A Research On The Mechanism Of Deoxycholic Acid Promoting Metaplasia Of Gastric Mucosa

Intestinal metaplasia and SPEM are two major types of metaplasia in the stomach,both of which are important risk factors for gastric cancer.Chronic inflammatory environment is an important driving force for the development of two metaplasia.An increasing number of studies have shown that bile acids play an important role in the development of intestinal metaplasia.Previous studies have suggested that intestinal metaplasia may originate from SPEM.Deoxycholic acid is a secondary bile acid,which has certain antibacterial and cytotoxic effects and may play a certain role in immune regulation.In this project,we investigated whether deoxycholic acid can affect the occurrence and development of intestinal metaplasia and SPEM by regulating macrophages.We conducted relevant research through two parts.Part Ⅰ : Deoxycholic acid-stimulated macrophage-derived exosomes to regulate intestinal metaplasia and proliferation in human gastric epithelial cellsAim: The crosstalk between macrophages and gastric epithelial cells has emerged as a player in chronic inflammation during intestinal metaplasia.Whether bile acids play a role in the above regulation remains to be studied.In this study,we hypothesized that deoxycholic acid-induced macrophages secreted exosomes to mediate intercellular communication and promote intestinal metaplasia in human gastric epithelial cells(GES-1 cells).Methods: Macrophage-derived exosomes(M-Exos)and deoxycholic acid-induced macrophage-derived exosomes(D-Exos)were isolated by ultracentrifugation.Ed U staining and CCK-8 assay were utilized to evaluate the effects of exosomes on the proliferation of GES-1 cells.Intestinal metaplasia was assessed by the expression of caudal-related homeobox transcription factor 2(CDX2)at both m RNA and protein level.Micro RNA sequencing revealed the micro RNA(mi RNAs)expression profiles of M-Exos and D-Exos.We used mi RNAs mimics,mi RNAs inhibitors and si RNAs to analyze the specific effects of mi RNAs and m RNA.Results: D-Exos promoted the expression of CDX2 and suppressed the proliferation of GES-1 cells,compared to M-Exos.The mi RNAs profiles and quantitative real-time PCR examination showed D-Exos enriched a higher level of hsa-mi R-30a-5p than M-Exos.Overexpressed has-mi R-30a-5p increased CDX2 expression and inhibited the proliferation in GES-1 cells via inhibiting the expression of downstream Forkhead Box D1(FOXD1,a potential regulatory factor in the process of intestinal metaplasia).Conclusion: D-Exos may promote intestinal metaplasia and suppress proliferation of GES-1 cells via hsa-mi R-30a-5p targeting FOXD1,which may be involved in the action mechanism of deoxycholic acid in promoting intestinal metaplasia of gastric mucosa.Part Ⅱ : Deoxycholic acid-stimulated macrophage-derived exosomes promote spasmolytic polypeptide-expressing metaplasia in the stomachAim: Spasmolytic polypeptide-expressing metaplasia(SPEM)is an important risk factor for the occurrence of gastric cancer and may be driven by a chronic inflammatory environment in which macrophage are involved.Previous studies have shown that intestinal metaplasia may originate from SPEM,and bile acid-induced chronic inflammation plays an important role in the process of intestinal metaplasia.However,the relationship between bile acids and SPEM is unclear.In this study,we speculated that the exosomes released from macrophages stimulated by deoxycholic acid participated in the development of SPEM.Methods: In vivo,mice were gavaged with deoxycholic acid for 4 weeks,and gastric tissues were harvested.In vitro,deoxycholic acid-induced macrophage-derived exosomes were isolated by ultracentrifugation and cocultured with the gastric organoids of mice.Immunofluorescence staining and quantitative real-time PCR were used to analyze markers of macrophages and SPEM.Results: In vivo,after 4 weeks of deoxycholic acid intragastric administration,macrophage markers(F4/80)and SPEM markers(TFF2 and GSII lectin)were increased in from treated mice compared with those from normal control mice.In vitro,macrophage-derived exosomes labeled with PKH67 were internalized by gastric organoids.Deoxycholic acid-induced macrophage-derived exosomes increased the expression of SPEM markers(TFF2 and GSII lectin)in gastric organoids compared to exosomes derived from macrophages without deoxycholic acid stimulation.Conclusion: Macrophage-derived exosomes may be a novel mechanism by which deoxycholic acid promotes SPEM.