The Repairment Function And Molecular Mechanism Of CBS-H₂S Axis In Intestinal Epithelial Injury Through Maintaining Crypt Basal Cell Stemness

Objective As the second largest epithelium of the human body,the intestinal epithelium plays a vital role in the digestive system.Intestinal epithelial cells are particularly susceptible to the regulation of homeostatic mechanisms,because mechanical and chemical stressors regularly destroy epithelial cells,and its exposure to intestinal pathogenic bacteria is common.Therefore,it is necessary for intestinal epithelium to regulate and manage the distribution of symbiotic microorganisms,maintain the epithelial barrier,and regularly renewal itself.However,the damage of the intestinal epithelium and the imbalance of the intestinal homeostasis mechanism are likely to be the initial cause of the development of intestinal diseases.Moreover,the pathology may in turn have a negative impact on epithelial regeneration and lead to abnormal intestinal epithelium,inflammatory diseases,and even cancer.In recent years,tissue stem cells are considered to be an important factor in the regeneration and repair of tissue damage.Therefore,understanding the repair mechanism of intestinal epithelial stem cells to intestinal epithelial injury can provide new treatment strategies for the occurrence,development and prognosis of intestinal diseases.Hydrogen sulfide(H2S),as an environmental toxin and toxic gas,is also an important endogenous signal molecule in cells and tissues,and is regarded as a member of the "gas transmitter" of biomolecules.Cystathionine-?-Synthase(CBS)is one of the important enzymes for the production of endogenous H2S,which dynamically regulates the level of H2S through the balance between production and degradation/elimination.Studies have shown that low concentrations of H2S can resist the invasion ability of intestinal cancer stem cells,and high concentrations of H2S can promote the invasion ability of cancer stem cells.However,the mechanism of H2S on the stem cells at the base of the intestinal epithelial crypts is also unknown.In addition,in the tissues of patients with digestive tract inflammation,the expression level of H2S markers is abnormal.The intestinal epithelium plays an important role in the occurrence and development of inflammation and cancer.Therefore,this study aims to explore the effect of CBS-H2S axis on intestinal epithelial crypt basal stem cells and its possible mechanism of action,and to explore its protective effect in the process of intestinal epithelial injury.Methods In order to clarify the role of CBS-H2S axis in intestinal epithelial injury,Dextran Sulfate Sodium Salt was used to construct a mouse intestinal epithelial inflammation model in this study,and an exogenous hydrogen sulfide donor dichloromethane complex GYY4137 was given at the same time,In this study,the CBS+/-mouse model was obtained by gene knockout technology,and the parallel control experiment was performed with the Wild Type(WT)group.In the study,the growth,survival and clinical manifestations of mice after treatments were observed to verify the protective effect and influence of the CBS-H2S axis on intestinal epithelial inflammation.Intestinal organoid culture technology was used to establish intestinal epithelial organoid models.WT and CBS+/-mouse intestinal epithelial crypt stem cells were cultured to observe the changes in the organoid culture process,and verify that the CBS-H2S axis is effective in the growth of intestine epithelial crypt stem cell.FD-4 was used to detect the permeability differences of organoids of different genotypes.The differences in fluorescence intensity were observed to understand the barrier function of mouse organoids of different genotypes,and verify the permeability or barrier function of the CBS-H2S axis to the intestinal epithelium.Using the constructed enteritis control model,we detected the characteristic cells of intestinal epithelium,colon lgr5+ stem cells,goblet cells,Paneth cells,and BMI-1+stem cell markers,and the immunohistochemical phenotype was used to verify that the CBS-H2S axis is important to the protective effect and influence of epithelial crypt basal cell population.At the same time,the expression level of the CBS-H2S axis on the intestinal epithelial ?-Catenin signaling pathway was detected to verify the influence of the CBS-H2S axis on the regulation of the intestinal epithelial ?-Catenin signaling pathway.Results In this study,we found that in the DSS colitis model,the total colon length after DSS treatment in the WT group and the CBS+/-group had a significant difference.In addition,we also found that the survival rate of mice lacking CBS is relatively low,and the clinical manifestations including blood in the stool,hair loss,weight loss,and anal prolapse were observed.At the same time,the lack of CBS deepens the inflammatory injury of the intestinal epithelium,and the damage can be alleviated by the exogenous H2S donor GYY4137.During the observation of organoid culture,we found that the number of organoids in the WT group was significantly higher than that of the CBS+/-group during the growth period.At the same time,we also found the appearance of cyst-like organoids.The number of cyst-like organoids in the CBS+/-group was significantly higher than that in the WT group.In addition,in terms of budding organoids,we found that not only the number of single budding organoids but also the number of multiple budding organoids,they were significantly higher in the WT group than in the CBS+/-group organoids,and the overall budding rate of the WT group organoids was also higher than that of CBS+/-group.In the FD-4 permeability test of organoids,we found that after adding FD-4 into the organoid samples,the permeability of the normal organoid barrier in the WT group remained.But FD-4 fluorescence signal appeared in the organoids of CBS+/-group.,the signal intensity was significantly higher than that in WT group.In the results of immunohistochemistry,we found that the lack of CBS led to the decrease of lgr5+cells,Paneth cells and goblet cells in the intestinal epithelium.At the same time,exogenous H2S can reverse the reduction of lgr5+cells,Paneth cells,and goblet cells caused by CBS deficiency from endogenous H2S.In the intestinal inflammation colitis model,the reduction of lgr5+cells,Paneth cells and goblet cells in CBS+/-gene mice is much more severe.In addition,we found that in the absence of CBS,the reduction of endogenous H2S will lead to the decrease in the expression of ?-catenin.Exogenous H2S can reverse the decrease in ?catenin expression caused by CBS deficiency.At the same time,for WT and CBS+/-mice,there is no significant difference in the expression of intestinal epithelium of BMI-1+cells in the DSS colitis model.But during the intestinal inflammation injury,after giving exogenous H2S donor,BMI-1+cells expressed,and the level of it in the WT group was higher than that in the CBS+/-group.Conclusion In the colitis model,the CBS-H2S axis affects the tolerance of the intestinal epithelium to the inflammation.Also,the exogenous H2S donors can improve this function,and alleviate the damage ability of the intestinal inflammation in the absence of CBS.In addition,CBS deficiency is more likely to increase the adverse prognostic outcome of intestinal injury.The results of organoid observation experiments reflect the promotion of the CBS-H2S axis on intestinal epithelial renewal.The lack of CBS may reduce the ability of intestinal epithelium to differentiate,proliferate and self-renew.In the FD-4 permeability test,we found that the lack of CBS leads to an increase in the permeability of intestinal epithelial stem cells,which has a damaging effect on the barrier function of the intestinal epithelium.The CBS-H2S axis may change the barrier function of the intestinal epithelium,to a certain extent,by affecting the basal stem cells of the intestinal epithelial crypt.The results of immunohistochemical experiments showed that CBS-induced endogenous H2S can increase the number of intestinal epithelial lgr5+cells,Paneth cells,and goblet cells,thereby enhancing the renewal,and proliferation and differentiation of intestinal epithelium.Exogenous H2S has a positive correlation effect on special types of intestinal epithelial cells.The CBS-H2S axis may activate BMI-1+cells through the exogenous H2S pathway in the process of intestinal injury,and promote the repair of intestinal injury.The CBS-H2S axis may promote the stemness of intestinal epithelial stem cells through the Wnt/?-catenin signaling pathway,and promote the repair of intestinal epithelial injury.