Mechanism Study Of Enteric Glial Cells And Intestinal Flora In Neuronal Intestinal Malformations

Part Ⅰ Mechanism Study of Intestinal Flora and Enteric Glial Cells in Improving Intestinal Nerve DysplasiaBackground:Intestinal neuronal dysplasia(IND)and HSCR are both Neuronal intestinal malformations(NIM).The incidence of IND is second only to HSCR,and its pathological manifestations are mainly non-functional hyperplasia or overdevelopment of localized plexuses in the submucosal and intermuscular ganglia of intestinal,and the clinical manifestations are refractory constipation or even intestinal obstruction,similar to HSCR.IND and HSCR are essentially two different outcomes for ENS to have abnormality in development.However,due to the unclear pathogenesis of IND and the lack of specific imaging and other auxiliary examination methods,the "gold standard" of diagnosis still depends on biopsy or postoperative pathology,which causes certain problems in clinical diagnosis and treatment.Therefore,exploring the pathogenesis of IND and understanding the interference and intervention factors during the development of ENS are of great significance for exploring new diagnostic and therapeutic and prevention methods of IND in the future.The traditional concept is that the embryonic development period is affected by a variety of pathogenic factors,resulting in abnormal ENS development,leading to intestinal dysfunction.However,with the deepening of embryological research,it has been found that with the delivery of the fetus,the development of ENS does not stagnate.ENCCs still migrate,differentiate and develop in the intestinal ducts of newborns,especially the occurrence and interconnection of synapses,which is an important developmental period in the neonatal period after birth.This period is also a critical period for the formation and establishment of the intestinal flora.The literature shows that the establishment and improvement of the early intestinal flora plays an important role in the continued development of ENS after birth,and the normal microbial flora plays an indispensable role in maintaining ENS function daily,regulating immunity,affecting hormone secretion and many other aspects.The intestinal flora is mainly divided into three types:probiotics,conditional pathogenic bacteria and pathogenic bacteria.Under normal circumstances,Bifidobacterium and Lactobacillus are the main normal dominant bacteria in the intestine,accounting for more than 90%.If the balance of the intestinal flora is broken,resulting in excessive proliferation of conditioned pathogenic bacteria or pathogenic bacteria,producing virulence factors,it will destroy the intestinal mucosal barrier and cause disease.Among them,Staphylococcal enterotoxins(SEs)belongs to the large family of toxins produced by Staphylococcus and Streptococcus,which can penetrate the intestinal mucosal barrier and cause toxin shock syndrome,food poisoning and a variety of autoimmune diseases.In previous studies,it is known that EGCs secrete cytokines and chemokines after being stimulated by bacteria and inflammation,which are involved in the inflammatory process of the intestine and the immune regulation of the intestine,and the loss of glial cells will lead to the destruction of the structure of the intestinal plexus and the damage of epithelial integrity.And EGCs can form a bidirectional signaling pathway from ENCs to other cells binding response with immune effector cells,intestinal endocrine cells,and epithelial cells.Therefore,EGCs can be considered to be an important intermediate medium for regulating the interaction between ENS and the intestinal microbiota.However,whether probiotics can achieve a protective effect on ENS by mobilizing the functional regulation of EGCs,and how the virulence factors produced by pathogenic bacteria affect the activity of EGCs,have not yet been clarified.Therefore,this work studied the susceptibility and response characteristics of EGCs to Staphylococcal enterotoxin A(SEA)through in vitro cell experiments,and further verified whether probiotics could have a protective effect on ENS by increasing the activity of EGCs in vivo experiments in animal models of IND.Objective:To investigate the response characteristics of EGCs to SEA and the mechanism of Bifidobacterium in improving colon motility in IND mouse models through EGCs.Methods:1.Cell experiment:Through SEA intervention at different concentrations in EGCs cell culture medium,by enzyme linked immunosorbent assay(Elisa),cellular immunocytofluorescent(ICF)et al.detected the changes of GFAP and GDNF of EGCs treated at different concentrations at different time points;2.Animal experiments:(1)Homozygoste Tlx2-/-IND model mice were screened by Southern Blot,and the development of EGCs in the intestine was assessed by WB,PCR,Elisa,IHC experiments;(2)By culturing living Bifidobacterium,the bacteria liquid was used to intervene in homozygous IND model mice,change the microflora structure of IND model mice,and verify the colonization of bifidobacteria in intestines of model mice by 16s rDNA Sequencing;(3)Confirm the improvement of colon motility by measuring and evaluating the symptoms and signs before and after intervention in IND model mice;(4)Re-evaluate the changes in EGCs markers in the model mice after intervention through WB,PCR and Elisa,and control with the other two group.Results:1.SEA can cause EGCs cytopathic effects in a dose-and time-dependent manner,and with the increase of SEA stimulation time and concentration,EGCs show retraction and rounding,cell light transmittance increases,and cytopathic effects are presented;2.EGCs reactively form an "activation state" under the stimulation of foreign antigens,and the cell volume increases under ICF staining,and the cell bulge is more obvious;3.EGCs produce GDNF response characteristics under bacterial antigen stimulation;4.Developmental abnormalities in EGCs cells in mice with homozygous Tlx2-/-mice;5.16s rDNA sequencing confirms that live bifidobacterium enema can increase the colonization of bifidobacterium in the colon of Tlx2/-mice;6.Tlx2-/-mouse model increased the number of EGCs in the colon after probiotic intervention,improved colon motility,increased expression of GFRa receptors in the colon,and increased GDNF content in serum(P<0.05)。Conclusion:1.SEA can cause the cytopathic effect of EGCs in a dose-and time-dependent manner,and promote the "activation state" of EGCs within a certain tolerance range and produce GDNF to help resist antigens,which in turn has a protective effect on ENCs;2.The responsiveness of EGCs to the intestinal flora makes them an important intermediate medium for interaction between the intestinal flora and ENS;Bifidobacterium may activate the process through the GDNF-GFRa pathway of EGCs and cause it to produce neurotrophic factors,which has an improving effect on symptoms and colon motility in animal models of IND.Part Ⅱ Aberrant Development of Enteric Glial Cells in the Colon of Hirschsprung’s DiseaseBackground:Hirschsprung disease(HSCR)is a developmental disorder of the enteric nervous system(ENS),which occurs due to the lack of ganglion cells at the distal end of the colon,resulting in the colonic segment without ganglion cells unable to move normally,resulting in colonic dyskinesia,resulting in functional intestinal obstruction.Children usually present with signs of distal intestinal obstruction shortly after birth,which are manifested by delayed meconium excretion,intractable constipation,bloating and vomiting,etc.,and if not timely intervention and treatment,there may even be serious complications such as Hirschsprung-associated enterocolitis(HAEC).The main treatment of HSCR is currently surgical treatment,meaning surgical treatment to remove the diseased intestinal segment.Although the surgical treatment method has obtained good treatment results in most children,there are still a considerable number of children who have chronic intestinal motility disorder or constipation obstructive problems after surgery,or both,and some children have recurrent enterocolitis after surgery,and the occurrence of these symptoms is inseparable from the congenital development of ENS congenital development in children themselves.Therefore,it is important to understand the biological process of ENS and explore the etiology mechanism of HSCR.ENS,the second largest nervous system in the human body,develops from embryonic neural crest-derived cells(ENCCs)and contains more than 100 million Enteric neuron cells(ENCs)(close to the total number of neurons contained in the spinal cord)and 4 to 10 times the number of Enteric glial cells(EGCs).It can regulate intestinal movement,nutrient absorption,immune response,blood circulation and other functions.In the 4th week of human embryonic development,ENCCs begin to colonize the intestinal wall and migrate along the development of the intestine to the tail end,in this process,ENCCs gradually proliferate and differentiate into different types of intestinal cell populations such as ENCs,EGCs and Cajar cells,and connect with each other to form a neural plexus,thereby regulating intestinal function.Among them,EGCs,as an important component of ENS,are similar in morphology to astrocytes in the central nervous system(CNS),distributed throughout the intestinal wall,composed of a variety of subtypes,and can be specifically expressed such as glial fibrillary acidic protein(GFAP),S100 calcium-binding protein β(S100β)and a variety of neurotrophic factors(NFs)play an important role in maintaining the intestinal epithelial barrier and mediating intestinal inflammatory responses and immune responses.In recent years,with the gradual deepening of people’s research on digestive tract diseases,the important role of EGCs in the regulation of intestinal has gradually been recognized,and in colonic motility diseases,it has been found that the number of EGCs in the intestinal canal of patients with slow transit constipation(STC)is reduced,so it is speculated that the reduction of EGCs may cause a decrease in intestinal motility,but its specific mechanism of action has yet to be explored.As a congenital colon motility disorder,HSCR has a typical lesion characteristic of"megacolon" with distal narrowing and proximal dilation due to its inability to move normally and the proximal feces accumulate.At the same time,in the operation of HSCR,accurate judgment of the range of the diseased intestinal tract and the complete removal of the lesion segment are essential to reduce the occurrence of postoperative complications and reduce the risk of secondary surgery,so in addition to the accurate identification and judgment of ENCs,the distribution and development of EGCs in the HSCR intestinal canal are evaluated.It is also of certain guiding significance to understand whether there is a correlation between the location of EGCs and ENCs in the lesion intestinal segment,which is also of certain guiding significance for the selection of surgical intestinal resection range.Objective:To investigate the abnormal development of EGCs in the HSCR lesions and the distribution relationship with ENCs.Methods:1.Specimen collection:35 cases of HSCR patients undergoing surgical treatment in pediatric surgery at Qilu Hospital of Shandong University from December 2018 to December 2020 were collected,and their clinical and imaging data were collected and summarized;2.Western Blot(WB)was used to detect the protein expression of EGCs markers GFAP and S100β in different segments of the diseased intestinal duct;3.Polymerase chain reaction(PCR)to detect the expression of GFAP and S100β mRNA levels in different segments of the diseased intestinal tract;4.Double immunofluorescence labeling(IF)of ENCs markers HuC/in different segments HuD and EGCs were fluorescently labeled to analyze the development and distribution of intercolonal EGCs and ENCs;5.The staining levels and intermuscular position distribution of GFAP and S100β in each segment of colon specimens were displayed by immunohistochemistry(IHC).Results:1.Compared with GAPDH,the expression levels of protein and mRNA of GFAP and S100β in the 4 parts of lesions(proximal segment,dilation segment,migration segment and stenosis segment)decreased from the proximal segment,dilated segment,transitional segment to aganglionic segment(P<0.05);2.GFAP and S100β and the classical intestinal neuron marker HuC/HuD IF in the lesion intestinal slices showed that there was no obvious EGCs and ENCs positive staining of aganglionic segments,and the positive staining morphology between the longitudinal muscle and the circumcisic muscle in the transitional and dilation segments was irregular and developmental deformity,while the proximal segment EGCs were located around ENCs,and the morphology of intermuscular ganglion was relatively complete,and the fluorescence intensity(AU)of gene of unit area was quantitatively analyzed.The trend of EGCs markers in four parts of the intestinal canal consistent with HuC/HuD was shown(P<0.05).3.In IHC staining,GFAP and S100β were not significantly positive stained in the ganglion-free cell segment,while between the longitudinal muscle and the circumferential muscle in the migration,dilation and proximal segments,a different number of positive staining particles were visible,among which the positive staining particles in the migration segment were significantly reduced,irregular in shape,and morphologically deformed.Positive quantitative results for IHC per unit area are consistent with quantitative results for proteins and mRNA.Conclusion:1.In the diseased intestinal canal of children with HSCR,the development and distribution of EGCs are consistent with ENCs,and there is a significant decrease in the number and developmental abnormalities of EGCs between the intermuscular segments of the ganglion;2.The markers of EGCs,GFAP and S100β,show a gradual trend of change in the intestinal canal of 4 parts of the lesion,and by detecting GFAP and S100β,it can provide clues to the developmental status of ENCs,and the normal EGCs at the excision edge can provide a reference for the scope of surgical resection,and also have certain guiding significance for the selection of the scope of surgical intestinal resection.