Mechanisms Of Zebrafish Inflammatory Bowel Disease In Pik3c3 Knockout Mutant

Inflammatory bowel disease(IBD)is a generic term for digestive system inflammation that is highly prevalent in Western countries.Because there is no clear conclusion about its pathogenesis,it is mainly through drug maintenance and surgical resection to maintain patients in remission.This type of disease does not affect the patient's life itself,but it will laed to physical and psychological torture.In this study,we knocked out the pik3c3/vps34 gene by CRISPR/Cas9 technology to obtain lethal zebrafish mutants.Unlike the mice mutants with homologous gene Pik3c3 knockout that died in gastralation,the early development of the zebrafish pik3c3 mutant was basically with no abnormality,but during from 8dpf to 9dpf the mutant would die soon with severely deformed small intestine.Through the next generation sequencing and histological analysis,we learned that the pik3c3 mutant showed inflammatory bowel disease-like characteristics.(1)Destruction of intestinal epithelial tissue structure(disappearance of intestinal folds,epithelial cell shedding,abnormal membrane protein localization and so on);(2)A large number of neutrophils migration to the intestine epithelium;(3)Inflammatory factors(tnfa,illb,cxcl8a and so on)was significantly increased.At present,there are not many IBD genetic models in zebrafish,and most of them are caused by excessive proliferation of microorganisms.However,by intestinal microbial CFU count and germ-free culture,we found that the inflammatory response in the pik3c3 mutant was not due to excessive proliferation of the intestinal microflora.In addition,based on the known functions of pik3c3,we discovered the innovation of this mutant.First,P catalyzed the production of phosphoinositide 3-phosphate(PI3P),whereas the small intestine epithelial tissue of this mutant was essentially free of PI3P expression.This demonstrated that the intestinal phenotype resulting from pik3c3 deletion was mediated by PI3P.Second,the inflammatory response resulting from pik3c3 deletion was epithelial cell autonomy.On the one hand,by artificially inducing caudal fin injury in zebrafish,we found that neutrophils could migrate to the site of injury and also migrate reversely,which indicated that the neutrophil injury response in vivo was not affected.On the other hand,in vitro 3D culture of human epithelial cell line Caco2 also demonstrated that inhibition of PIK3C3 could disrupt epithelial cell integrity,and this was mediated through endosomal trafficking.Third,the inflammatory response resulting from pik3c3 deletion was not dependent on autophagy or ER stress.TEM and protein expression analysis revealed that autophagy in intestinal epithelial cells was not significantly inhibited.This ruled out the effects of autophagy and endoplasmic reticulum stress on the inflammatory response in this mutant.In addition,the pik3c3 mutant has great application significance.A large number of GWAS screens for IBD patients have identified many genes associated with epithelial cell barrier(cdh1,hnf4a,ttc7a,etc.),and we have also found that these genes were significantly down-regulated in epithelial cells of this mutant.At the same time,we have found that tnfa was not significantly activated in the intestine,which explained why TNFa inhibitors had no effect on some patients.Therefore,this mutant also had great value in the screening of IBD drugs.In summary,the pik3c3 mutant not only explained the role of pik3c3 in the maintenance of epithelial cell homeostasis,but also established an IBD model of epithelial cell injury,which had a bright future in the study of IBD mechanism and clinical treatment.