Molecular Mechanism Of Gastric Mucosal Injury Induced By Targeted Deletion Of Slc26a9 Gene In Parietal Cells

Objective:Gastric mucosal injury is divided into two categories:local and diffuse.Local injury is a repairable injury caused by toxin intake,bile and some infectious factors that does not change the cell differentiation pattern.Diffuse injury refers to chronic injury caused by abnormal differentiation（metaplasia）of gastric mucosal cells,which is most commonly caused by parietal cell loss（autoimmune atrophic gastritis,AIG）or chronic Helicobacter pylori infection and is closely related to the occurrence of gastric neoplastic lesions including intestinal gastric cancer and gastric type 1neuroendocrine tumor（G-NET）.After gastric mucosa injury,the repair response to reestablish the epithelial barrier is rapid and efficient,including cell proliferation and migration,immune cell infiltration,angiogenesis and Spasmolytic polypeptide/trefoil factor 2（TFF2）-expressing metaplasia（SPEM）.Among them,SPEM appears at the ulcer margin and exists as a reparative lineage.At the same time,in diffuse injury,after the loss of parietal cells,chief cells reprogramming fails and cell transformation occurs,resulting in the evolution of chief cells to SPEM,which in turn progresses to intestinal metaplasia and ultimately leads to gastric cancer.Therefore,SPEM is an important pathophysiological process for the repair of gastric mucosal injury and is of great significance for the prevention and control of gastric mucosal injury diseases.In the normal gastric corpus,parietal cells and acidic environment play a crucial role in the regulation of gastric mucosal autoimmunity and cell differentiation and transformation.Slc26a9,an anion transporter of the Slc26a family,is highly expressed in the stomach and is essential for parietal cell survival and function.We report for the first time that Slc26a9 loss leads to the development of gastric cancer.However,there are few studies associating this ion channel with the pathophysiology of gastric mucosal injury.Therefore,the aim of this study was to explore the role and specific mechanism of Slc26a9 in gastric mucosal injury and mucosal repair and remodeling.This provides a new direction for the prevention and treatment of gastric mucosal diseases.Methods:1)HE staining and immunohistochemistry（IHC）were used to detect the phenotype of gastric mucosa in gastric parietal cell-specific Slc26a9 knockout mice(Slc26a9^fl/fl/Atp4b-Cre)and control mice(Slc26a9^fl/fl).2)q PCR was used to detect inflammatory cytokines in the gastric mucosa of Slc26a9^fl/fl/Atp4b-Cre mice and Slc26a9^fl/fl mice,including IFN-γ、IL-1β、IL-2、IL-4、IL-6、IL-11 and IL-17.3)WB and IHC techniques were used to detect the expression of proteins of gastric mucosal injury-related pathways,namely Fas/Fasl pathway and Shh/Ptch1 pathway.4)The expression of Slc26a9 in normal gastric mucosa,AIG gastric mucosa and type1 G-NET gastric mucosa was detected by q PCR and IHC.5)In GES-1 cells,Slc26a9 was silenced using a lentiviral vector carrying a specific sh RNA.WB technique was used to detect the expression of Slc26a9 and Shh/Ptch1pathway proteins in GES-1 cells of Slc26a9 gene silencing group（GES-1+Sh Slc26a9）and GES-1 cells of the lentiviral empty transfer control group（GES-1+Shctr）.6)In the gastric ulcer model,HE staining and IHC were used to detect the phenotype of mouse gastric mucosa.7)The proliferation ability of GES-1+Shslc26a9 and GES-1+Shctr cells was compared by CCK-8 proliferation assay.WB was performed to examine the role of Wnt pathway regulating Slc26a9 in gastric epithelial repair.8)q PCR and IHC were used to detect the expression of Slc26a9 in gastric mucosa of normal subjects and patients with gastric ulcer.Results:Compared with Slc26a9^fl/flmice,Slc26a9^fl/fl/Atp4b-Cre mice exhibited autoimmune atrophic gastritis（strongly loss of parietal cells,oxyntic atrophy followed with chronic inflammation）,accompany with activation of CD4⁺T cell mediated inflammatory cytokines release,as well as activation of Fas/Fas L signaling pathway.Moreover,downregulation of Shh/patch signaling pathway caused two gastric mucosal diffused injury phenotypes,including SPEM and intestinal metaplasia,as well as neuroendocrine cell hyperplasia.Consistent with animal experiments,Slc26a9 m RNA and protein expression was significantly decreased in the autoimmune gastritis,gastric cancer and I type gastric neuroendocrine tumor tissues when compared with health control.In the local injury study（gastric ulcer model）,SPEM clearly appeared on gastric ulcer margin 14 days after ulcer in Slc26a9^fl/fl/Atp4b-Cre mice and 1 month after ulcer in Slc26a9^fl/flmice,respectively.At the same time,Ki67 was strongly expressed near gastric ulcers in both ulcer model mice,but compared with Slc26a9^fl/fl mice,the expression of Ki67 near ulcers was increased to a lesser degree in Slc26a9^fl/fl/Atp4b-Cre mice.The above results indicated that loss of Slc26a9 slows gastric mucosal repair.In addition,silencing the Slc26a9 gene in GES-1 cells decreased cell proliferation,and Slc26a9 promoted ulcer healing through the Wnt pathway.In addition,compared with normal gastric mucosa,Slc26a9 expression was significantly down-regulated in gastric mucosa of patients with gastric ulcer.Conclusion:Targeted deletion of Slc26a9 in the parietal cells caused gastric mucosa autoimmune dysregulation and epithelia cell transformation,and also inhibited proliferation in neighboring units and migration of surface cells,thus hindering the rapid repair of gastric mucosa.