| BackgroundThe gastric mucosal epithelium is in direct contact with gastric contents and is under constant attack by potential pathogens,including drugs,alcohol,salivary enzymes,bacteria,viruses,parasites,etc.In order to maintain homeostasis and integrity,the gastric barrier has special structures and functions that coordinate simultaneous defense against physical and chemical damage,these structures or functions include:microbiota,tight junctions between epithelial cells and intrinsic immunity.Helicobacter pylori(HP)-induced gastritis is generally considered to be mucosal inflammation caused by the combination of bacterial virulence factors and host immune activation,which can also cause dysbiosis and epithelial barrier defects.Antimicrobial peptides(AMP)are a class of small molecule peptides with antimicrobial functions that are induced in humans and widely expressed in a variety of human mucosa,and their production is associated with possible infection or inflammation.Regenerating islet-derived 3α(REG3A),a member of the REG family,has been shown to be involved in the pathogenesis of several gastrointestinal or systemic inflammation-driven diseases,including inflammatory bowel disease(IBD)and rheumatoid arthritis(RA),but the relationship between REG3A and HP infection has not been studied.S100A9 is actively released in the inflammatory response and plays a key role in the regulation of the inflammatory response by stimulating leukocyte recruitment and inducing cytokine secretion.Human P-defensin 2(HBD2)is closely related to the inflammatory response.HBD2 not only has anti-inflammatory effects,but also increases the expression of pro-inflammatory factors under specific conditions and has a selective role in regulating the inflammatory response.Understanding the alteration of gastric mucosal microecology,epithelial barrier and antimicrobial peptides by HP will help us to take a more effective approach to treat HP induced gastritis as well as to prevent further gastric disease progression.ObjectivesIn this study,we investigated the effects of HP on the structure,abundance and diversity of gastric microbiota of patients with gastritis;we clarified the changes of HP on the expression of antimicrobial peptides REG3A,HBD2 and S100A9,and observed the changes of villi structure and cell junctions in gastric epithelial cells in patients with gastritis.MethodsPatients infected or uninfected with HP gastritis were clinically recruited and divided into HP positive/negative groups based on rapid urease,13C breath test and pathological findings,and their gastric antral mucosal specimens were collected.The diversity and structural alterations of the microbiota were analyzed by 16S rRNA sequencing.mRNA of antimicrobial peptides REG3A,HBD2 and S100A9 in gastric mucosa of HP-induced gastritis were analyzed by RT-qPCR,Western blotting and immunohistochemical were used to detect the altered mucosal expression of the antimicrobial peptides REG3A,HBD2 and S100A9.Transmission electron microscopy was used to observe the changes of gastric epithelial villi and cellular tight junctions in patients with HP infection.ResultsWe observed a large number of bacteria which belong to 33 phyla and 603 genera in gastric microorganisms.The dominant phylum types in the gastric flora were mainly five major groups,Proteobacteria、Firmicutes、Bacteroidetes、Cyanobacteria and Actinobacteria,with the Proteobacteria accounting for 40%overall,Firmicutes 18%,and Bacteroidetes 12%,Cyanobacteria 11%,Actinobacteria 5%,Acidobacteria 4%,Fusobacteria 2%,Chloroflexi 1%,the rest were less than 1%in HP negative group.Epsilonbacteraota 50%,HP positive group Epsilonbacteraota 50%,Firmicutes 9%,Bacteroidetes 5%,Cyanobacteria 4%,Actinobacteria 3%,Acidobacteria 2%,Fusobacteria 1%in HP positive group.HP is predominant in the stomach,with an average abundance of 50%in infected individuals;HP infection does not change the type of dominant flora,but its presence significantly reduces the abundance of bacteria other than HP.Alpha diversity was lower in the HP-positive group than in the HP-negative group,and the Shannon,Simpson,ACE,and Chao1 indices were 4.38 ±0.33.0.63± 0.04,1060.35±37.79 and 1063.73± 40.16,respectively,in the HP-positive group 7.28±0.11,0.95±0.01,1198.61± 54.68、,1182.43±65.68,where P(Chaol)=0.12.P(ACE)=0.04.P(Shannon)<0.01,P(Simpson)<0.01.PCA(Principal Component Analysis).PCoA(Principal coordinates analysis),and NMDS(Non-Metric Multi-Dimensional Scaling)analysis indicated some aggregation within the two sample groups.PERMANOVA or Anosim box plot analysis was performed to test whether the samples of different groups were significantly different from each other.Beta diversity was tested for significant differences between samples of different groups,and the distance between groups was greater than within groups in both weighted and unweighted analyses(R>0,P all less than 0.01).LEfSe(Line Discriminant Analysis(LDA)Effect Size)(LDA was set to 4)analysis was used to screen for statistically different groups between the two groups,and the dominant microbial communities in the HP negative group:Proteobacteria、Gammaproteobacteria、Vibrionales、Vibrionaceae、Vibrio、uncultured_bacterium_g_Vibrio、Firmicutes、Chloropast、Cyanobacteria、Oxyphotobacteria、uncultured_bacterium_o_Chloroplast、Bacteroidetes、Bacteroidia、Bacteroidalea、Bacilli、Clostridia、Clostridiales、Alphaproteobacteria、Lactobacillales、Prevotellaceae,while Epsilonbacteriota、Helicobacter、Campylobacterales were dominant in the HP-positive group.RT-qPCR analysis of REG3A,S100A9,HBD2 in gastric mucosa of HP-positive patients Western blotting analysis of REG3A and S100A9 protein expression was significantly elevated in the gastric mucosa of HP-positive patients(P<0.01).Immunohistochemical staining observed expression of all three antimicrobial peptides in gastric epithelial secretory cells,inflammatory cells,and darker staining in HP-positive gastric mucosa.The destruction of the epithelial cell membrane and the entry of mucus particles into the gastric lumen were observed by transmission electron microscopy in HP-positive patients.Gastric epithelial cell gaps were widened in HP-infected patients,but tight junction structures were intact.measuring the length of tight junctions in both groups,the HP-negative group 393.75±121.64 nm The tight junction length was measured in the HP negative group 393.75± 121.64 nm and the HP positive group 448.28 ± 152.89 nm,and there was no significant difference in the tight junction length between the two groups(P=0.184).ConclusionHP caused microecological changes in the stomach,and the abundance and diversity of gastric flora were reduced;the expression of antimicrobial peptides REG3A,HBD2 and S100A9 could be induced by HP,and the expression of these antimicrobial peptides was closely related to secretory cells and inflammatory cells;HP damaged the gastric mucosa and caused ultrastructural changes in epithelial cells. |
