Epigenetic Regulation Of ASIC1 In Gastric Hypersensitivity Of Adult Offspring With Prenatal Maternal Stress

Functional dyspepsia(FD)is a common functional disorder of the upper gastrointestinal tract associated with belching,early satiety,nausea,vomiting and epigastric pain.It afflicts 40%?70% of the population according to various estimates.These patients feel pain in the absence of any structural,morphologic,or known organic abnormality.However,clinical studies have identified gastric hypersensitivity(GHS)to gastric distension as a major contributor to pain of epigastric origin.The etiology and the cellular mechanisms of GHS remain unknown,primarily owing to the lack of availability of visceral tissue from FD patients and normal human subjects.Animal models that mimic specific pathologies of FD,such as GHS,are essential to identify therapeutic targets to relieve the morbidity of visceral pain.Early life events,such as severe psychological stress,inflammation and trauma,are established risk factors for the development of FD,in adulthood.Stressors during gestational period could have effect on the offspring's tissue structure and function,which may predispose to gastrointestinal diseases.Epigenetic programming is highly sensitive to changes in cellular microenvironment during in utero development.At present,the pathogenesis of FD has not been fully elucidationand treatment is much insufficiency.The quality of life FD patients are affected severely.We will explore the epigenetic mechanism of FD,and provide a theoretical basis for clinical treatment of chronic visceral pain.1.Specific Aims:(1)To establish an animal model of PMS-induced gastric hypersensitivity and to explore the electrophysiological properties of dorsal root ganglion(DRG)neurons innervating the stomach.(2)To investigate the role and expression of acid-sensing ion channels(ASICs)in PMS-induced gastric hypersensitivity.(3)To investigate the methylation of Cp G island in the promoter of asic1 gene.(4)To investigate the mechanism of demethylation of upregulated ASIC1.(5)To investigate the binding of nuclear factor-?B(NF-?B)with the Cp G islands of asic1 gene promoter.(6)To investigate the role of NF-?B in PMS-induced gastric hypersensitivity.2.Methods:(1)Pregnant Sprague Dawley(SD)rats were divided into PMS and CON group.PMS rats were exposed to heterotypic intermittent stress(HIS)from gestational 7 to delivery.Behavioral response to graded gastric balloon distension(GD)was detect by Electromyographic(EMG)recordings to evaluate gastric hypersensitivity.(2)Gastric-specific DRG neurons were labeled with Dil and acutely dissociated for measuring excitability and ASICs current with patch-clamp techniques.(3)RT-PCR analysis and Western Blot were performed to detect the expression of ASICs,NF-?B,DNMT1,DNMT3 a,DNMT3b,Gadd45?,MBD1,MBD2,MBD4 and TDG in gatric DRGs of rats.(4)Immunofluorescence double staining was performed to detect the cellular distribution of ASICs and NF-?B in gastric DRGs of rats.(5)Methylation specific PCR(MSP)was performed to detect the status of DNA methylation on the Cp G islands of asic1 gene promoter.(6)Chromatin immunoprecipitation assay(Ch IP)were performed to detect the binding of NF-?B with asic1 gene promoter in gastric DRGs of rats.(7)Intrathecal injection of Amiloride and NF-?Bp65 sh RNA was applied to investigate the roles of ASICs and NF-?B in PMS-induced gastric hypersensitivity.3.Results:(1)PMS induced significant gastric hypersensitivity of adult offspring rats.The excitability and ASICs current density of gastric-specific DRG neurons increased.(2)Intrathecal injection of Amiloride,an inhibitor of ASICs,could significantly attenuate PMS-induced gastric hypersensitivity.Amiloride could also reverse the hyperexcitability and ASICs current density of gastric-specific DRG neurons.(3)ASIC1,ASIC2 and ASIC3 expressed in gastric-specific DRG neurons.PMS significantly increased ASIC1 protein and m RNA expression of adult offspring,while m RNA of ASIC2 and ASIC3 was not changed.ASIC1 co-localized with Neu N-positive neurons and mostly in the CGRP-positive(peptide)and NF200-positive neurons.ASIC1 did not co-localized with GS and IB4-positive(non-peptide)neurons.(4)The methylation of Cp G island of asic1 gene promoter was significantly demethylated in PMS adult offspring.DNMT1 and DNMT3 b m RNA expression of gastric DRGs in PMS adult offsring were greatly decreased,while the m RNA expression of DNMT3 a,Gadd45?,MBD1,MBD2,MBD4 and TDG was not significantly altered.(5)NF-?B expression in gastric DRGs of PMS adult offspring was greatly enhanced.NF-?B and ASIC1 were co-localized in the gastric DRG neurons(6)There were 2 binding sites of NF-?B in the Cp G island of asic1 gene promoter by online software prediction.The chromatin immunoprecipitation assay confirmed that there was a significant increase in binding activity of p65 with the two binding sites of asic1 gene promoter in PMS adult offspring compared to age-matched CON rats.(7)Intrathecal injection of NF-?B p65 sh RNA lentivirus significantly inhibited the expression of NF-?B and ASIC1,and could reduce the neuronal excitability.NF-?Bp65 sh RNA lentivirus could also attenuate PMS-induced gastric hypersensitivity.4.Conclusions(1)PMS can successful induce gastric hypersensitivity in adult rat offspring,which is associated with the enhanced excitability of gastric-specific DRG neuron and increased ASICs currents.(2)The enhanced expression of ASIC1,which is attributed to asic1 gene promoter DNA demethylation,would contribute to PMS-induced gastric hypersensitivity.(3)Asic1 gene promoter DNA demethylation is likely caused by decreased expression of DNMT1 and DNMT3 b m RNA levels.It suggested that epigenetic mechanisms were involved in PMS-induced gastric hypersensitivity.(4)The increased expression of NF-?B and enhanced interaction with asic1 gene promoter contributed to PMS-induced gastric hypersensitivity.