Erbin Inhibits The Progression Of Inflammantory Bowel Disease By Suppressing Autophagic Cell Death

Increasing patients suffered from inflammatory bowel disease?IBD?are at risk for development of colorectal cancer?CRC?.However the pathogenesis and the molecular mechanisms of IBD remains unclear.Erbin?also known as Erbb2ip?was first discovered as an Erbb2 interacting protein in 1997.Erbin,belonging to the LAP family?leucine rich repeat and PDZ domain?,plays an important role in the regulation of cell proliferation and differentiation,cardiac development,myelination,and progression and metastasis of cancer.Nevertheless,the role of Erbin in IBD is still uncharacterized..Previously,we found that the expression of Erbin was significantly decreased in colonic mucosa of patients with IBD,mice with acute IBD and mice with spontaneous chronic IBD?IL-10-/-mice?,compared with that in relatively normal colonic mucosa of control groups.Moreover,using an Erbin?c/ ? c?Erbin PDZ domain was knocked out?mouse model of IBD,we found that Erbin?c/?cmice were more susceptible to injury for experimental IBD.To further study the role of Erbin in IBD,we established another Erbin full knockout mice by a CRISPR/cas9 System,in which a deletion of base in Erbin exon 16 causes frameshift resulting protein translation termination.Erbin^-/-mice were used to study the role and the potential mechanisms of Erbin in IBD.In DSS-induced mice colitis model,which was similar with patients suffered from ulcerative colitis,the body weight,the ratio of intestinal weight to body weight,the extent of intestinal epithelial inflammation and crypt damage were used to evluate the injury of IBD,we found that Erbin^-/-mice are more susceptible to IBD than Erbin+/+mice.Furthermore,in vitro and in vivo experiments were used to study the potential mechanisms of Erbin inhibiting the progression of IBD.In the colon of DSS-induced colitis mice and IL-10-/-mice,decreseased Erbin expression,increased expression ofautophagic marker LC3 B,were observed when compared with that of control mice.Furthermore,the expression of LC3 B was significantly upregulated,while the expression of ATG16L1 was downregulated in Erbin^-/-mice compared that with Erbin+/+mice.Electron microscopy showed increased formation of autophagic/lysosomal vacuoles in the colon of Erbin^-/-mice compared with Erbin+/+ mice.Autophagic/lysosomal vacuoles in DSS-induced Erbin^-/-mice was significantly increased accompanied with extensive mitochondrial swelling.Then,293 T cells were treated with TNF-? to mimic the in vitro model of cells stimulated by inflammatory microenvironment.Immunofluorescence showed more autophagosomes formation in cells after TNF-? treatment.Increased formation of autophagosomes were detected in cells knocked down of Erbin and treated with TNF-?compared with that in cells treated with TNF-? merely.And RT-PCR showed upregulated expression of LC3 B accompanied with the increased formation of autophagosomes.Flow cytometry showed that about 40% CT26 cells died 22 hours after TNF-?treatment.However,almost 90% CT26 cells died 22 hours after TNF-?treatment when Erbin expression was knocked down,suggested that knockdown of Erbin aggravates TNF-?inducing cell death.Finally,autophagy inhibitor chloroquine?CQ?was used when establishing DSS-induced IBD model.HE staining results showed CQ treatment significantly attenuated inflammatory cell infiltration into mucosa and extensive damage of epithelium along with crypt destruction in DSS induced Erbin^-/-mice.In accordance with above results,the expression of apoptosis-related biomarkers could be fell in the colon of DSS induced Erbin^-/-mice after CQ treatment.In summary,our study uncovers a crucial role of Erbin in IBD,and elucidates that Erbin inhibits the progression of IBD by supressing autophagic cell death,providing a new clue for clinical treatment of IBD.