Study On The Mechanism Of Helicobacter Pylori Inhibiting AMPK In Spasmolytic Polypeptide-expressing Metaplasia

Backgrounds:Helicobacter pylori(Hp)is a Gram-negative bacterium and one of the major pathogenic factors for gastric mucosal carcinogenesis.It can cause gastritis,duodenal ulcers,gastric ulcers,and gastric cancer.H.pylori infection leading to gastric mucosal carcinogenesis is mainly the result of the interaction between its virulence factors and host cells.Its cytotoxin-associated gene A(Cag A)is one of the main pathogenic virulence factors of H.pylori.Early diagnosis and treatment are the most effective means of preventing and controlling gastric mucosal carcinogenesis.However,the molecular mechanisms and molecular markers of H.pylori infection leading to gastric cancer are mostly based on late-stage gastric cancer tissue samples,and the molecular mechanisms of Correa cascade in early gastric mucosal premalignant lesions are unclear.Spasmolytic polypeptide-expressing metaplasia(SPEM)is a gastric mucosal metaplastic lesion that is parallel to intestinal metaplasia and is considered an important precursor lesion of gastric cancer.Studies have shown that the initial factor that triggers SPEM is often due to loss of parietal cells caused by various reasons such as drugs,H.pylori infection,etc.The occurrence of SPEM reflects the reprogramming of gastric mucosal epithelial cells and can progress to gastric cancer under sustained inflammatory conditions.It is an important new target for the prevention and control of gastric cancer.AMP-activated protein kinase(AMPK)is an evolutionarily conserved serine/threonine protein kinase and is the most important energy sensor in cells.Dysfunction of AMPK is closely related to the occurrence of diseases such as diabetes,cardiovascular disease,and non-alcoholic fatty liver.However,the role and mechanism of H.pylori in regulating AMPK in SPEM need to be elucidated.Clarifying the role and molecular mechanism of H.pylori in regulating AMPK in SPEM is of great significance for preventing gastric cancer.Methods:1.PCT-DIA microproteomics was used to obtain proteomic data of gastric mucosal precancerous lesions,stage I gastric cancer and its paracancerous tissues and stage II gastric cancer and its paracancerous tissues.Heat map and volcano map were used to demonstrate the proteomic changes during the Correa cascade reaction.UMAP,machine learning and Mfuzz were used to analyze the proteomic changes of gastric mucosal precancerous lesions.Heat map,volcano map and GSEA pathway enrichment to analyse changes in protein expression profiles in the gastritis intestinalisation group and the heterogeneous hyperplasia group.Heat map,five-fold cross-validation and GSEA pathway enrichment to analyse changes in protein expression profiles in the gastritis combined intestinalisation group.2.C57 mice were divided into a control group and a tamoxifen group,and a SPEM model with parietal cell apoptosis was constructed by intraperitoneal injection of tamoxifen.Hematoxylin-eosin(HE)staining was used to observe the pathological damage of the gastric mucosa.Real-time PCR(RT-PCR)was used to examine the transcript levels of SPEM-related genes in the mouse gastric mucosa.Immunofluorescence co-staining of GIF and GSII was used to detect the severity of SPEM.Immunofluorescence co-staining of ATPase and Caspase-6 was used to detect the co-localization of Caspase-6 and ATPase.3.Immunoblotting and enzyme-linked immunosorbent assay(ELISA)were used to detect the protein levels of Caspase-6 in cells infected with H.pylori.4.Immunoblotting was used to detect the protein levels of phosphorylated AMPK in cells infected with H.pylori.Activation of AMPK with metformin and TUNEL was used to detect the apoptosis of cells infected with H.pylori.5.The binding potential of Cag A with LKB1 and AMPKα was explored by molecular docking.Immunoprecipitation was used to detect the effect of Cag A on the binding of LKB1 and AMPKα.Finally,immunoblotting was used to detect the protein levels of phosphorylated AMPK and Caspase-6 in H.pylori-infected cells that were either Cag A-negative or Cag A-positive.6.A SPEM model was constructed in C57 mice by gavage with Cag A-negative or Cag A-positive H.pylori strains.HE staining was used to observe the pathological damage of the gastric mucosa.RT-PCR was used to examine the transcript levels of SPEM-related genes in the mouse gastric mucosa.IImmunofluorescence co-staining of GIF and GSII was used to detect the severity of SPEM.Finally,ELISA was used to detect the protein activity of Caspase-6.Results:1.PCT-DIA mass spectrometry was used to detect 6345 proteins in gastric mucosal pre-cancerous lesions.Differential protein analysis revealed that the difference in protein abundance in the early stages of gastric mucosal lesions was much smaller than that in the late stages.UMAP dimension reduction and mfuzz clustering analysis revealed that gastric mucosal pre-cancerous lesions could be distinguished through proteomics.Comparison of protein differences between the gastritis metaplasia and dysplasia groups showed that necrosis-related genes were generally expressed at higher levels in the gastritis metaplasia group and at lower levels in the dysplasia group,as indicated by GSEA analysis.The expression of Caspase-3,Caspase-6,Caspase-8,and Caspase-10 in the Caspase family of proteins showed an upward trend,with Caspase-6 exhibiting the most significant upregulation.Additionally,downstream controlled apoptotic protein BID was significantly increased,while the level of gastric mucosal parietal cell marker H+/K+ATPase was significantly decreased,and AMPK downstream protein HNF4α expression was downregulated.Correlation analysis showed that BID was positively correlated with Caspase-6,H+/K+ATPase was negatively correlated with Caspase-6,HNF4α was positively correlated with Caspase-6,and BID was positively correlated with Caspase-6.HNF4α was positively correlated with BID,but negatively correlated with H+/K+ ATPase.Comparison of protein differences between the gastritis and metaplasia groups,a group of proteins was screened by random forest,and GSEA analysis showed that the AMPK signaling pathway was inhibited in metaplasia.2.RT-PCR analysis revealed that the transcription levels of Atp4 b,Gif,and Msit1 were significantly downregulated in tamoxifen-treated mouse gastric mucosa,while the transcription levels of HE4,Dmbt1,and Clusterin were significantly upregulated.Immunofluorescence analysis showed that the severity of SPEM was high in tamoxifen-treated mouse gastric mucosa and Caspase-6 protein increased in areas where wall cell apoptosis occurred.3.Immunoblotting showed that H.pylori infection led to an increase in Caspase-6cleavage,while ELISA assay revealed that H.pylori infection resulted in an increase in Caspase-6 activity.4.Immunoblotting showed that H.pylori infection led to a decrease in AMPK phosphorylation.TUNEL assay revealed that H.pylori infection resulted in cell apoptosis,and activation of AMPK by metformin could alleviate H.pylori-induced cell apoptosis.5.Molecular docking revealed the potential binding of Cag A to LKB1 and AMPKα.Immunoprecipitation showed that Cag A could inhibit the binding of LKB1 to AMPKα.Immunoblotting revealed that Cag A-positive H.pylori could inhibit AMPK phosphorylation and increase Caspase-6 cleavage,while activation of AMPK by metformin could attenuate Caspase-6 cleavage.6.RT-PCR analysis revealed that the transcription of Atp4 and Gif was significantly downregulated in mouse gastric mucosa infected with Cag A-positive H.pylori,while the transcription levels of HE4,Tff2,Dmbt1,and Cd44v9 were significantly upregulated.Treatment with metformin led to an upregulation of the transcription levels of Atp4 b and Gif,while the transcription levels of HE4,Tff2,Dmbt1,and Cd44v9 were downregulated.Immunofluorescence analysis showed that SPEM was more severe in mouse gastric mucosa infected with Cag A-positive H.pylori,but treatment with metformin significantly alleviated H.pylori-induced SPEM.Additionally,ELISA assay revealed that Caspase-6 activity increased in mouse gastric mucosa infected with Cag A-positive H.pylori,while treatment with metformin led to a decrease in Caspase-6 activity.Conclusion:In summary,we found that the AMPK signaling pathway was suppressed during the intestinal metaplasia of gastric mucosa.The virulence factor Cag A of H.pylori could inhibit the binding of LKB1 to AMPKα,inhibit AMPK phosphorylation,and thus lead to the occurrence of SPEM.These results suggest that Cag A promotes SPEM through the AMPK-Caspase-6 axis,and AMPK may be one of the important targets for the prevention and treatment of gastric cancer.