| Background Helicobacter pylori（H.pylori）is a gram-negative bacterium that affects human health.H.pylori was first discovered in 1982 by Australian doctors Barry Marshall and Robin Warren.Its spiral-shaped shape（hence the name Helicobacter pylori）is thought to have evolved to penetrate the stomach lining and cause infections.H.pylori is usually located in the stomach,and lymphomas（also known as organoexodal marginal zone B-cell lymphoma）have been reported affecting mucosa-associated lymphoid tissues of the stomach,esophagus,colon,rectum,or periocular tissues.Stomach infections of H.pylori in humans usually have no symptoms but can sometimes cause gastritis.Long-term infection with H.pylori can cause atrophic gastritis,intraepithelial neoplasia and gastric cancer.H.pylori infection is an important factor in the occurrence of gastric cancer,but it has not been paid enough attention to,the world has 42.8% of adults infected with H.pylori.In addition,34.0 percent of children are infected.It is worth noting that H.pylori infection in adults mainly originated in children.However,the pathogenesis of H.pyloriassociated pediatric gastritis remains unclear.Macrophages,a type of innate immune cell,have been reported to be involved in the pathogenesis of H.pylori induced gastritis.However,the role and mechanism of macrophage dysfunction in H.pylori associated pediatric gastritis remains unclear.Vacuolated cytotoxin A（Vac A）from Helicobacter pylori disrupts the endocytosis pathway leading to macrophage apoptosis by inhibiting IFN‐β signaling.In addition,Vac A stimulates caspase 8,Bid,and mitochondrial dependent cascades in order to induce apoptosis.This mechanism alters the host’s immune response to H.pylori,leading to chronic infection.In addition,the internalization and phagocytosis of macrophages against H.pylori was delayed by the involvement of Vac A.However,how the function of macrophages changes after H.pylori infection remains unknown.Src homology 2 domain-containing Phosphatase 2（SHP2）is a non-receptor protein tyrosine phosphatase encoded by PTPN11,which plays an important role downstream of growth factor receptor signal transduction.H.pylori can inhibit IFN-G signaling by activating SHP2.H.pylori infection is known to be one of the main causes of gastritis and stomach cancer.IFN-G is an important inflammatory cytokine through JAK2/STAT1 signaling pathway.In helicobacter pylori infection,cytotoxin-associated gene A（Cag A）is delivered to host cells through the CAG type IV secretion system.Cag A activates SHP2 and causes conformational changes,thereby blocking phosphorylation of STAT1.In addition,polymorphisms in host IL-1B and TNF-α,as well as common single nucleotide polymorphisms in SHP2 encoding PTPN11,may be associated with gastric atrophy in patients with CGA-positive H.pylori infection.Therefore,activation of SHP2 promotes gastritis caused by H.pylori infection.In addition,the absence of SHP2 in macrophages alleviates inflammation.However,the role of SHP2 activation in the regulation of macrophages is not clear at present.In this study,the molecular characteristics of children’s gastritis tissues infected with or without H.pylori were established by RNA sequencing analysis.We found a large number of differentially expressed genes（DEG）in H.pylori infected gastritis tissues compared with uninfected gastritis tissues.In addition,H.pylori infected gastritis tissue shows an overactive immune response.We found that the tyrosine protein phosphatase nonreceptor 11（PTPN11）encoding SHP2 was significantly increased in macrophages from h.pylori infected gastritis tissues.Furthermore,we found that SHP2 could activate the glycolysis function of macrophages and promote H.pylori induced inflammation.Transcription factor Spi1,as a downstream molecule of SHP2,may be responsible for regulating the expression of metabolism-related genes.In conclusion,our study elucidate the molecular structure of H.pylori infected gastritis tissue in children,suggesting that the SHP2/Spi1 axis may be a new therapeutic target for H.pylori induced gastritis in children.ObjectiveTo investigate the role and mechanism of SHP2-mediated macrophage dysfunction in H.pylori-associated pediatric gastritis.MethodsNormal gastric antrum（8 cases）,gastritis tissue from children without helicobacter pylori infection（8 cases）,and gastritis tissue from children with helicobacter pylori infection（8cases）were collected.RNA-sequencing was used to detect the expression of different genes in normal antrum,non-helicobacter pylori infection and helicobacter pylori infection of pediatric gastritis.Repeatability and gene expression of each sample were analyzed by bioinformatics method.The number of differential genes and their corresponding functions and intermolecular interactions were further analyzed.In addition,Top5 differential genes were screened out,and the expression of target genes was detected by q PCR.HE staining was used to detect the occurrence of inflammation in normal gastric antrum,non-helicobacter pylori infection and helicobacter pylori infection in children with gastritis.The expression of SHP2 in pediatric gastritis was detected by Western blot.Metabolic profiling of macrophages（including oxidative phosphorylation and glycolysis of macrophages）was performed by hippocampal metabolic analysis.Immunofluorescence was used to detect the localization of SHP2 and its co-localization with macrophages in normal gastric antrum,non-helicobacter pylori infected and helicobacter pylori infected pediatric gastritis tissues.The levels of reactive oxygen species（ROS）in macrophages were analyzed by flow cytometry.The expression of Spi1 and inflammatory factors was also detected by knocking down SHP2 in vivo.Furthermore,Spi1 can regulate the expression of inflammatory factors by knocking down Spi1.Results1.There were significant differences in genes expression in gastritis tissues infected with or without H.pylori compared with normal gastric antrum mucosa.2.Compared with H.pylori-positive gastritis tissue,H.pylori-positive gastritis tissue showed a large number of differentially expressed genes（DEGs）.3.H.pylori-infected gastric tissues exhibited more serious inflammation than non-H.pylori infected gastritis tissue.4.PTPN11 gene was highly expressed in the tissues of H.pylori associated gastritis,and the encoding-protein SHP2 expression was significantly higher than that of H.pylori–negative gastritis tissue and normal group.5.SHP2 is highly expressed in macrophages of H.pylori-associated gastritis tissues.6.Macrophages infected with and without H.pylori showed higher glycolysis function than normal macrophages.7.The glycolysis function of H.pylori infected macrophages was significantly stronger than that of non-H.pylori infected macrophages.8.Abnormal glycolytic function of macrophages infected with H.pylori is closely related to excessive inflammation.9.Knockdown of SHP2 can inhibit glycolytic function and inflammatory response of H.pylori-infected macrophages.10.SPI1 is the transcription factor controlling the most differentially expressed genes,and most of the differentially expressed genes are closely related to metabolism and immune response.11.The expression of transcription factor Spi1 was significantly increased in H.pylori infected macrophages.12.Spi1 in macrophages was also decreased after SHP2 knockdown.13.After SPI1 knockdown,the expression of inflammatory factors in macrophages decreased.Conclusion1.Macrophages in H.pylori infection-associated pediatric gastritis promote glycolysis function and induce severe inflammatory response through high expression of SHP2.2.SHP2 may regulate glucose degradation and inflammation of H.pylori infected macrophages through regulating the expression of transcription factor SPI1. |
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