure of gastric mucosa and gastric fluid microbiota.A pen-shaped p H meter was used to detect the gastric fluid p H.The naphthalene ethylenediamine hydrochloride method was performed to detect the nitrite contents,the enzymatic cycling assay was performed to detect the content of total bile acid,and the gas chromatography method was performed to detect the content of short-chain fatty acids in the gastric fluid.2.Hp PMSS1-infected INS-GAS mouse model was constructed.The control group was INS-GAS mice gavaged with Brucella broth.Both groups were given either water with probiotics(Lactobacillus salivarius and Lactobacillus rhamnosus)or drinking water.Mouse feces were collected before sacrificed.After the mice were sacrificed,the gastric p H of the mouse was measured with a pen-shaped p H meter,and the mouse stomach tissues were collected.Steiner staining method was applied to examine Hp colonization in mouse stomach.HE staining method was used to observe pathological changes in mouse stomach.Immunohistometaplasia staining was used to detect the expression of CD45 in gastric tissue.Extract sample DNA and amplify the target 16 S r RNA V3-V4 hypervariable region segments.After sample mixture,purification and library construction,it is ready for subsequent sequencing.Paired-end sequencing was performed using the Illumina Mi Seq high-throughput sequencing platform.When the sequencing has finished,bioinformatics methods are used to analyze the structure of gastrointestinal microbiota in mice.Extract mouse gastric tissue RNA,enrich m RNA,fragment the m RNA and reverse synthesize c DNA,connect the adaptor to perform fragment screening and library enrichment,and the subsequent sequencing was performed on Illumina Hi Seq Xten high-throughput sequencing platform.Bioinformatics methods were applied to analyze the sequencing data.The RT2 Profiler PCR Array method was performed to analyze the gene expression related to "inflammatory response and autoimmunity" in gastric tissues.Western blotting was used to detect TLR2 and IL-17 expression in gastric tissue.3.Recruit asymptomatic young adult volunteers,detect the Hp infection status of volunteers by 13 C urea breath test,and collect volunteers' feces.Endoscopy was performed on Hp-positive volunteers and gastric mucosal samples were collected.Then,Hp-positive patients were given a bismuth quadruple regimen for 14 days(esomeprazole 20 mg,bismuth potassium citrate 220 mg,amoxicillin 1000 mg,furazolidone 100 mg,bid)to eradicate Hp.Four weeks after eradication,13 C urea breath test was performed to confirm Hp status.We collected the feces of volunteers received.Endoscopy was performed on volunteers received Hp eradication therapy and gastric mucosal samples were collected.Twenty-four weeks after the eradication,the feces of Hp-eradicated volunteers were collected,and the Hp-eradicated volunteers were subjected to endoscopy and gastric mucosal samples were taken.Extract sample DNA and amplify the target 16 S r RNA V3-V4 hypervariable region fragments.After sample mixture,purification and library construction,it is ready for subsequent sequencing.Paired-end sequencing was performed using the Illumina Mi Seq high-throughput sequencing platform,and bioinformatics methods were used to analyze the structure of the gastrointestinal microbiota in volunteers.4.Hp-infected C57BL/6 mouse model was built using Hp PMSS1 strain.The control group was C57BL/6 mice gavaged with Brucella broth.Both groups were given either eradication drugs or normal saline for 14 days.Probiotics(Enterococcus faecium R0026 and Bacillus subtilis R0179)were given to mice that had been treated with eradication drugs for 4 weeks.Collect mouse feces,gastric mucosa and colon samples at the endpoint of eradication treatment and probiotic treatment,respectively.Giemsa staining was used to detect Hp colonization in mouse stomach,and HE staining was used to observe the inflammation and pathology of the mouse gastric tissue.Extract sample DNA and amplify the target 16 S r RNA V3-V4 hypervariable region fragments.After sample mixture,purification and library construction,it is ready for subsequent sequencing.Paired-end sequencing was performed using the Illumina Mi Seq high-throughput sequencing platform,and bioinformatics methods were used to analyze the structure of the gastrointestinal microbiota in mice.The Gas chromatography was performed to detect the contents of SCFA in mouse feces,and Western blotting was used to detect Occuludin and Claudin1 expression in mouse colon tissue.Results: Changes in the structure of gastric microbiota and the content of specific metabolites in gastric carcinogenesis and their relationship with Hp infection.1.Alterations in gastric mucosal microbiota during gastric carcinogenesis(1)? diversity analysis: the Chao and Shannon indexes of the gastric mucosal microbiota in gastritis patients were not significantly different from those of patients with intestinal metaplasia(P>0.05),and the Chao index of gastric mucosal microbiota in gastric cancer patients was significantly higher than that in patients with gastritis(P<0.01)and intestinal metaplasia(P<0.001),Shannon index was also significantly higher than that in patients with gastritis(P<0.01)and intestinal metaplasia(P<0.01).There was no significant difference in the Chao index between the adjacent gastric tissue and the cancerous tissue microbiota,while the Shannon index of the cancerous tissue microbiota was significantly higher than that of the adjacent gastric mucosal microbiota in gastric cancer(P<0.05).In addition,we also compared the ? diversity of mucosal microbiota adjacent to gastric cancer tissues and gastritis and found that the Chao index of the adjacent mucosal group was significantly higher than that of the gastritis group(P<0.05),while the Shannon index was not significantly different.The Chao index(P<0.001)and Shannon index(P<0.001)of gastric mucosal microbiota in Hp-positive patients were significantly reduced.The Chao index and Shannon index of the Hp positive group in the gastritis group and the intestinal metaplasia group were significantly lower than the Hp-negative group(all P<0.001).However,there was no significant difference in the Chao index and Shannon index between the Hp-positive and Hp-negative gastric mucosal microbiota in gastric cancer patients.(2)? diversity analysis: PCo A analysis based on different distance algorithms found that the gastritis gastric mucosa group and the intestinal metaplasia mucosa group cannot be effectively distinguished,while the gastric cancer gastric mucosal group and the other two groups have a clear trend of separation(ANOSIM,Bray-curtis P=0.001,weighted Uni Frac P=0.001).Gastric mucosal samples from gastric cancer tissues tend to separate from adjacent samples(ANOSIM,Bray-curtis P = 0.07,weighted Uni Frac P=0.02).There are significant differences in the structure of gastric mucosal microbiota between adjacent cancerous tissues and gastritis gastric mucosa(ANOSIM,Bray-curtis P=0.01,weighted Uni Frac P = 0.03).The Hp-positive and Hp-negative gastric mucosa samples were clearly separated(Adonis,Euclidean P=0.001;ANOSIM,weighted Uni Frac P=0.001).Among them,the gastritis group(Adonis,Euclidean P=0.001;ANOSIM,weighted Uni Frac P=0.001)and the intestinal metaplasia group(ANOSIM,Bray-curtis P=0.001,weighted Uni Frac P=0.001)have a good separation regarding Hp negative and positive samples,following with the gastric cancer group(ANOSIM,Bray-curtis P = 0.057,weighted Uni Frac P = 0.002).(3)LEFSe analysis: Bacillus in the gastritis group was significantly higher than that in intestinal metaplasia group and gastric cancer group.In the stage of gastric cancer,the relative abundance of Lactobacillus,Prevotella,Veillonella,Haemophilus,Fusobacterium and Neisseria were significantly increased.The relative abundances of Clostridia,Prevotella7,Prevotella,Johnsonella and Solobacterium in gastric cancer tissues were significantly higher than those in adjacent tissues.The relative abundance of Bacillus in the gastritis gastric mucosa group was significantly higher than that in the adjacent cancerous tissue group,while the relative abundance of Lactobacillus,Prevotella7,Veillonella,Fusobacterium,and Neisseria were significantly lower.The relative abundance of Helicobacter in the Hp-positive gastric mucosa group was significantly higher than that in the Hp-negative gastric mucosa group,while the relative abundance of Bacillus,Lactobacillus,Rothia,Streptococcus,Fusobacterium was significantly reduced.Among stages of gastritis,intestinal metaplasia,and gastric cancer,the relative abundance of Helicobacter in the Hp-negative subgroup was significantly lower than that of the Hp-positive subgroup,while the relative abundances of Lactococcus,Bacillus,Pseudomonas,Streptococcus and Neisseria in the Hp-negative gastritis gastric mucosa group were significantly higher than those in the Hp positive subgroup.The relative abundance of Lactococcus,Bacillus,Acinetobacter,Streptococcus and Ralstonia in the Hp-negative intestinal metaplasia gastric mucosa group was significantly higher than that in the Hp-positive intestinal metaplasia gastric mucosa group.Compared with the Hp-positive gastric cancer gastric mucosa group,the relative abundances of Streptococcus,Brevibacterium,Ochrobactrum,Ralstonia,and Rothia in the Hp-negative gastric mucosa group were significantly increased.2.Alterations in gastric fluid microbiota during gastric carcinogenesis(1)? diversity analysis: Shannon index of gastritis gastric fluid group is not significantly different from that of intestinal gastric fluid group,Shannon index of intestinal gastric fluid group is not significantly different from that of gastric cancer gastric fluid group,and Shannon index of gastric cancer gastric fluid group is significantly lower than that of gastritis gastric fluid group(P<0.01).There was no significant difference in Chao index and Shannon index between the gastric fluid microbiota of Hp-negative and Hp-positive patients.There was no significant difference in Chao index and Shannon index between the Hp-positive gastritis gastric fluid group and the Hp-negative gastric fluid group;the Chao index and Shannon index of the Hp-positive intestinal metaplasia gastric fluid group were not significantly different from the Hp-negative intestinal metaplasia gastric fluid group,but the Simpson index Significantly lower than the Hp-negative intestinal metaplasia gastric fluid group(P<0.05).(2)? diversity analysis: PCo A analysis based on the unweighted Uni Frac distance shows that the gastric fluid samples of the gastritis and intestinal metaplasia groups are relatively close,while the gastric cancer group shows a trend of separation(ANOSIM,unweighted Uni Frac P=0.001).However,weighted Uni Frac distance-based PCo A analysis showed that samples in three groups are difficult to distinguish(ANOSIM,weighted Uni Frac P=0.17).The PCo A analysis based on the unweighted Uni Frac distance showed that samples in Hp-positive and Hp-negative groups are very close(ANOSIM,weighted Uni Frac P=0.399),while the PCo A analysis based on the weighted Uni Frac distance shows significant differences between Hp-positive and negative gastric fluid microbiota(ANOSIM,Weighted Uni Frac P=0.001).PCo A analysis based on Euclidean distance and weighted Uni Frac distance showed significant differences in the distribution of Hp positive and negative gastritis gastric fluid groups(ANOSIM,Euclidean P=0.02,weighted Uni Frac P=0.018).Similarly,based on the Euclidean distance and weighted Uni Frac distance,the PCo A analysis of intestinal metaplasia and gastric cancer gastric fluid samples showed that the distribution of Hp-positive and Hp-negative intestinal metaplasia gastric fluid samples was significantly different(ANOSIM,Euclidean P=0.001,weighted Uni Frac P=0.001),while there was no significant difference in the distribution of gastric fluid samples from Hp-positive and Hp-negative gastric cancer(ANOSIM,Euclidean P=0.06,weighted Uni Frac P=0.17).(3)LEFSe analysis: the relative abundances of Pseudomonas,methylobacterium,Treponema2,Pelomonas and Acidovorax in the gastritis group are significantly higher than those in intestinal metaplasia and gastric cancer groups,while the relative abundances of Ochrobactrum,Campylobacter,Acinetobacter,Paracoccus and Staphylococcus in the intestinal metaplasia group are higher than the gastritis and gastric cancer groups,the relative abundance of Lactobacillus,Brevundimonas,Enhydrobacter and Megasphaera in the gastric fluid microbiota of gastric cancer groups was significantly higher than that of the gastritis and intestinal metaplasia groups.The relative abundance of Helicobacter,Selonomonas4,Corynebacterium and DefluviitaleaceaeUCG011 in the gastric fluid microbiota of the Hp-positive group was significantly higher than that of the Hp-negative group,while the relative abundance of Streptococcus,Haemophilus and Gemella was significantly reduced.The relative abundances of Helicobacter in the Hp-positive gastritis gastric fluid group were significantly higher than that in the Hp-negative gastritis gastric fluid group,while the relative abundances of Bosea,Corynebacterium and Stenotrophomonas were significantly decreased.Compared with the Hp-negative intestinal gastric fluid group,the relative abundances of Helicobacter and Achromobacter in the Hp-positive intestinal gastric fluid group were significantly increased,while the relative abundance of Streptococcus,Veillonella,Gemella,Rothia and Granulicatella were significantly decreased.In the stage of gastric cancer,the relative abundance of Helicobacter and RikenellaceaeRC9gutgroup in the Hp-positive gastric fluid group was significantly higher than that of the Hp-negative gastric fluid group,while the relative abundance of Corynebacterium was significantly lower than that in the Hp-negative gastric fluid group.3.Comparison of the characteristics of gastric mucosa and gastric fluid microbiota in gastric carcinogenesis(1)Overall comparison(a)? diversity analysis: Observed species index(P<0.05)and Shannon index(P<0.001)of gastric mucosal microbiota were significantly lower than those of gastric fluid microbiota.The Chao index of gastric cancer gastric mucosal group was significantly higher than that of the other five groups(P<0.001).In the process of gastritis? intestinal metaplasia ? gastric cancer,Shannon index showed a gradual decrease in gastric fluid,while a gradual increase in gastric mucosa.(b)? diversity analysis: There is a significant difference in the microbiota between gastric mucosa samples and gastric fluid samples(ANOSIM,unweighted Uni Frac P=0.001,weighted Uni Frac P=0.001).Based on the Euclidean distance and Bray-curtis distance,the PCo A analysis of gastric mucosa and gastric fluid samples among the stages of gastritis,intestinal metaplasia,and gastric cancer found that the gastric mucosa samples in the gastritis and intestinal metaplasia groups were significantly separated from the gastric fluid samples in the gastritis and intestinal metaplasia groups,while gastric fluid samples in different disease stages cannot be effectively distinguished.The gastric cancer gastric mucosal group samples have a significant tendency to separate from the gastritis and intestinal gastric mucosal groups,and exhibit a tendency closer to the gastric fluid samples(ANOSIM, Euclidean P=0.001,Bray-curtis P=0.001)(c)LEFSe analysis: The relative abundances of Helicobacter,Lactococcus,Bacillus,Pseudomonas and Geobacillus in the gastric mucosal microbiota is significantly higher than that in the gastric fluid microbiota,while the relative abundances of Nesseria,Fusobacterium,Prevotella,Streptococcus and Veillonella were significantly lower than the gastric fluid microbiota.The 6 groups of LEFSe analysis of gastric mucosa and fluid microbiota among stages of gastritis,intestinal metaplasia and gastric cancer showed that Bacillus in the gastritis gastric mucosa group was significantly higher than other groups,and the relative abundance of Pesudomonas and Thermus in the intestinal metaplasia gastric mucosa group was significantly increased,the relative abundance of Acinetobacter and Johnsonella in the gastric cancer gastric mucosa group was significantly increased.The relative abundance of Alloprevotella,Veillonella,Peptostreptococcus,Granulicatella and Actinomyces in the gastritis gastric fluid group was significantly increased,and the relative abundance of Haemophilus,Neisseria,Ochrobactrum,Campylobacter,and Rothia in the gastric cancer gastric fluid group was significantly increased.The relative abundance of Prevotella,Fusobacterium,Leptotrichia,Brevundimonas and Megasphaera was significantly increased.(2)Paired sample analysis(a)? and ? diversity analysis: The difference in the Shannon index between gastric mucosa and gastric fluid microbiota in the gastric cancer group was significantly smaller than that in the gastritis group(P<0.001)and the intestinal metaplasia group(P<0.001).Based on the analysis of Bray-curtis distance and the weighted Uni Frac distance,we observed that the difference of distances between the paired gastric mucosa and fluid samples in the gastric cancer stage was smaller than that in the gastritis stage(P<0.001)and the intestinal metaplasia stage(P<0.05),while in the gastritis and intestinal metaplasia stage,there was no significant difference in the distance difference between the gastric mucosa and gastric fluid samples.(b)Analysis of bacterial differences: There are 103 different bacterial genera between paired gastric mucosa and fluid samples in patients with gastritis.The relative abundance of Helicobacter,Bacillus,Clostridiumsensustricto1,Enterococcus and Lactococcus in the gastritis gastric mucosal group was significantly higher than gastritis gastric fluid group.However,the relative abundance of Rothia,Streptococcus,Neisseria,Fusobacterium and Peptostreptococcus in the gastric fluid of patients with gastritis was significantly increased.There are 53 different bacterial genera between the paired gastric mucosa and fluid samples of patients with intestinal metaplasia.Compared with the intestinal metastatic gastric fluid microbiota,the relative abundance of Lactobacillus,Geobacillus and Clostridiumsensustricto1 in the intestinal metaplasia gastric mucosal microbiota was significantly higher,while the relative abundance of Neisseria,Prevotella,Fusobacterium,Rothia,and Veillonella was significantly reduced.Comparison of gastric mucosa and gastric fluid microbiota in gastric cancer patients revealed 53 different bacterial genera.Among them,the relative abundance of Lactococcus,Bacillus,Acinetobacter,Enterococcus and Geobacillus in gastric cancer gastric mucosa was significantly higher than that in gastric fluid,while the relative abundance Veillonella,Prevotella,Neisseria,Rothia and Fusobacterium is significantly lower than that in gastric fluid.4.Changes in p H and specific metabolites in the stomach in gastric carcinogenesis(1)Changes in p H in the stomach in gastric carcinogenesis: the p H of the gastric fluid in the Hp-positive intestinal metaplasia group was significantly higher than that in the Hp-negative intestinal metaplasia group(P<0.05).However,in the gastritis and gastric cancer groups,the p H of the Hp-positive subgroup exhibited an increasing trend,but no statistically difference when compared to the Hp-negative subgroup.The p H of the Hp-negative gastric cancer group was significantly higher than that of the Hp-negative gastritis group(P<0.01)and the Hp-negative intestinal metaplasia group(P<0.05).Although the p H of the Hp-positive gastric cancer group was higher than that of the Hp-positive gastritis and intestinal metaplasia group,the p H of the gastric fluid in the Hp-positive gastritis,intestinal metaplasia and gastric cancer group had no statistical difference.(2)The alterations of nitrite content in the stomach in gastric carcinogenesis: during the process of gastritis ? intestinal metaplasia ? gastric cancer,the nitrite content in the gastric fluid gradually increased,and the content of nitrite in the intestinal gastric fluid group was significantly higher than that of the gastric fluid from gastritis.In the gastric cancer group(P<0.05),the p H of the gastric cancer gastric fluid group was significantly higher than that of the gastritis gastric fluid group(P<0.001)and the intestinal metaplasia gastric fluid group(P<0.05).Hp infection had no significant effect on the nitrite content in each disease stage.The nitrite content of Hp-positive gastric cancer gastric fluid group was significantly higher than that of Hp-positive gastritis group,and the nitrite content of Hp-negative gastric cancer gastric fluid group was also significantly higher than that of Hp-negative gastritis group.The nitrite content in Hp-postive gastritis group(P=0.086)and intestinal group(P=0.078)was higher than Hp-negative gastritis group and intestinal metaplasia,respectively.(3)The alterations of bile acids in the stomach in gastric carcinogenesis: the total bile acid content of gastric cancer gastric fluid group was significantly higher than that of gastritis gastric fluid group(P<0.05).Compared with the intestinal metaplasia gastric fluid group,the total bile acid content of the gastric cancer gastric fluid group had an increasing trend(P=0.062).The total bile acid content of Hp-negative gastric cancer gastric fluid group was significantly higher than that of Hp-negative gastritis(P<0.01)and intestinal metaplasia(P<0.01)gastric fluid group.In the gastritis and intestinal metaplasia stage,the total bile acid content in the Hp-negative and positive gastric fluid was not significantly different,while in the gastric cancer stage,we found that the total bile acid content in the Hp-positive gastric cancer gastric fluid was significantly lower than that in the Hp-negative gastric cancer gastric fluid group(P<0.05),while the total bile acid content of the Hp-positive gastric cancer gastric fluid group was not significantly different from the Hp-positive gastritis and intestinal gastric fluid groups.(4)Alterations of short-chain fatty acids in the stomach in gastric carcinogenesis: the levels of total short-chain fatty acids(P<0.01),acetic acid(P<0.05),propionic acid(P=0.076),butyric acid(P<0.05),isobutyric acid(P=0.053),valeric acid(P<0.01),and isovaleric acid(P=0.051)were higher than those in the gastritis group.The level of total short-chain fatty acids(P<0.01)acetic acid(P<0.05),propionic acid (P=0.065),and butyric acid(P<0.05)in the gastric cancer group was higher than those in the intestinal metaplasia group.However,there was no significant difference in the content of various short-chain fatty acids between the gastritis group and the intestinal metaplasia group.The content of total acid,acetic acid,propionic acid,butyric acid and isovaleric acid in Hp-positive gastric cancer gastric fluid group was significantly higher than that of Hp-positive gastritis(all P<0.05)and intestinal metaplasia(all P<0.05)gastric fluid group;The content of isobutyric acid in Hp-positive gastric cancer gastric fluid group was significantly higher than the Hp-positive intestinal gastric fluid group(P<0.05),and the content of valeric acid in the Hp-negative gastric cancer gastric fluid group was significantly higher than that of the Hp-negative gastritis gastric fluid group.The content of isobutyric acid in the Hp-negative intestinal gastric fluid group was significantly higher than that in the Hp-positive intestinal gastric fluid group(P<0.05).The content of total acid(P<0.05),acetic acid(P<0.05)and isobutyric acid(P<0.05)in the Hp-negative intestinal metaplasia gastric fluid group was significantly higher than that in the Hp-positive intestinal metaplasia gastric fluid group.However,in the stage of gastritis and gastric cancer,there is no significant difference in content of various short-chain fatty acid between Hp positive and negative in the gastric fluid.Impact of Hp infection and probiotic intervention on gastrointestinal tract of INS-GAS mice.1.The impact of Hp infection and probiotic supplementation on gastric mucosal lesions in INS-GAS mice Obvious inflammatory cell infiltration and intestinal metaplasia have been seen in the gastric mucosal tissue of mice infected Hp for 4 months.In the Hp infection combined with probiotic treatment group(4M group),the inflammation of the gastric mucosal tissue was significantly reduced,and the gastric mucosal lesions were also significantly improved.Compared with the mice in the control group(7M group),the Hp-infected mice in the 7-month group had significantly increased gastric mucosal inflammatory cell infiltration,and worsened pathological conditions of the gastric tissue.The expression of CD45 in gastric mucosa of Hp infection group was significantly higher than that of control group,while the expression of CD45 in gastric mucosa of Hp infection combined with probiotic treatment group was significantly lower than that of Hp infection group.The scores of gastric mucosal inflammation(P<0.01),oxyntic gland atrophy(P<0.01),metaplasia(P<0.01)and dysplasia(P<0.05)of mice in the Hp infection group were significantly higher than those of control mice.Compared with the mice in the Hp infection group(4M group),the scores of gastric mucosal inflammation(P<0.01),oxyntic gland atrophy(P<0.05),and metaplasia(P<0.01)in the Hp infection combined with probiotic treatment group were significantly decreased,The score of dysplasia(P=0.06)has a downward trend.There was no significant difference in the histopathological scores of gastric mucosa between the probiotic treatment group and the control group.The gastric p H of Hp-infected mice was significantly higher than that of control mice,and the gastric p H in Hp-infected mice was significantly reduced after probiotic intervention.2.The impact of Hp infection and probiotic supplementation on the gastrointestinal microbiota of INS-GAS mice(1)? diversity analysis: There was no significant difference in the Chao index among control group,Hp infection group,probiotic treatment group and Hp infection combined with probiotic treatment group(P>0.05).The Shannon index of the control group,Hp infection group and probiotic treatment group was not significantly different(P>0.05),while the Shannon index of the Hp infection combined with probiotic treatment group was significantly higher than that of the Hp infection group(P<0.01).(2)? diversity analysis: based on the weighted Uni Frac distance,result of PCo A analysis(PC1 explained 40.76% of the difference in microbiota,and PC2 explained 22.83% of the difference in microbiota)showed that there are different degrees of confounding factors between the control group and Hp infection group samples,which cannot be effectively distinguished.The samples of the probiotic treatment group and the Hp infection combined probiotic treatment group are more similar and cannot be effectively distinguished.A supervised PLS-DA analysis was performed(Comp1 accounted for 16.18% of the difference in interpretation weight,Comp2 accounted for 13.42% of the difference in interpretation weight),the control group and Hp infection group samples had a clear trend of separation,while the probiotic treatment group cannot be effectively distinguished from the Hp infection combined with probiotic treatment group.(3)Microbial composition and LEFSe analysis: The dominant bacteria phyla in each group mainly include Firmicutes,Bacteroidota,Proteobacteria and Actinobacteriota.The average relative abundance of Firmicutes in the probiotic intervention group(probiotic treatment group,Hp infection combined with probiotic treatment group)was lower than that of the non-probiotic intervention group(control group and Hp infection group).The average relative abundance of Bacteroides in the probiotic treatment group and the Hp infection combined with probiotic treatment group was higher than that of the control group and the Hp infection group.The average relative abundance of Campilobacterota phylum in the Hp infection group was higher than that in the control group,the probiotic treatment group and the Hp infection combined with probiotic treatment group.The number of Hp-related OTU reads in the gastric mucosal microbiota of mice in the Hp infection group was significantly higher than that in the control group(P<0.05)and the probiotic treatment group(P<0.05);Hp OTU reads in the Hp infection combined with probiotic treatment group were significantly higher than that in the control group(P<0.01)and the probiotic treatment group(P<0.01),the number of Hp OTU reads in the Hp infection group was not significantly different from the Hp infection combined with probiotic treatment group(P>0.05).The relative abundance of Lactobacillus rhamnosus(P<0.01)and Lactobacillus salivarius(P<0.001)in the mouse stomach microbiota of Hp infection combined with probiotic treatment group was significantly higher than that of the Hp infection group.The relative abundance of Lactobacillus salivarius in the mouse stomach of the probiotic treatment group was significantly higher than that in the control group(P<0.05).Compared with the control group,the relative abundance of Helicobacter in the stomach of mice in the Hp infection group was significantly increased,while the relative abundance of Anaerocolumna,Anaerovorax,NK... |
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