Preliminary Study Of The Muscularis Macrophage In Hirschsprung’s Disease

BackgroundHirschsprung disease(HSCR)is a common developmental malformation of digestive tract in neonatal period.The main pathogenesis is related to the proliferation,apoptosis/death,migration and differentiationof enteric neural crest cell(ENCC)during embryonic period,.This resulting resulted in persistent abnormal contraction of the affected bowel segment,compensatory expansion and thickening of the proximal colon,and ultimately was manifested as delayed meconium excretion,intractable constipation and abdominal distension.This disease seriously affects the life quality of children,and even threatens lives.Therefore,Further further studying the pathogenesis of HSCR is important and urgent in order to intervene early to improve the prognosis of the disease.At present,the research is mainly focused on the regulation mechanism of enteric nevous system development.The formation of the enteric nervous system is a very complex process.In addition to being precisely regulated by its own genes and regulatory factors,it is also affected by the surrounding microenvironment.Macrophages are an important component of the intestinal microenvironment.Among them,the muscularis macrophage(MM)is closely related to the myenteric nerve plexus.Studies have shown that there is an obvious crosstalk and even developmental interdependence between MM and enteric neual cells.Moreover,MM mainly exhibits M2 macrophage phenotype.But it is not clear whether MM is related to Hirschsprung’s disease.ObjectiveTo explore the distribution and quantity of MM in the aganglionic segment of Hirschsprung’s disease and the mechanism of MM participating in the pathogenesis of Hirschsprung’s disease.MethodThe colon specimens were collected retrospectively from 14 Hirschsprung’s disease patients who underwent surgery at our institution from August 2018 to January 2021 and the patients’age is under 6-month-old.The specimens were divided into aganglionic group and ganglionic group.The samples were prepared for immunohistochemistry.We analyzed the expression of marker of MM(CD 163)in the HSCR samples of the two groups.The human mononuclear leukemia cell line(THP-1)was induced into MO type macrophages and then induced into M1 type macrophages using lipopolysaccharide and interferon yor induced into M2 type macrophages using interleukin-4 and interleukin-13.M1/M2 macrophages were co-cultured with neural stem cell line(SH-SY5Y)or SH-SY5Y cells were treated with conditioned culture medium from M1/M2 macrophages.The proliferation,apoptosis/death and migration of SH-SY5Y cells were detected with cell counting Kit-8,flow cytometry,ethidium bromide/acridine orange fluorescence double staining and cell migration test.ResultsImmunohistochemistry showed that in the HSCR ganglionic segment CD 163-positive muscularis macrophage were mainly located around the myenteric ganglion cells and there were also scattered CD 163-positive MMs in the.circular muscle.In the aganglionic segment of HSCR,CD 163-positive MMs were significantly reduced.The cell count of CD 163-positive MMs was statistically different between the two groups(t=8.288,P<0.05).The results of in vitro cell experiments showed that the cell proliferation of SH-SY5Y was significantly enhanced when treated with M2 macrophage conditioned culture medium compared with the group treated with M1 macrophage conditioned culture medium(1.21±0.04 vs.0.81±0.05,P<0.05).The mortality rate of SH-SY5Y was significantly reduced(16.67±4.04%vs.58.67±7.23%,P<0.05)and the apoptotic rate(early apoptosis and late apoptosis)of SH-SY5Y was also significantly decreased(3.23±0.45%vs.17.17 ±1.75%,P<0.05)when co-cultured with M2 macrophages in comparison to the group co-cultured with M1 macrophages.At the same time,M2 macrophages could significantly promote the migration ability of neural stem cells.When SH-SY5Y was co-cultured with M2 macrophages,the cell migration count after 48 hours was significantly increased than that co-cultured with M1 macrophages(143.67±40.13 vs.72.67±16.29,P<0.05).ConclusionsThe number of the muscularis macrophage with M2 macrophage phenotype in the aganglionic segment of HSCR was significantly reduced,and M2 macrophages could promote the proliferation and migration of neural stem cells in vitro,and inhibit their apoptosis/death.It is speculated that the muscularis macrophage might participate in the pathogenesis of Hirschsprung’s disease by regulating the development of enteric neural cells.