Effects Of Morphine Preconditioning On NGF-induced TRPV1 Sensitization In Neurocytes And Its Signaling Mechanisms

Objective: To investigate the effects of morphine preconditioning on nerve growth factor(NGF)-induced transient receptor potential vanilloid 1(TRPV1)sensitization in neurocytes and its signaling mechanisms,explaining the potential mechanism of morphine regulating myocardial ischemic nociceptive signaling transmission.Methods: dorsal root ganglia(DRG)cells isolated from 2 days old SD rats or pheochromocytoma cells(PC12)were seeded into 24 or 6-well plates respectively,and randomly divided into 8 groups: control group(group CON),CAP stimulation group(group CAP),NGF sensitization group(group NGF),and morphine preconditioning groups at different concentrations(group MPC0.3,group MPC1.0 and group MPC3.0),TRPV1 antagonist group(group I-RTX),TRPV1 antagonist +NGF group(group I-RTX+NGF).The cells in group CON were cultured in normal culture condition,and the cells in group CAP were stimulated by 100nmol/L capsaicin while in group NGF the cells were treated with 100nmol/L NGF for 1 hour.In addition,in group MPC0.3,MPC1.0 and MPC3.0,the cells were pretreated with 0.3?mol/L,1.0?mol/L or3.0?mol/L morphine for 10 min,followed by wash-out for 30 min,respectively,prior to the addition of NGF.Meanwhile,the cells in group I-RTX and I-RTX+NGF were stimulated by 100nmol/L I-RTX for 20 second,followed by the co-stimulation of100nmol/L capsaicin and I-RTX.Afterwards,the whole cell recording technology was employed to detect the inward current,and the expressions of substance P(SP)and calcitonin gene-related peptide(CGRP)in DRG cells were observed by bi-fluorescence in all groups,while the level of TRPV1,phosphorylated(p)TRPV1,extracellular signal-regulated kinase1/2(ERK1/2),p ERK1/2,protein kinase B(AKT),p AKT of PC12 cells were determined by Western Blot.Results: The whole cell patch clamp recording results demonstrated that the inward current amplitude of group CAP was enhanced with compared with group CON(P<0.05),and this enhancement was inhibited by I-RTX(P<0.05),meanwhile,the inward current amplitude of group NGF was also enhanced while compared with group CAP(P<0.05),both the I-RTX and morphine preconditioning at all different concentrations repressed this enhancement(P<0.05).The results of bi-fluorescence showed the co-existence of SP and CGRP in each groups.Compared with CAP group,the transmission of SP and CGRP from the cell body to synaptic was significantly increased,and morphine preconditioning blocked this increase.Further,the co-existence of SP and CGRP was attenuated in group I-RTX+NGF;The results of western blot indicated that,the relative expressions of TRPV1,p TRPV1,p AKT,p ERK1/2 were up-regulated in group CAP with compared with group CON(P<0.05),and I-RTX suppressed this up-regulation(P<0.05),meanwhile,the relative expressions of TRPV1,p TRPV1,p AKT,p ERK1/2 in group NGF were increased while compared with group CAP(P<0.05),and this increase was alleviated by I-RTX(P<0.05).Moreover,the relative expressions of p TRPV1,p AKT,p ERK1/2 were also reduced in all MPC groups,and only in group MPC0.3 did the relative expression of TRPV1 was reduced(P<0.05).Conclusion: NGF induces the sensitization of TRPV1 channel in neurocytes and the activation of downstream ERK1/2 and AKT signaling pathways,leading to the release of neuronal peptides.In addition,the regulation of morphine preconditioning on myocardial ischemic nociception transmission may be mediated by inhibiting the NGF-induced TRPV1 sensitization.