The Role Of Substance P And NK Cells In The Pathogenesis Of Hirschsprung Disease Associated Enterocolitis

PART ONE Study of change of substance P level in Hirschsprung disease model mice(Ednrb knockout mice)and its role in the function of natural killer cells and their mechanism in pathogenesis of Hirschsprung disease associated enterocolitisObjective: This study is to find the change of substance P(SP)level and natural killer cells(NK cells)in Hirschsprung disease(HD)model mice(Ednrb knockout mice)with and without Hirschsprung's disease associated enterocolitis(HAEC).Besides,we study the interaction between SP and NK cells to illuminate the mechanism of HAEC.Methods: Ednrb knockout mice were bought from Jackson laboratory and were feed in SPF environment.They were divided into HD group and wide type group and then divided by age-postnatal 1 week(w),2w and 3w.3w HD mice had HAEC.Haematoxylin-eosin(HE)staining was used to evaluate intestinal inflammation.RNA sequencing of intestine tissues was to find difference of gene expression.Immunohistochemistry(IHC)and immunofluorescence(IF)were to show expression of SP and NK cells.Western-blot and real-time quantitative Polymerase Chain Reaction(RT-q PCR)analyzed the protein and m RNA level of SP?Nkp46?RORgammat?NK1R?IFNgamma?perforin?Granzyme B and IL22.Compouds of feces were studied by metabonomics study.Results: HAEC happened in 3w HD mice.And dilated intestine showed more severe degree than narrow intestine.RNA sequencing found the most differential intestine was dilated intestine of 3w HD mice.SP elevated in narrow colon of 3w HAEC mice.In IHC and IF results,SP and NK cells(including NK-22 cells)elevated in narrow intestine of 3w HAEC mice.Both protein and m RNA levels showed increase of SP?Nkp46?RORgammat?NK1R?IFNgamma and IL22 in narrow colon of 3w mice.Metabonomics found that the starch and sucrose metabolomics pathway and the pentose and glucoronate interconversions pathway show significant difference and were correlated to many nerve related genes.Conclusion: Pathology of HAEC is more obvious in dilated intestine than the narrow part.However,SP,NK cells and related factors increase in narrow intestine of 3w HD mice.This hints that interaction between SP and NK cells may do good to the maintenance of intestinal hemeostasis.PART TWO Study of change of substance P level in HD patients and its role in the function of NK cells and their mechanism in pathogenesis of HAECObjective: This study is to find the change of SP level and NK cells in HD patients with and without HAEC.Besides,we study the interaction between SP and NK cells to illuminate the mechanism of HAEC.Methods: Samples were collected from HD patients and were divided by with and without HAEC.Samples of control group were from congenital anorectal malformation patients without enteral abnormality.HE staining was used to evaluate intestinal inflammation degree.IHC and IF were to show expression of SP and NK cells.Western-blot and real-time quantitative RT-q PCR analyzed the protein and m RNA level of SP,Nkp46,NK1 R,IFNgamma,perforin,Granzyme B and IL22.Results: Dilated intestine showed more severe degree than narrow intestine in HAEC patients.In IHC and IF results,SP and NK cells elevated in narrow intestine of 3w HAEC mice.Both protein and m RNA levels showed increase of SP,CD16,RORgammat,NK1 R,IFNgamma and IL22 in narrow colon of HAEC group.Conclusion: Pathology of inflammation is more severe in dilated intestine than the narrow part of HAEC patients.However,SP,NK cells and related factors increase in narrow intestine of HAEC group.This means that interaction between SP and NK cells may be beneficial to intestinal hemeostasis.PART THREE Study of interaction between SP and NK cells in vitroObjective: After sorting mice intestinal NK cells through magnetic beads and culturing NK92 MI cell line,function change of NK cells with SP stimulation will be studied and the mechanism will be discussed.Methods: Percoll discontinuous density gradient centrifugation combined with magnetic bead separation was used to obtain purified primary NK cells in the colon of mice,and their purity was observed by immunofluorescence staining.The NK cells were divided into control group,SP gradient concentration stimulation group and NK1 R antagonist Aprepitant + SP gradient concentration stimulation group.The killing activity of NK cells was detected by lactate dehydrogenase(LDH)release method.Enzyme-linked immunosorbent assay(ELISA)was used to detect the IFNgamma concentration in the supernatant.the expression of IFNgamma and IL22 in NK cells were observed by IF.NK92 MI cell line was cultured into logarithmic growth state for experiment,and were grouped the same as mice NK cells.The killing activity of NK cells and the concentration of IFNgamma in the supernatant was detected.The NK cells stimulated by SP were analyzed by transcriptome sequencing.The possible mechanism was analyzed.The intestinal epithelial barrier model was established by using Caco2 cells,and the effect of NK cells stimulated by SP on tight junction protein ZO-1 was observed by IF and western-blot.Results: The purity of NK cells from mice intestine reached 93.50%.SP could enhance the killing activity of Nkp46(+)NK cells in mice and promote the secretion of IFNgamma and il-22,which was related to the SP concentration.The optimal concentration was 10-8M,which could be blocked by Aprepitant.SP could enhance the killing activity of NK92 MI cell line and promote its secretion of IFNgamma.The optimal concentration was 10-8M.Transcriptome sequencing showed that MAPK signaling pathway showed the greatest change in SP stimulation NK92 MI group.In the group of NK92 MI cells treated with SP,the expression level of zo-1 in Caco2 cells decreased most significantly.Conclusion: SP can enhance the activity of NK cells and promote the secretion through receptor NK1 R.Related pathways may be MAPK signaling pathway.NK92 MI cell lines only with function of traditional NK cells have damage to intestinal epithelium.However,Nkp46(+)NK cells purified from mice colon,due to include NK-22 cells,the secretion of IL22 can help intestinal epithelium repaired.The mutual balance of both make intestine has stronger effect against pathogens and at the same time,protect the intestinal epithelial integrity and maintain intestinal hemeostasis.