A Study Of The Therapeutic Effect Of Interfering Prostaglandin E2 Biosynthetic Pathway In A Mouse Model Of Hirschsprung’s Disease

Objective:Hirschsprung’s disease（HSCR）,also known as loss of the neuronal in the intestinal tract,is a congenital disorder that occurs mostly in the infant.The main reason is the lack of ganglia cells in the distal intestinal wall,which is caused by the inability of neural crest cells to migrate,proliferate and differentiate during early embryogenesis,leading to absence of the enteric nervous system.Multi-omics data analysis showed that prostaglandin E2（PGE2）and its synthetase m PGES（Microsomal Prostaglandin E2 Synthase）were significantly increased in the intestinal tube of HSCR lesions.It has been reported that PGE2 signaling pathway affects the cytoskeleton of F-actin,thereby inhibiting the migration of cells.Therefore,we speculate that PGE2 may play a role in the pathogenesis of HSCR.The aim of this study was to explore the relationship between PGE2 and Hirschsprung’s disease based on HSCR animal model,and determine whether PGE2 biosynthetic pathway could be a potential target for the treatment of HSCR disease.Methods:1.Screening the siRNA of targeting m PGES.The siRNA sequence of targeting m PGES gene was designed and transfected CT26.WT cells in vitro,then the expression level of m PGES m RNA were detected by q PCR,and m PGES protein was detected by Western blot.2.Preparation and characterization of siRNA and polyethyleneimine（PEI）nanocomposites.PEI/siRNA complexes were prepared with different N/P ratios,then the optimal N/P ratio was determined by gel retardation assay.And siRNA was mixed with PEI with this ratio,then zeta potential and particle size of this sample were measured by dynamic light scattering（DLS）.3.Determine the effect of intraperitoneal injection of the pregnant mouse.PEI and Cy5-siRNA complexes（1mg siRNA/kg）were prepared according to the optimal N/P ratio,and the compound was injected into pregnant mice via intraperitoneally.After 24 h,optical images of the whole body,maternal gastrointestinal,kidneys,liver,pancreas,uterus,and fetal mouse were acquired with an IVIS 200 imaging system.4.Analysis of in vivo interference efficiency of sim PGES.PEI/sim PGES complexes（1mg siRNA/kg）were prepared according to the optimal N/P ratio were administered via the intraperitoneally.Then liver,spleen,lung,kidney,rectum,colon,and cecum tissues of the treated mouse were dissected.Analyze the expression of m PGES in tissues by q PCR and Western blot to determine the in vivo interference efficiency of PEI/sim PGES.5.Construction of HSCR mouse model.The Endothelin receptor-B（Ednrb）gene of C57BL/6J mouse was functionally knocked out by CRISPR/Cas9 technology to obtain Ednrb^+/-mouse.Ednrb^+/-male and female mice were mated to obtain Ednrb^-/-mice.Analysis of the symptoms,signs and distribution of gastrointestinal ganglion cells in Ednrb^-/-mice confirmed the success of HSCR mouse modeling.6.For therapeutic treatment studies,Pregnant rats were injected intraperitoneally with PEI/sim PGES 606 complex at 1 mg siRNA/kg in E8.5 and E12.5,and the PEI/si NC complex was used as a negative control.PEI/sim PGES 606 complex（1mg siRNA/kg）were administered via the intraperitoneally to pregnant mouse in E8.5 and E12.5,meanwhile,the PEI/si NC complex was used as a negative control.Through genotyping to identify Ednrb^-/-mice,observed the digestive tract phenotype of Ednrb^-/-mice in different treatment groups and performed HE staining and immunohistochemistry to evaluate the effect of treatment HSCR disease model.Results:1.The designed 6 pairs of siRNA screening results showed that sim PGES 606 has obvious inhibitory effect on m PGES in vitro;2.Intraperitoneal injection of sim PGES 606 can effectively inhibit the expression of m RNA and protein of m PGES in tissues such as the rectum,and the PEI/siRNA complex can penetrate the placental barrier.3.PCR,DNA sequencing,Ednrb^-/-mouse phenotypic identification and immunohistochemical analysis confirmed the successful modeling of HSCR mice.And the mouse model is different from the size of the Ednrb gene fragment knockout reported in the literature.4.The in vivo efficacy analysis of sim PGES in HSCR mouse model showed that PEI/sim PGES606 complex can effectively relieve the stenosis of the distal portion of the colon and rectum in Ednrb^-/-mice,and increase the number of ganglion cells which is the distal portion of the colon in Ednrb^-/-mice.Conclusion:1.The siRNA which effectively inhibits mouse m PGES has been obtained.The complex formed by PEI and siRNA at N/P=8 can be transported to intestinal epithelial cells to exert RNA interference effect.2.PEI/siRNA can be effectively transported to pregnant mice by intraperitoneal administration.3.The mouse model of Hirschsprung’s disease has been successfully constructed.4.Inhibition of PGE2 biosynthesis can alleviate the degree of digestive tract lesions in HSCR mice,increase the distribution of ganglion cells in intestinal tissues,and have a better therapeutic effect on Hirschsprung’s disease.