The Effects And Mechanism Of Par4 For Visceral Hypersensitivity And Pain Mediated By Mast Cell

Objective Protease-activated receptor 4?PAR₄?,a member of G-protein-coupled receptors,is Involved in many pathophysiological processes.Accumulating evidence has demonstrated that PAR₄ as a modulator of visceral nociception has a role as inhibitor of visceral pain and hypersensitivity.Because PAR₄ operates via G-protein-coupled receptors,it seems feasible to look for its downstream effectors among the pain-transducing signal in primary afferent.Recent studies suggested that mast cells exerted a key role in the regulation of irritable bowel syndrome?IBS?visceral sensitivity.It is also found that the down-regulation of PAR₄ in mast cells followed by the increase in the activity of mast cells in IBS patients.Therefore,mast cells appear to be the potential targets for expressing the analgesic action of PAR₄.PAR₄ may directly act on mast cells mediating visceral pain signal transduction pathways.However,Whether PAR₄ could directly influence the mast cell activation in gastrointestinal tract to inhibit the visceral pain and hypersensitivity remains to be clarified.The present subject is designed to investigate whether the effect of PAR₄ on visceral hypersensitivity is mediated by mast cells by using morphological,electrophysiological and molecular metheds.Using IBS rats as a visceral hypersensitive model,we investigate the expression of PAR₄ in mast cells in gastrointestinal tract;the effect of PAR₄ on mast cells activation,the effect of PAR₄ on visceral pain and hypersensitivity mediated by mast cell,to explore the effects of PAR₄ activation on gastrointestinal pain signal transduction induced by visceral hypersensitivity.The research results will provide a new target for the treatment of visceral hypersensitivity in IBS patients.Materials and methods 1.Animal model of visceral hyperalgesia.Animal model of visceral hyperalgesia were established by colorectal distension?CRD?methods.Rat IBS models were screened by AWR score and semi quantitative grading and electromyography?EMG?recordings were performed.The magnitude of EMG activity was measured by a BL420-F multi-channel physiological signal acquisition and processing system?the instrument,made in Chendu,China?.2.Intracolonic administration.IBS rat models received intracolonic administration of 100 ?g of PAR-4-activating peptide?AYPGKF-NH2,PAR₄-AP?or control peptide?YAPGKF-NH2?diluted in in 0.15 ml 0.9% Na Cl.AWR score,semi quantitative grading and electromyography?EMG?recordings were performed at 6 h following the end of intracolonic administration.3.Histology and Immunohistochemistry.Expression of PAR₄,NOS,and P2X7 in mast cells of IBS rat intestinal tissues were examined by using immunohistochemistry conbined with toluidine blue dyeing or confocal laser scanning microscopy technique.4.Bone marrow derived mast cells?BMMC?preparation.Rat bone marrow derived mast cells were prepared both by recombinant rat IL-3?rr IL-3?and by recombinant mouse stem cell factor?rm SCF?,Expression of PAR₄,NOS,and P2X7 in cultured mast cells were examined by immunohistochemistry and flow cytometry after trearment of BMC with PAR₄-AP.5.Real-time polymerase chain reaction?RT-PCR?.Effects of PAR₄ on IL-1? and NOS m RNA levels in mast cells were assessed by using real-time PCR after PAR₄-AP treated with rat IBS models or cultured mast cells.6.Western blot analysis.Effects of PAR₄ on IL-1? and NOS protein levels in mast cells were measured by using western blot analysis after PAR₄-AP treated with rat IBS models or cultured mast cells.Results 1.PAR₄ agonist inhibit nociceptive response to colorectal distension?1?Visceromotor response?VMR?to colorectal distension.Visceromotor response?VMR?to colorectal distension was measured at 1h after the intracolonic administration of the PAR₄ agonist?AYPGKF-NH2?or the control peptide?YAPGKF-NH2,inactive on PAR₄?.1h after one single administration of the PAR₄ agonist AYPGKF-NH2,a significant decreased intensity of VMR was observed in response to the 40 and 60 mm Hg pressures of colonic distension,compared with basal?time 0?measurements.?2?Electromyogram?EMG?recording of abdominal muscles.EMG signals to colorectal distension were recorded at 1h after the intracolonic administration of the PAR₄ agonist?AYPGKF-NH2?.Basal area under the curve?AUC?of contraction of the average peak EMG amplitude was calculated as the area under the rectified EMG signal trace for the 2 minute period immediately preceding each 2 minute CRD.The AUC values of the EMG during the first and second distensions were computed and basal AUC subtracted to obtain the net AUC in response to CRD.1h after the intracolonic administration of the PAR₄ agonist,the AUC of contraction of the average peak EMG in response to the 40 and 60 mm Hg pressures of colonic distension,was significantly decreased,compared with control.2.PAR₄,NOS,and P2X7 immunoreactivity in the colon of IBS rats.PAR₄,NOS,P2X7 and mast cells were assessed by immunohistochemical staining for PAR₄,NOS,P2X7 and Tryptase in colon and caecum from IBS rat models and control.Numbers of PAR₄ and NOS positive cells were substantially higher after the intracolonic administration of the PAR₄ agonist in the IBS rats than those of controls.However,Numbers of Tryptase immunoreactive mast cells and P2X7 positive cells were substantially decreased in the IBS rat after the intracolonic administration of the PAR₄ agonist in the IBS rats,compared with control.3.Mast cells expressing PAR₄,NOS and P2X7 immunoreactivy in IBS rats.Double labeling and flow cytometry showed that mast cells express PAR₄,NOS and P2X7 immunoreactivity in IBS rats or in cultured bone marrow-derived mast cells.4.The effects of PAR₄ activation on mast cells.?1?The PAR₄ agonist up-regulation of PAR₄ protein.Effects of the PAR₄ agonist on PAR₄ protein in cultured mast cells were assessed by measurement of PAR₄ expression using flow cytometry and western blotting.PAR₄ level was significantly increased in cultured mast cells after PAR₄ agonist treatment for 60 min.?2?The PAR₄ agonist up-regulation of NOS expression.Flow cytometry and western blotting showed that the expression of NOS in cultured mast cells was significantly increased at 1 h after AYPGKF-NH2 treatment compared with controls.5.The activation of PAR₄ up-regulation of IL-1? m RNA in IBS rats and cultured mast cells.Effects of the PAR₄ agonist on IL-1? expression in IBS rats and cultured mast cells were assessed by measurement of IL-1? m RNA expression using real-time PCR.IL-1? m RNA level was significantly increased in cultured mast cells or IBS rats after PAR₄ agonist treatment for 60 min.Conclusion 1.Activation of PAR₄ can modulate nociceptive responses in IBS rat models by reduced visceral hypersensitivity.2.Activation of PAR₄ probably decreased the number of mast cells in IBS rat models.3.PAR₄ may directly act on MCs mediating visceral pain signal transduction pathways through its regulation of IL-1? processing and release,expression of NOS and P2X7 recptors.