Functional Study Of Beclin1 In The Intestinal Inflammation In Zebrafish

Zebrafish(Danio rerio)is a type of subtropical teleost fish belonging to the Cyprinidae.Zebrafish is characterized by complete genome annotation,short sexual maturity,in vitro fertilization and development,and transparency at larva stage.At present,many transgenic lines in which immune cells are labeled have been developed which makes it easy to track the immune response in various tissues and organs.In addition,innate immunity plays a major role at larva stage of zebrafish,eliminating interference from acquired immunity.And the function of intestine in zebrafish is very similar to that in mammals.Therefore,zebrafish is an ideal animal model to investigate the molecular mechanisms of intestinal inflammation.There are two main methods for the construction of zebrafish enteritis model: chemical reagent induction and genetic defect.So far,more and more zebrafish enteritis models have been established,providing a lot of valuable information for research work on enteritis.The morphogenesis of zebrafish intestine can be divided into three stages: intestinal cavity formation,intestinal epithelial cell polarization,and intestinal epithelial cell remodeling and differentiation.Yolk is completely absorbed and the intestine is functional after 5 days post fertilization(dpf).The intestine of larva zebrafish can be divided into three segments based on morphology and gene expression.They are intestinal bulb,mid-intestine and posterior intestine.The intestinal bulb and mid-intestine are mainly responsible for the absorption of nutrients,while the function of posterior intestine is to absorb water and regulate the osmotic pressure.There are four main cell types in zebrafish intestinal epithelium: enteroendocrine cell,enterocyte,goblet cells,and vacuolated cells.Among them,enterocytes are the predominant cell types.Zebrafish do not have M cells(membranous cell)that is classical in mammals.Compared to mammals,the structure of zebrafish intestine is much simpler.The intestinal epithelium of zebrafish lacks crypt and muscular layer is directly adhered to the mucosal layer.It also do not have organized lymphoid structure,and the nerve cell body is distributed between the smooth muscle layer.The structure and function of Beclin1 protein is highly conserved in many species.In zebrafish,beclin1 is located on chromosome 12 and contains 12 exons.Beclin1 protein has 447 amino acids and it is about 52 kDa.Beclin1 protein has four domains:BH3(Bcl-2 homolog3)domain,coiled-coiled domain(CCD),evolutionarily conserved domain(ECD),and nuclear export domain(NES).Bcl-2,Vps34,UVRAG,ATG14 all can bind to different domains of Beclin1.Beclin1 is a recognized autophagy-related protein.It forms complexes with other proteins and participates in regulating the occurrence and maturation of autophagy.Beclin1 can also perform autophagyindependent functions.It regulates the endosome trafficking,which involves the endocytosis and reuse of some receptors on the cell membrane.In addition,it can also regulate the occurrence and development of tumors by regulating chromosome stabilization,DNA repair,cell cycle,and apoptosis.Autophagy is one of the important degradation systems in the cell.It can remove damaged or aging organelles and invading pathogens,degrade longevity proteins,and also adapt to external environmental changes such as starvation,hormone imbalances and oxidative stress.Many studies have shown that autophagy is involved in maintaining intestinal homeostasis.Autophagy can enhance the function of tight junctions in intestinal epithelial cells,participate in the degradation of connexins,and maintain the first barrier of intestine against pathogenic microorganisms.Autophagy can negatively regulate the inflammatory response by inhibiting inflamosome.In addition,it can also regulate the development and differentiation of T cells and B cells,and regulate antigen presentation of MP cell(mononuclear phagocytic cell).The normal function of intestinal epithelial cells is inseparable from autophagy.Both the secretory function of goblet cells and the natural resistance to bacteria require autophagy.In addition,autophagy is closely related to human inflammatory bowel disease(IBD).According to the genome-wide association studies,autophagy-related genes such as Atg16 L is human IBD susceptible gene.The purpose of this project is to study the function of Beclin1 in regulating intestinal inflammation in zebrafish.The experiment are mainly relied on two transgenic lines Tg(mpeg1:GFP)and Tg(lyz:GFP)to detecte intestinal inflammation-related indicators via immunohistochemistry and quantitative real-time PCR.At the same time,chloroquine,an autophagy inhibitor,is used to investigate whether Beclin1's function to regulate enteritis depends on the autophagy pathway.In this project,we used CRISPR/Cas9 technology to knock out beclin1 in zebrafish,and a zebrafish mutant was obtained.The early intestinal morphology and structure of the beclin1 mutant are comparable to sibling.However,at 6.5dpf,the intestinal epithelial barrier function was impaired,and a small number of mutants have a narrow intestine.While at 7.5dpf,the proportion of zebrafish with the narrow intestine is significantly increased.The histological changes of the intestinal blub are mainly the destruction of the intestinal villi structure and loose arrangement,the distribution of epithelial cells on the basal side,and the changes of nucleus from the original spindle to the round.Thickening of the intestinal muscle layer can also occur in severe phenotypes.Inflammatory reactions in the intestinal tissue of the mutants were manifested in the massive accumulation of neutrophils and macrophages,infiltration of neutrophils,and up-regulation of transcription levels such as immune-related factors mmp9 and tnf-?.The number of immune cells and the narrow intestine were rescued after antibiotic treatment in mutant,but the intestinal epithelial barrier function could not be improved.Treatment of wild-type zebrafish with the autophagy inhibitor-chloroquine failed to mimic the impaired intestinal epithelial barrier.So impaired intestinal epithelial barrier function induced by Beclin1 deletion may not depend on autophagy.However this part of the conclusion needs to be further verified.In this project,the most direct effect caused by Beclin1 deletion is to disrupt the function of the intestinal epithelial barrier.After intestinal epithelium is damaged,intestinal microbe invasion leads to inflammation.According to the immune response and the individual differences,the proportion of zebrafish with narrow intestine varies.The role of Beclin1 in the intestinal epithelial barrier function needs further study.According to the current data,Beclin1 is more likely to regulate the intracellular circulation of cell-cell junction proteins in an autophagy-independent manner.Inflammatory bowel disease has two subtype,Ulcerative colitis and Crohn's disease.The phenotype of the beclin1 mutant is similar to the phenotype and pathogenesis of Crohn's disease,so the beclin1 mutant can provide some information for the study of Crohn's disease.In addition,the research on the autophagy-independent function of Beclin1 is currently limited to tumor cells.This subject can enrich the content of autophagyindependent function of Beclin1 in maintaining intestinal homeostasis.