A Study About Changes And Mechanism Of VEGF, BMP-2, BMP-4 In Neuronal Intestinal Malformation

ObjectiveNeuronal intestinal malformation(NIM) is one of the most common disease,Its pathological characteristics are the abnormity on quantity,quality and distribution of intestinal ganglion cells,which result in a series of clinical symptoms,such as constipation,enterocolitis and others.This kind of disorders was usually divided into Hirschprung's disease and allied disorders.Some studies indicate that the abnormal ENS in anorectal malformation(ARM)increase,which may be the cause of dysfunction of rectum and the cause of constipation.Recently a lot of cases with their colonic ganglion cells abnormal at 12 or 18 years old and later were found.Many studies show that the abnormity of colonic ganglion cells is accompanied by many adult colonic disorders. Some concluded that acquired factors,such as ischemia,anoxia,inflammation,infection, toxin et al,caused the delayed disorders.Although there are marked absence of ganglion cells and proliferation of nerve fibers in the colon of patients,the precise mechanism of NIM is currently unknown,and it is one hot spot of research work in surgery.Animal experiments indicated that BMP had important roles in allelotaxy and central neural development.By immunochemistry,real-time quantitative polymerize chain reaction(PCR) and Western-blot,we discussed the role of BMP and VEGF in the occurrence of NIM through study of their expression in NIM diseases.We want to know the functions of BMP and VEGF during the enteric nervous system development.To further elucidate the mechanism and determine the biomarker of NIM,intestinal neuropathy rats are used in the study.The ganglion of the rats' colon are studied by the use of HE staining,then we performed a longitudinal observational study of reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH),and total antioxidative capacity(T-AOC) in rats' enteric tissues and nerve tissues,which would give the direction for later mechanism research of NIM.MethodsIn this study,all specimens were attained from 62 patients with confirmed pathological diagnosis of NIM,pre-operatively or post-operatively,in the surgery department of affiliated Hospital of Shandong University between 2006 and 2008.The narrow segment of HD was taken as the aganglionic segment,the transitional segment conformed by pathological diagnosis was taken as the ganglionic segment,and the dilated segment was taken as the proximate normal segment.One portion of colonic tissue from each of the three segments was taken respectively and two was stored in -84℃.Similar groups of tissue from the three were fixed in 10%neutral-formalin and embed with paraffin following.All were divided into three groups with different ages, included baby group,children group and adult group.32 patients who had no neuronal disease,were taken as control group.Specimen of the abnormal ganglionic segment and normal segment of colon were stained by polmer detection system for immunochemistry staining,photos were taken by RS Image system,and the area of every image of BMP-2,-4 and VEGF were judged by Image analysis system.The qualitative expressions of BMP gene and VEGF gene of these segments were detected by real-time quantitative PCR,and the difference of the expression was concluded by SDS software and the 2^{-ΔΔCt} method.The correlation between the expression of BMP gene and VEGF gene was calculated by Bivariate-person way of SPSS for windows 17.0 Software.At last,the qualitative expressions of BMP protein and VEGF protein of these segments in 62 cases were detected by Western blot. The relation between the expression of BMP and VEGF and the cause of NIM and the correlation of BMP and VEGF would be concluded.Adult male rats(Wistar,180-220g;Laboratory Animal Center of Shandong University,China) were used in this study.Rats were randomly divided into two groups, i.e.,experimental group(n=40) and corresponding time-matched control group(n=50). In order to evaluate abnormal ENS and the time-dependent changes of LPO,CS2 was administrated to Wistar rats by inhalation at a single concentration of 1250mg/m3 for 12 weeks.Age-matched control rats received an equivalent volume of air.On completion of 0,2,4,8 and 12 weeks of CS2 administration,rats(n=10 per experimental and control group) were chosen at random and sacrificed by cervical decapitation.The distal colon was taken out and fixed in 10%neutral buffered formalin before routine processing for paraffin sectioning.Histological sections were stained with hematoxylin and eosin for routine examination.The enteric tissues,cerebral cortex,hippocampus and spinal cord were quickly dissected and frozen in liquid nitrogen.The levels of MDA,ROS GSH and activities of SOD,GSH-Px,CAT,T-AOC were measured using commercial assay kits. All indexes were examined in enteric tissues,cerebral,hippocampus,spinal cord and serum of experimental and corresponding time-matched control rats.Results1.ImmunochemistryPositive expression of VEGF,BMP-2,-4 were found in abnormal ganglionic segment and normal control group.The stain of anti-VEGF,anti-BMP-2,4 was all low in myenteric nerve plexuses of abnormal ganglionic segment(P＞0.05),however there were no obvious difference in muscular layers(P＞0.05).VEGF,BMP-2,4 were expressed in ganglion cells of HD,the expression in strictural segment was highly decreased to the dilated segment(P＜0.05),and it was also different between transitional segment and normal segment(P＜0.05),no evident different between normal segment and dilated segment.In baby group,children group and adult group,the expression in strictural segment or transitional segment was decreased to normal segment(P＜0.05).The expression in normal segment of children group or adult group was decreased to the expression in the baby group(P＜0.05),but there was no different between the expression in strictural or transitional segment of baby group and other group(P＞0.05).2.Real-time quantitative PCRThe expression in strictural segment showed highly decreasing as compared to the dilated segment(P＜0.05).It was showed that the expression of VEGF,BMP-2,-4 in strictural segment was 0.09,0.07,0.19 times respectively of that in dilated segment by the real-time quantitative PCR,and the expression of VEGF,BMP-2,-4 in transitional segment was 0.21,0.22,0.33 times respectively of that in dilated segment.The expression from normal to strictural segment was decreased.The expression of VEGF or BMP-2,-4 in normal segment showed decreasing with age,thus their expression shows time-dependent and space-dependent changes.There was positive correlation between VEGF and BMP(r=0.586,P＜0.01).There was positive correlation between BMP-2 and BMP-4 too(r=0.762,P＜0.01).3.Western-blotThe expression of VEGF,BMP-2,-4 in strictural segment was highly decreased to the dilated segment(P＜0.05),and the expression in transitional segment was highly decreased to the dilated segment(P＜0.05).The expression in dilated segment of children group or adult group showed decreasing.Their expression shows time-dependent and space-dependent changes.4.Changes of lipid peroxidation in intestinal neuropathy ratsThe cells became lager and vacuole apomorphosis through the time course of CS2 exposure.The number of myenteric plexus ganglion cells decreased significant in the end. In our present study,changes of these indexes of oxidant system and antioxidant status in nerve tissues of rats occurred.The levels of ROS significantly increased in enteric tissues,cerebral cortex, hippocampus,spinal cord and serum in experimental group.In enteric tissues,ROS significantly increased by 11.8%,34.9%,38.5%and 42.7%after 2,4,8 and 12 weeks.In cerebral cortex,ROS significantly increased by 19.0%,30.5%and 31.5%after 4,8 and 12 weeks,respectively.Spinal cord ROS levels significantly increased(P＜0.01) on 8 and 12 weeks of exposure and corresponded to 134.7%and 164.4%of control, respectively.Compared to the control,the levels of ROS in serum increased(P＜0.01)by 71.9%,85.3%,and 87.9%after 4,8 and 12 weeks,respectively.MDA levels in enteric tissues significantly increased by 33.3%,52.4%,,66.7%and 76.2%after 2,4,8 and 12 weeks.Cerebral cortex MDA levels significantly increased after 2,4,8 and 12 weeks(P＜0.01) and corresponded to 117.1%,122.2%,128.6%and 134.8%of control, respectively.Serum also showed a significant increase in MDA levels after 2(14.0%),4 (19.0%),8(33.0%) and 12(53.6%) weeks of exposure.CS2 caused a significant decrease in GSH-Px activities in enteric nerve,cerebral cortex,hippocampus,spinal cord and serum after different time points.The activities of GSH-Px decreased(P＜0.01) by 24.8 %,18.2%,29.9%,16.2%and 33.3%in enteric tissues,cerebral cortex,hippocampus, spinal cord and serum after 12weeks of intoxication,respectively.A reduction in GSH levels and CAT and T-AOC activities in nerve tissues was showed on different time points,while SOD activities significantly increased.Compared to the control,the contents of GSH and activities of CAT and T-AOC began to decrease after 4,2,and 4 weeks in enteric tissues,respectively,after 8,8 and 2 weeks in cerebral cortex, respectively,after 8,2 and 2 weeks in hippocampus,respectively,after-,8 and 8 weeks in spinal cord,respectively,and after 8,4 and 8 weeks in serum,respectively(The(-) indicated that GSH levels were not changed in spinal cord after 0,2,4,8 and 12 weeks of CS2 treatment).After 12 weeks,GSH contents and CAT,T-AOC activities decreased(P＜0.01) by 9.1%,20%and 19.4%in enteric tissues,by 15.2%,22.6%and 23.6%in cerebral cortex,respectively,by 25.6%,46.4%and 32.3%in hippocampus,respectively, by -2.2%(P＞0.05),35.0%and 15.5%in spinal cord,respectively,and by 16.3%,48.6% and 30.7%in serum,respectively.However,SOD activities significantly increased in nerve tissues and serum.After 12 weeks,SOD activities significantly increased(P＜0.01) by 20.8%,31.5%,44.4%,64.4%and 12.1%in enteric tissues,cerebral cortex, hippocampus,spinal cord and serum,respectively.The levels of ROS and MDA significantly increased in enteric tissues,cerebral cortex, hippocampus,spinal cord and serum after intoxication.However,the levels of GSH and the activities of GSH,GSH-Px,CAT and T-AOC in nerve tissues of rats decreased after 0,2,4,8 and 12 weeks.Ca²⁺ concentrations and CaM contents significantly increased (P＜0.01) after 4,8 and 12 weeks.Time-dependent changes of ROS,MDA and antioxidant status in nerve tissues were found.ConclusionsVEGF,and BMP-2,-4 can be found in human ENS of normal or NIM diseases.Their expression shows time-dependent and space- dependent changes.They reached to the peak in newborn period.The decrease in the guts of adults and the abnormal ganglionic bowl implies that they maybe participate in the formation and maturation of ENS segment.The expression of two genes was intrinsic,and there was positive correlation between VEGF and BMP.They may cause early differentiation and early maturation,so the change may be the cause of disease.CS2 exposure leads to the morphologic change in the intestinal ganglion cells.This study shows that elevation of lipid peroxidation and reduction of antioxidant status in rats' nerve tissues,and changes of these indexes suggested that ROS and concomitant LPO,at least in part,were involved in neuropathy.The misbalance in oxidative and antioxidant status might be involved in the occurrence and development of ENS neuropathy.