The Mechanism Underlying The Inhibitory Effect Of Oxytocin On The Mast Cell Degranulation And DRG Neuron Excitability

Pain affects all aspects of people’s lives, although modern medicine has made great progress, chronic pain is still a major challenge. In the digestive system, chronic visceral pain is mainly seen in functional bowel disorders (for example, chronic idiopathic dyspepsia, functional abdominal pain, irritable bowel syndrome), most commonly in irritable bowel syndrome (IBS). At present, it is found that both central and peripheral mechanisms play important roles in the process of visceral pain. In the central nervous system, the mechanism of the brain-gut axis becomes more and more prominent. In the peripheral, the immune mechanism and the nerve-immune association play an important role in the occurrence of visceral pain.Oxytocin (OT), a nine amino acid neuropeptide, is mainly synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. In clinical practice, OT is mainly used in labor induction, postpartum hemorrhage, parturition and lactation. It has been demonstrated that OT and oxytocin receptor (OTR) are expressed in bowel. OT/OTR signaling is physiologically significant in the regulation of gastrointestinal motility and sensation, modulation of intestinal inflammation, regulation of the permeability of the mucosa to macromolecules, and maintenance of the mucosa. Some studies have also demonstrated that OT applied in the peripheral (intravenous injection, intraperitoneal injection, subcutaneous injection or colon perfusion) plays a crucial role in visceral hypersensitivity/pain inhibition. Further study of the effect and mechanism of OT on visceral pain can provide new insights for the prevention and treatment of visceral pain. This study aims to investigate the peripheral analgesic mechanisms of OT in immune and nervous aspects.Part IThe mechanism underlying the inhibitory effect of oxytocin on the mast cell degranulation in colonic hypersensitivity ratsObjectivesIt has been demonstrated that OT and OTR are expressed in bowel and that OT plays an important role in visceral hypersensitivity/pain inhibition. However, the mechanisms underlying the inhibitory effect of OT on visceral hypersensitivity/pain have not yet been fully elucidated. The severity and frequency of abdominal pain are correlated with the number of mast cells in close proximity to colonic nerves in IBS, and when activated, mast cells degranulate and release mediators that enhance the excitability of enteric and primary afferent neurons, leading to visceral hypersensitivity. This study was conducted to investigate the effects of OT on mast cells degranulation and the underlying mechanisms, which could contribute to find a peripheral immune mechanism of the inhibition of OT on visceral hypersensitivity.MethodsThe expression of OTR on mast cells was assessed with immunofluorescence assays and Western blot. The colon perfusion of 2,4,6-trinitrobenzene sulfonic acid (TNBS) was to used to induce visceral hypersensitivity. OT was instilled into the lumen of the colon after TNBS in OT+TNBS group. Tissue sections were stained by toluidine blue for mast cell counting and degranulation. The levels of histamine were measured by a commercial enzyme linked immunosorbent assay (ELISA) kit. The electrical properties of mast cells were tested by whole-cell patch clamp recording. The intracellular Ca2+ was determined by fluorescent microscopy using an indicator dye.Results1. OTR expression in mast cellsImmunostaining of human and rat colon tissues revealed that OT receptors were expressed in human and rat colonic mast cells. The ratio of tryptase-positive mast cells expressed OTR from human ulcerative colon sections or TNBS induced colonic hypersensitivity rat colon sections significantly increased compared with that of control group. We investigated if OTR was expressed in human and mouse mast cell lines, human mast cell line-1 (HMC-1) and mouse mastocytoma cell line (P815) cells. OTR immunoreactivity was observed in both HMC-1 and P815 cells, and all of the HMC-1 and P815 cells expressed OTR. Additionally, we found that the OTR protein was identified in HMC-1 and P815 cells.2. Effects of OT on mast cell degranulationOT remarkablely decreased TNBS-induced colonic mast cell degranulation in rats. OT significantly reduced the rate of TNBS-evoked mast cell degranulation from 81.9%(59/72) to 53.5%(38/71).3. Effects of OT on mast cell histamine releaseIn P815 cells, compound 48/80 (C48/80) caused a marked increment in histamine release compared with that of the control group, which was attenuated by 10-6 M OT pretreatment.4. Effects of OT on C48/80-induced inward currents in mast cellsIn order to test the inward currents on the membrane caused by C48/80 following OT administration in HMC-1 and P815 cells, whole cell patch-clamp recordings were performed. Under voltage-clamp configuration, the HMC-1 cell or P815 cell was initially held at a -60 mV command potential. After addition of C48/80, the inward currents were markedly increased compared with that of the baseline group, which was attenuated by OT pre-treated before C48/80 in HMC-1 cell or P815 cell.5. Effect of OTR antagonist or the NOS inhibitor on OT induced the histamine release and inward currentsPretreatment of the OTR antagonist atosiban or the nonselective nitric oxide synthase (NOS) inhibitor NG-Methyl-L-arginine acetate salt (L-NMMA) significantly attenuated the inhibitory effect of OT on C48/80-evoked histamine release in P815 cells and inward currents in HMC-1 and P815 cells.6. Effects of OT on intracellular Ca2+ response in mast cellsEffects of OT on the Fura-2 fluorescence ratio (F340/F380), due to changes in intracellular Ca2+ concentration, were tested by calcium imaging technique. OT produced a significant increase in intracellular Ca2+ in HMC-1 and P815 cells.Conclusion1. OTR is expressed in mast cells, and the ratio of tryptase-positive mast cells expressed OTR from human ulcerative colon sections or TNBS induced colonic hypersensitivity rat colon sections significantly increased compared with that of control group.2. OT inhibits mast cell degranulation, which may contribute to the inhibition of colonic hypersensitivity.3. OT might exert the inhibition of mast cell degranulation via Ca2+ -NOS pathway.Part ⅡThe mechanism underlying the inhibitory effect of oxytocin on dorsal root ganglia neuron excitability in ratsObjectivesOT plays an important role in pain modulation and antinociception in the central nervous system. However, little is known about its peripheral effects. This study was conducted to investigate the effect of OT on the electrical properties of neurons in the dorsal root ganglia (DRG) and the underlying mechanisms.MethodsDRG neurons from adult rats were acutely dissociated and cultured. Intracellular Ca2+ was determined by fluorescent microscopy using an indicator dye. The electrical properties of DRG neurons were tested by patch-clamp recording. OTR and neuronal nitric oxide synthase (nNOS) on DRG neurons were assessed with immunofluorescence assays. Immunofluorescence assays and nitrate reductase method were used to detect the content of nitric oxide.Results1. Effects of OT on the excitability of DRG neuronsOT decreased the excitability, increased the outward current, and evoked the membrane hyperpolarization in cultured small-sized (19-27 μM diameter) DRG neurons. After OT application, the rheobase of small-sized DRG neurons was significantly increased. In addition, OT significantly decreased the number of action potentials (AP) evoked by 2×and 3×rheobase current stimulation.2. Effect of the selective nNOS inhibitor on OT induced the membranehyperpolarizationPre-treatment of the selective nNOS inhibitor NPLA significantly attenuated the hyperpolarization effect evoked by OT.3. Effect of OT on NO production in DRG neuronsOT induced the production of NO was examined using the NO-fluorescent indicator DAF in acutely dissociated DRG neurons and NO assay kit in rat DRGs. Pre-treatment of the OTR antagonist atosiban or the selective nNOS inhibitor NPLA significantly reversed the NO production caused by OT.4. Colocation of OTR and nNOS in Isolectin B4 (IB4)-labeled DRG neuronsConfocal analysis of trinal immunofluorescence experiments revealed that＞85% (±1%) of IB4-labeled nonpeptidergic nociceptor DRG neurons expressed OT receptors and nNOS.5. Effects of the blocker of ATP-sensitive K+ (KATP) channel on electrical properties of DRG neuronsOT-evoked membrane hyperpolarization and increase of outward current were distinctly attenuated by glibenclamide, a blocker of KATP channel.6. Effects of OT on intracellular Ca2+ response in DRG neuronsOT caused a dose-dependent increase in intracellular Ca2+ in small-diameter DRG neurons.Conclusion1. Colocation of OTR and nNOS in IB4-labeled DRG neurons related to algesia.2. OT evoked the membrane hyperpolarization and decreased the excitability in cultured small-sized DRG neurons, then exerting the peripheral antinociceptive effects.3. OT might evoke the membrane hyperpolarization by activation of the Ca2+/nNOS/NO/KATP pathway in DRG neurons.