The Role And Its Mechanism Of ErbB4 Receptor In Gut Homeostasis Within Intestinal Tissue

Gut is one of the largest neuroendocrine organs within the human body,with a microbiome composed of trillions of microbes.Intestinal microorganisms participate in many physiological activities including normal intestinal function,neuroregulation and colonization of antagonistic pathogenic microorganisms to maintain intestinal homeostasis through mucosal epithelial cell barrier,neuronal immune regulation and cytokine interaction.Previous studies have found that inflammatory bowel disease,irritable bowel syndrome and depression are all related to the imbalance of intestinal flora homeostasis,and can affect the disease process through neurotrophic factors and neurotransmitters,which indicated that the homeostasis of intestinal flora plays an important role in the regulation of the occurrence and development of diseases,but the mechanism of action is still unclear.Intestinal mucosal epithelial cells express a variety of pattern recognition receptors,such as EGFR receptor.EGFR has been found to play an important role in the regulation of gastrointestinal mucosal epithelial inflammation and intestinal flora in recent years.ErbB4 is a member of the epidermal growth factor receptor family,which has four members:EGFR（ErbB1）,ErbB2（Neu）,ErbB3,and ErbB4.As an important member of the epidermal growth factor family,ERb B4 is mainly expressed in cerebral cortex GABA can inhibit interneurons and regulate the proliferation of central nervous system,tumor and heart.So far,the expression,distribution and role of ErbB4 in intestinal tissues are not clear.On the other hand,whether ErbB4 interacts with intestinal flora is still unclear.Therefore,this study takes this as an entry point and comprehensively adopts genomics,morphology,molecular biology and other methods to elucidate the role of ErbB4 signaling pathway in intestinal flora homeostasis from the perspective of intestinal regulation,so as to provide a scientific basis for the prevention and new treatment of diseases.Methods:（1）The expressions of NRG1 and ErbB4 in intestinal tissues were detected by RT-PCR in C57BL/6 mice.（2）The expression and spatial distribution of NRG1 and ErbB4 in the colon of mice were observed by multi-label immunofluorescence staining.The C57BL/6 mice were used to observe the relationship between NRG1 and ErbB4 expression and the spatial location of interneurons in colon.（3）The expression and distribution of ErbB4 protein in Villin-positive epithelial cells were further detected by fluorescence labeling visualization method in colon tissue of Vil1-Cre-Ai9 mice.（4）Using Cre-Lox P gene editing technology,ErbB4 conditional gene knockout mice(Vil1-Cre,ErbB4^-/-),litter control mice(ErbB4^flox/flox,Vil1-Cre),ErbB4 gene knockout mice(ErbB4^+/-)and homotypic control wild-type mice (ErbB4^+/+)were used to analyze the knockout efficiency and apparent morphological characteristics of ErbB4 in colon by gene identification,immunohistochemistry,RT-PCR and Western blotting assay.（5）16S r DNA high-throughput sequencing was performed on the colonic flora of ErbB4^+/+mice and ErbB4^+/-mice in the same litter to analyze the composition of the microflora of mice.（6）16S r DNA high-throughput sequencing was performed on the colonic flora of Vil1-Cre mice and their litter Vil1-Cre-ErbB4^-/-mice to analyze the composition of the microflora of mice.（7）The ulcerative colitis model was established by using Vil1-Cre mice and their litter Vil1-Cre;ERBB4^-/-mice.Colon tissues and colonic microflora of mice were collected at different time points,and the appearance,morphological changes,m RNA and protein levels of mice after modeling were analyzed by general observation,RT-PCR and immunohistochemistry.16Sr DNA high-throughput sequencing was performed on the colonic flora of Vil1-Cre and their litter Vil1-Cre;ErbB4^-/-model mice,respectively,and the composition of the microflora was analyzed.（8）C57BL/6 mice were used to construct ulcerative colitis model,and the expression of ErbB4 in the inflammatory epithelium during the injury and recovery stages were analyzed by RT-PCR and western blotting.Results:（1）NRG1-ErbB4 signaling is widely expressed in mouse intestinal tissues:RT-PCR results showed that the expression of ErbB4 and NRG-1 were detected in the stomach,duodenum,jejunum,ileum,and colon of normal mice.The results of multiple immunofluorescences showed that NRG1 was expressed in the lamina propria of the colonic mucosa and co-expressed with PGP9.5 in the colonic mucosa of mice,ErbB4 is not only expressed in intestinal neurons,but also expressed in the colonic mucosal epithelium.ErbB4,Ch AT and Tuj-1 were co-expressed in the lamina propria of colonic mucosa.NRG1,Ch AT and CGRP were co-labeled in the lamina propria of colonic mucosa in interneurons,and a small amount of NRG1 and TH were co-labeled in the submucosa of colonic mucosa.Further labeling with Ai9 transgenic mice revealed the expression of ErbB4 in Villin-positive epithelial cells.（2）ErbB4 can regulate intestinal microflora homeostasis in mice:Compared with ErbB4^+/+,the proportion of norank-f-Muribaculaceae、Lachnospiraceae-NK4A136-group,unclassified-f-Lachnospiraceaeand Akkermansiaare in ErbB4^+/-mice intestinal microflora are decreased,the proportion of Prevotellaceae-UCG-001,Helicobacter,Lactobacillus,Quinella and Alloprevotella are increased in ErbB4^+/-mice.Compared with Vil1-Cre,the proportions of norank-f-Muribaculaceae,Lachnospiraceae-NK4A136-group,Enterococcus,Dubosiella,Bacteroides and unclassified-f-Lachnospiraceae are decreased in the community composition of Vil1-Cre-ErbB4^-/-mice.While,the proportion of Lactobacillus,Odoribacter in the community composition of Vil1-Cre-ErbB4^-/-are increased.Through the analysis of the above differential microflora,it can be concluded that norank-f-Muribaculaceae is the common differential microflora,and their proportions in the differential comparison decreased.（p<0.05）.（3）The loss of ErbB4 intensifies the pathogenetic process of ulcerative colitis:A mouse model of ulcerative colitis was established.The pathological damage of ulcerative colitis mice with ErbB4 knockout was more serious.RT-PCR results showed that the m RNA levels of ErbB4 and NRG1 were up-regulated in different degrees.Western Blot results showed that NRG1 was expressed in colon,and ErbB4 was increased after modeling.The relative abundance（%）of the three groups of Vil1-cre control group（Vehicle）,injury group（4d DSS）,and Recovery group（3d Recovery）in normal mouse ulcerative colitis model showed that:Compared with the control group,no significant change in norank-f-Muribaculaceae.But compared with the control group,the relative abundance（%）of the norank-f-Muribaculaceae is decreased in the Vil1-Cre-ErbB4^-/-injury group（c KO-4d DSS）（p<0.05）.Conclusions:（1）ErbB4 was widely expressed in intestinal tissues and highly expressed in Villin-positive cells.（2）ErbB4 plays a regulatory role on the intestinal flora,and mainly participates in the regulation of intestinal flora by Villin-positive cells.（3）ErbB4 is involved in the occurrence and development of ulcerative colitis mainly by controlling the intestinal inflammatory susceptible flora norank-f-Muribaculaceae.In conclusion,our study revealed the influence and expression pattern of ErbB4 signaling pathway on intestinal flora changes,and revealed that ErbB4signaling pathway is a key endogenous regulatory factor affecting intestinal flora homeostasis,among which Villin-positive colon mucosal epithelial cells are the main cellular target and action vector.Furthermore,it has deepened our understanding of the internal regulation and mechanism of peripheral nerves affecting intestinal flora homeostasis,and provided molecular targets and pathologic and pathogenic regulatory basis for the treatment of related diseases,and provides a new perspective and scientific basis for exploring the molecular mechanism of neuro-intestinal regulation.