Study On The Molecular Mechanisms Of ANKRD22-mediated Lgr5~+ Cell Proliferation Promoting Gastric Mucosal Repair

Background:Due to the location and functional characteristics of the stomach,the gastric mucosal barrier is prone to suffer from various endogenous and exogenous damages.Gastric mucosal epithelial damage has become a common pathology in gastrointestinal diseases.Stem/progenitor cells exist in gastric epithelial,which play an important role in the epithelial cell proliferation and tissue healing after gastric mucosal injury.Rapid gastric epithelial stem/progenitor cell proliferation and inflammatory response inhibition play key roles in promoting the repair of gastric mucosal damage.However,specific targets inducing both the abovementioned effects keep elusive.Our previous research suggests that Ankyrin repeat domain-containing protein 22（ANKRD22）may have a potential role in the repair of gastric mucosal injury.Objective:The purpose of the study is to explore the regulatory effect of ANKRD22 on the repair of gastric mucosal injury,to clarify the molecular mechanism of ANKRD22involved in the rapid proliferation of gastric epithelial stem/progenitor cells and local inflammation,to explore the possibility of the inhibitory small-molecule lead compound targeting ANKRD22 as a novel protective agent for gastric mucosal repair.Methods:The expression of ANKRD22 in human gastric epithelium was detected by biological analysis and immunohistochemistry,and its subcellular localization was determined by immunofluorescence and mitochondrial separation.ANKRD22expression in gastric disease and inflammatory injury was detected by RT-q PCR and Western blot.We constructed Ankrd22^-/-mouse based on CRISPR/Cas9 and established an acute gastric mucosal injury model in mice via intragastric Et OH/HCl administration.The percentage of Lgr5⁺cells in gastric mucosa damaged region was observed in Lgr5-EGFP-IRES-cre ERT2 mice,and the multi-directional differentiation potential of Lgr5⁺cells was identified by PCR.Organoid culture and flow cytometry were performed to evaluate the effects of ANKRD22 on Lgr5⁺gastric epithelial stem/progenitor cell proliferation.RT-q PCR was used to explore the expression of other gastric stem/progenitor cell markers after ANKRD22 deletion.The effect of ANKRD22on mitochondrial Ca²⁺concentration was observed by confocal fluorescence.Western blot was used to detect the expression changes of downstream proteins in Wnt-Ca²⁺pathway after ANKRD22 deletion.Inflammatory cell infiltration in Ankrd22^-/-gastric tissue was detected by flow cytometry.Luminex antibody chip and ELISA were used to explore the regulatory effect of ANKRD22 on the secretion of inflammatory factors and M1/M2 polarization of macrophages.Immunofluorescence and Western blot were used to detect the changes in macrophage mitochondrial Ca²⁺level and NFAT expression caused by Ankrd22 knockout.Furthermore,ANKRD22 inhibitory lead compound was obtained by virtual screening with its site of action identified by Ca²⁺influx,and verified in vivo and in vitro through luciferase reporter and mouse models.Results:1.ANKRD22 is downregulated in acute gastric mucosal injuryANKRD22 was highly expressed in human gastric epithelium as indicated by bioinformatics analysis and immunohistochemistry.Fluorescence colocalization and mitochondrial separation showed that most of ANKRD22 was localized in mitochondria.The expression of ANKRD22 showed a downward trend in the disease states of Hp infection,gastric cancer,and gastric inflammatory injury.Ankrd22 knockout has a repair effect on the acute gastric mucosal injury induced by HCl/Et OH in mouse model.2.ANKRD22 deletion results in rapid proliferation of Lgr5⁺gastric epithelial stem/progenitor cellsANKRD22 deletion promotes the expression of gastric epithelial stem/progenitor cell marker Lgr5.The Lgr5⁺cells below the area of gastric mucosal damage were increased compared to the non-injured area,and PCR detection showed that these Lgr5⁺cells have multidirectional differentiation potential.After chemical injury,the number of Lgr5⁺gastric epithelial stem/progenitor cells in Ankrd22^-/-mice was significantly increased compared with that in Ankrd22^+/+mice.There is a negative correlation between ANKRD22 and LGR5 in chronic gastric tissue inflammation.3.ANKRD22 deletion suppresses the noncanonical Wnt-Ca²⁺pathwayBy using Ca²⁺fluorescent staining,we observed that Ankrd22 deletion reduced mitochondrial Ca²⁺influx in gastric epithelial cells.Based on the identification that inhibiting the non-canonical Wnt-Ca²⁺pathway can upregulate the activity of canonical Wnt pathway,we found that the ANKRD22 deletion inhibited the expression of DAG,p-Ca MKII and NFAT downstream of the non-canonical Wnt-Ca²⁺pathway,thereby indirectly up-regulating the canonical Wnt pathway.4.ANKRD22 deletion reduces gastric mucosal inflammation through inhibition of macrophage activationFlow cytometry revealed that the percentage of CD45⁺leukocytes was significantly lower in Ankrd22^-/-damaged gastric epithelium than in that of Ankrd22^+/+mice.Chip analysis and ELISA showed that Ankrd22 knockout reduced the levels of inflammatory factors IL-1αand TNF-α.ANKRD22 expression was increased in activated macrophages,and ANKRD22 deletion inhibited inflammatory factors secretion and M1polarization of macrophages.In terms of mechanism,Ankrd22 knockout inhibited macrophage activation by reducing mitochondrial Ca²⁺and cytoplasmic NFAT level.5.Screening and functional verification of the inhibitory small-molecule lead compound targeting ANKRD22 in vivo and in vitroWe screened an ANKRD22 inhibitory lead compound-AV023 with L122/D132 as the action site.It can promote the proliferation of Lgr5⁺gastric epithelial stem/progenitor cells,reduce inflammation,and up-regulate Wnt pathway activity in vivo and in vitro.Intraperitoneal injection of AV023 promoted the repair of injured gastric mucosa in mice.Conclusions:We have identified a mitochondrial molecule ANKRD22,the expression of which is down-regulated when the gastric mucosa is injured.Loss of ANKRD22 indirectly activates the canonical Wnt pathway by reducing mitochondrial Ca²⁺level and cytoplasmic NFAT expression in gastric epithelial cells,thereby promoting Lgr5⁺stem/progenitor cells proliferation in the context of gastric mucosal damage.Ankrd22knockout also reduces gastric mucosal inflammation through inhibiting the secretion of cytokines by macrophages.Finally,we have identified the inhibitory small molecule lead compound targeting ANKRD22 with repair effects of gastric mucosal damage.These studies suggest that ANKRD22 is a novel potential molecular target for gastric mucosal repair,and ANKRD22 inhibitory small molecule lead compound AV023 has potential clinical application prospects.