Cytochrome P45026A1 Contributes to the Maintenance of Neuropathic Pain

The cytochrome P450 proteins(CYP450s)have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases.CYP26A1,a member of the CYP450 family,carries out the oxidative metabolism of retinoic acid(RA),the active metabolite of vitamin A.Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation(SNL).CYP26A1 was mainly expressed in spinal neurons and astrocytes.HPLC analysis displayed that the content of all-trans-RA(at-RA),the substrate of CYP26A1,was reduced in the spinal cord on day 7 after SNL.Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain.Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine(IL-10)production.The RA receptors RARα,RXRβ,and RXRγwere expressed in spinal neurons and glial cells.The promoter of Il-10 has several binding sites for RA receptors,and at-RA directly increased Il-10 mRNA expression in vitro.Finally,intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia.Collectively,the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation.CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

Increased CXCL13 and CXCR5 in Anterior Cingulate Cortex Contributes to Neuropathic Pain-Related Conditioned Place Aversion

Pain consists of sensory-discriminative and emotional-affective components.The anterior cingulate cortex(ACC)is a critical brain area in mediating the affective pain.However,the molecular mechanisms involved remain largely unknown.Our recent study indicated that C-X-C motif chemokine 13(CXCL13)and its sole receptor CXCR5 are involved in sensory sensitization in the spinal cord after spinal nerve ligation(SNL).Whether CXCL13/CXCR5 signaling in the ACC contributes to the pathogenesis of pain-related aversion remains unknown.Here,we showed that SNL increased the CXCL13 level and CXCR5 expression in the ACC after SNL.Knockdown of CXCR5 by microinjection of Cxcr5 shRNA into the ACC did not affect SNL-induced mechanical allodynia but effectively alleviated neuropathic painrelated place avoidance behavior.Furthermore,electrophysiological recording from layer Ⅱ-Ⅲ neurons in the ACC showed that SNL increased the frequency and amplitude of spontaneous excitatory postsynaptic currents(sEPSCs),decreased the EPSC paired-pulse ratio,and increased the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio,indicating enhanced glutamatergic synaptic transmission.Finally,superfusion of CXCL13 onto ACC slices increased the frequency and amplitude of spontaneous EPSCs.Pre-injection of Cxcr5 shRNA into the ACC reduced the increase in glutamatergic synaptic transmis sion induced by SNL.Collectively,these results suggest that CXCL13/CXCR5 signaling in the ACC is involved in neuropathic pain-related aversion via synaptic potentiation.

Sphingosine 1-phosphate acts as an activator for the porcine Gpr3 of constitutively active G protein-coupled receptors

We cloned the complete coding sequences of porcine Gpr3,Gpr6,and Gpr12 genes.Further,on the basis of their high levels of sequence similarity,these genes are identified as a subfamily of G protein-coupled receptors.These putative protein sequences also showed high sequence identity with other mammalian orthologs,including several highly conserved motifs.A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction(RT-PCR)and real-time PCR,specially in the brain,pituitary,fat,liver and oocyte,where its strong expression was observed.The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open-reading frame.Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase(AC)similar in amplitude to that produced by fully stimulated Gs-coupled receptors.Moreover,sphingosine 1-phosphate(S1P)could increase AC activation via the constitutively active Gpr3 receptor.When a Gpr3-green fluorescent protein(GFP)construct was expressed in HEK293 cells,GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes.After S1P treatment,agonist-mediated internalization could be visualized by confocal microscopy.In short,our findings suggest the porcine Gpr3,Gpr6,and Gpr12 genes as a subfamily of G protein-coupled receptors,and porcine Gpr3 was a constitutively active G protein-coupled receptor.Constitutive activation of AC and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1P,suggesting that S1P might act as an activator for porcine Gpr3 receptor.