Spinal Mechanism Of Purinergic Receptor P2X7Rs In Neonatal Colonic Inflammation-induced Chronic Visceral Hypersensitivity In Adult Rats

Background:Irritable bowel syndrome(IBS)is one of functional gastrointestinal disorders,characterized by abdominal discomfort or pain.At present,the pathogenesis of IBS has not been clear.The clinical treatment regarding to IBS has not been comprehensive.Therefore,we need to further explore the molecular mechanisms of IBS.In this study,we aimed to probe into the effects of tumor necrosis factor receptor related factor 6 and purinergic receptors P2X7 Rs in the process of visceral pain induced by neonatal colonic inflammation(NCI).Experimental methods:1.Following the injection of 0.5% acetic acid solution solved in normal saline,visceral hypersensitivity in adult rats was induced.2.Colorectal distension response was measured by observing pain threshold.3.The protein and mRNA expressions of TRAF6 and P2X7 Rs in the spinal cord dorsol horn from control and NCI rats at different ages were examined by western blotting and Q-PCR.4.To verify whether P2X7 Rs participated in visceral pain,pain threshold was measured after intrathecally injecting P2X7 Rs inhibitor A438079.5.To determine whether TRAF6 was involved in visceral hypersensitivity,visceral pain was tested following intrathecal injection of TRAF6 si RNA.6.Cellular distributions in the spinal cord dorsol horn of P2X7 Rs and TRAF6 were examined using immunofluorescence localization.Experimental results:1.At the age of 6wk after the treatment of NCI,the distension threshold was significantly decreased in NCI rats compared with control rats and maintained to 8wk.However,at the age of 4wk and 10 wk,the thresholds were not obviously different between control and NCI rats.2.NCI enhanced the expression of P2X7 Rs in colon-related spinal cord dorsol horn at the age of 6wk and 8wk.The application of P2X7 Rs inhibitor A438079 prominently decreased the CRD threshold evoked by NCI time-and dose-dependently.3.The expression of TRAF6 was obviously upregulated in spinal cord dorsol horn of NCI rats at the age of 6wk and 8wk.Intrathecal injection of TRAF6 siRNA reduced the expression of P2X7 Rs and remarkably alleviated hypersensitivity induced by NCI in a time-dependent manner.4.Immunofluorescence showed that P2X7 Rs were expressed in astrocytes and neuron,but TRAF6 were only expressed in astrocytes.Moreover,P2X7 Rs and TRAF6 were co-located in astrocytes.Conclusions:Our results suggested that P2X7 Rs and TRAF6 were involved in the development of visceral pain hypersensitivity induced by neonatal colonic inflammation.In addition,P2X7 Rs were regulated by TRAF6.Hence,to some extent,P2X7 Rs might be a potential target for the treatment of visceral pain in IBS patients.