Epigenetic Regulation Of Visceral Hyperalgesia In Adult Rats Induced By Neonatal Somatic Inflammation

Background:Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by abdominal pain and bloating in association with altered bowel movements. Recent studies have shown that hydrogen sulfide (H2S), synthesized by cystathionine-β-synthetase (CBS), has become recognized as an important endogenous molecular in nociceptive signaling and inflammation and that neonatal period is vulnerable time for development of IBS. However, the molecular and epigenetic mechanisms of visceral nociceptive processing are not fully understood.Specific Aims:Therefore, the present studies were designed to investigate the following specific aims:(1) To establish a novel animal model for IBS induced by neonatal somatic inflammation (NSI). To examin whether NSI could produce visceral hypersensitivity and enhance the expression of the endogenous hydrogen sulfide producing enzyme CBS and Transient receptor potential vanilloid1(TRPV1) expression in dorsal root ganglion (DRG) in adult rats.(2) To define the properties of colon-specific DRG neurons and possible ionic or molecular bases for the enhanced neuronal excitability.(3) To examine the methylation status of CBS gene in primary sensory neurons after NSI.(4) To examine the pain-related gene expression level in colon tissues both from rats and human.Methods:(1) Animal model of IBS was induced by giving different stimulation intensity such as complete Freund’s adjuvant, carrageenan or formalin at different time points (born after10d,3w,6w). Behavioral testing methods of colorectal distension was employed to determine visceral hyperalgesia in adult rats.(2) Patch clamp technology was used to study excitability of colon specific neurons of dorsal root ganglion (DRG T13-L2).(3) Real-time PCR and Western blotting wre employed to study both transcription and protein level of hydrogen sulfide synthase CBS gene and TRPVl gene in colon related DRGs and colon mucosa/colon muscle.(4) Fluorescent-immunohistochemistry was employed to detect co-expression of CBS and TRPV1in colon specific DRG neurons.(5) Primary cultured rat DRG neuron cells, given NaHS,a hydrogen sulfide donor, Western blotting wre employed to observe the expression of TRPV1.(6) Methylation specific PCR and bisulfite sequencing (BSP) were employed to observe methylation status of CBS gene promoter. Real-time PCR was used to study the specific methylation modification related genes expression changes, including the DNA methyltransferase Dnmts (Dnmt3a, Dnmt3b), methyl CpG binding domain protein MBDs (MBD2, MBD4) and DNA repair enzyme thymine DNA glycosylase (TDG).(7) Western blotting was used to study the expression level of CBS and TRPV1in rat intestinal tissue and human mucosa. Meanwhile, immunohistochemistry staining was employed to detect the distribution of CBS and TRPV1molecules in intestinal mucosa of normal and clinical IBS patients.Results:(1) NSI at postnatal day (PND)10with CFA injection induced visceral hypersensitivity in adult rats from6weeks to9weeks, recovered to normal level at12weeks. But neonatal somatic inflammation by carrageenan or formalin did not induce visceral hypersensitivity in adult rats. Meanwhile, neonatal somatic inflammation at PND21and PND42had no effect on visceral sensitivity at adult.(2) NSI at PND10by CFA changed the resting membrane potentials, enhanced rheobase and markedly decreased the number of action potentials (APs) evoked by2X and3X rheobase current stimulation when compared with the NS-treated group, thus indicating the enhancement of neuron excitability of colon specific neurons of dorsal root ganglion (DRG T13-L2). However, AOAA treatment reversed these effects.(3) NSI up-regulated CBS expression level from treatment of CFA by8hour,3weeks and6weeks in colon DRGs.(4) NSI up-regulated TRPV1expression level from treatment of CFA by6weeks in colon DRGs. However, AOAA treatment after7-day10mg/kg injection intraperitoneally reversed both visceral hypersensitivity in6weeks adult rats and the up-regulated TRPV1expression level in the DRGs of rats.(5) CBS and TRPV1were co-labelled in colon specific DRG neurons of rats.(6) Incubation primary culturesd DRG neurons with NaHS, a donor of H2S, increased TRPV1expression of DRG neurons.(7) NSI demethylated CpG sites of cbs gene promoter region of3weeks and6weeks old rats using methylation specific PCR (MSP) and BSP sequencing. Meanwhile neonatal somatic inflammation led to a decrease in mRNA level of Dnmt3α and Dnmt3b gene, but not MBD2, MBD4and TDG genes in DRGs of6weeks rats.(8) NSI induced both CBS and TRPV1expression level up-regulation in colon mucosa and colon muscle of rats.(9) Both CBS and TRPV1expression level were up-regulated in colon mucosa of IBS patients.Conclusion:(1) Neonatal somatic inflammation stimulation induced visceral hypersensitivity in adult rats, which showed characteristics of stimulus intensity-dependent, stimulus time-dependent and stimulus site-independent.(2) Neonatal somatic inflammation-induced visceral hypersensitivity is, at least partially, mediated by activation of CBS-H2S signaling pathway in DRG neurons.(3) Methylation status of CBS gene caused up-regulation of CBS gene expression, leading to increased syhthesis of hydrogen sulfide, thus induced visceral hypersensitivity.(4) Neonatal somatic inflammation stimulation can induce increased expression of CBS and TRPV1in adult rat colon tissue and colon mucosa of IBS patients.(5) These studies might shed light on the pathogenesis of IBS and may have important implications for treatment of functional gastrointestinal disorders such as IBS.