BackgroundMu opioid receptor (MOR) plays a crucial role in mediating analgesic effects of opioids and is closely associated with the pathologies of neuropathic pain. Previous studies have reported that peripheral nerve injury downregulates MOR expression, but the epigenetic mechanisms remain unknown.ObjectiveTherefore, we investigated DNA methyltransferase3a (DNMT3a) expression or methylation changes within MOR promoter in the spinal cord in a neuropathic pain induced by a chronic constriction injury (CCI) mouse model and further determined whether these injury-associated changes are reversible by pharmacological interventions.MethodsA CCI mouse model was established and tissue specimens of lumbar spinal cords were collected. The nociception threshold was evaluated by a Model Heated 400 Base. DNMT3a and MOR mRNA and protein level were detected by real-time-polymerase chain reaction and Western blot, respectively. Methylation of DNMT3a gene was measured by methylation-specific PCR.ResultsOur data showed that chronic nerve injury led to a significant upregulation of DNMT3a expression that was associated with increased methylation of MOR gene promoter and decreased MOR protein expression in the spinal cord. Inhibition of DNMT3a catalytic activity with DNMT inhibitor RG108 significantly blocked the increase in methylation of the MOR promoter, and then upregulated MOR expression and attenuated thermal hyperalgesia in neuropathic pain mice.ConclusionThis study demonstrates that an increase of DNMT3a expression and MOR methylation epigenetically play an important role in neuropathic pain. Targeting DNMT3a to the promoter of MOR gene by DNMT inhibitor may be a promising approach to the development of new neuropathic pain therapy.

BackgroundCumulating evidence has revealed the effectiveness of acupuncture therapy in relieving pain via immunoregulation. However, its underlying mechanism remains unknown. The present study was designed to determine the changes of immunogenic responses at different time-points of electroacupuncture (EA) interventions in neuropathic pain rats.MethodsThe neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EA was applied at Zusanli (ST36) and Yanglingquan (GB34) for the EA groups. The thermal pain threshold was detected with an algesia-detector. The subgroups of plasma and splenic lymphocytes were determined via fluorescence-activated cell sorting. Specific inflammatory cytokines were assayed using an ELISA-based bead multiplex assay. The activities of splenic natural killer (NK) cells and cytotoxic T lymphocytes were detected by methyl thiazolyl tetrazolium colorimetric method. For confirming the involvement of NK cell in EA-analgesia, anti-asialo-ganglio-N-tetraosylceramide (anti-asialo-GM1) antibody was given to CCI rats before EA.ResultsFollowing CCI, the thermal pain threshold of the affected hind footpad was significantly decreased, and increased from the 3rd day to the 12th day after EA interventions, presenting a time-dependent tendency from the 5th day on. From day 3 to 5 of EA interventions, the percentages and activity of splenic NK cells, concentrations of splenic interleukin-2 (IL-2) and beta-endorphin (β-EP) were significantly increased. Meanwhile, the concentrations of plasma IL-2, IL-1β and gamma-interferon (IFN-γ) were significantly decreased and returned to the normal level on day 12 following EA. Plasma transforming growth factor-β (TGF-β) levels were considerably upregulated on day 5 and 12 following EA. The CD4+/CD8+ T cell ratio was markedly downregulated compared with the control and CCI groups on day 5 and returned to the normal level on day 12 following EA. After depleting NK cells by anti-asialo-GM1 antibody, the increased thermal pain threshold following EA intervention was obviously reduced.ConclusionsRepeated EA interventions have a time-dependent cumulative analgesic effect in neuropathic pain rats, which is closely associated with its regulatory effects on NK cells, splenic IL-2, β-EP, and plasma IL-2, IL-1β, IFN-γ and TGF-β levels.

BackgroundEvidence is building steadily on the effectiveness of acupuncture therapy in pain relief and repeated acupuncture-induced pain relief is accompanied by improvement of hippocampal neural synaptic plasticity. To further test the cellular and molecular changes underlying analgesic effect of acupuncture, the global change of acupuncture associated protein profiles in the hippocampus under neuropathic pain condition was profiled.MethodsThe chronic constrictive injury (CCI) model was established by ligature of the unilateral sciatic nerve in adult Wistar rats. Rats were randomized into normal control (NC) group, CCI group, and CCI with electroacupuncture (EA) stimulation group. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) in the EA group. Differentially expressed proteins in the hippocampus in the three groups were identified by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The functional clustering of the identified proteins was analyzed by Mascot software.ResultsAfter CCI, the thermal pain threshold of the affected hind footpad was decreased and was reversed gradually by 12 sessions of acupuncture treatment. Following EA, there were 19 hippocampal proteins identified with significant changes in expression (>2-fold), which are involved in metabolic, physiological, and cellular processes. The top three canonical pathways identified were “cysteine metabolism”, “valine, leucine, and isoleucine degradation” and “mitogen-activated protein kinase (MAPK) signaling”.ConclusionsThese data suggest that the analgesic effect of EA is mediated by regulation of hippocampal proteins related to amino acid metabolism and activation of the MAPK signaling pathway.

BackgroundCumulating evidence has shown a close correlation between electroacupuncture stimulation (EAS) frequency-specific analgesic effect and central opioid peptides. However, the actions of hippocampal acetylcholinergic receptors have not been determined. This study aims to observe the effect of different frequencies of EAS on the expression of hippocampal muscarinic and nicotinic acetylcholinergic receptors (mAChRs, nAChRs) in neuropathic pain rats for revealing their relationship.MethodsForty male Wistar rats were randomly and equally divided into sham, CCI model, 2, 2/15 and 100 HzEA groups. The neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EAS was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 30 min, once daily for 14 days except weekends. The mechanical pain thresholds (withdrawal latencies, PWLs) of bilateral hindpaws were measured. The expression levels of hippocampal M1 and M2 mAChR, and α4 and β2 nAChR genes and proteins were detected by quantitative RT-PCR and Western blot, separately. The involvement of mAChR and nAChR in the analgesic effect of EAS was confirmed by intra-hippocampal microinjection of M1mAChR antagonist (Pirenzepine) and α4β2 nAChR antagonist (dihydro-beta-erythroidine) respectively.ResultsFollowing EAS, the CCI-induced increase of difference values of bilateral PWLs on day 6 and 14 was significantly reduced (P < 0.05), with 2/15 Hz being greater than 100 Hz EAS on day 14 (P < 0.05). After 2 weeks’ EAS, the decreased expression levels of M1 mAChR mRNA of both 2 and 2/15 Hz groups and M1 mAChR protein of the three EAS groups, α4 AChR mRNA of the 2/15 Hz group and β2 nAChR protein of the three EAS groups were considerably increased (P < 0.05), suggesting an involvement of M1 mAChR and β2 nAChR proteins in EAS-induced pain relief. No significant changes were found in the expression of M2 mAChR mRNA and protein, α4 nAChR protein and β2 nAChR mRNA after CCI and EAS (P > 0.05). The analgesic effect of EAS was abolished by intra-hippocampal microinjection of M1mAChR and α4β2 nAChR antagonists respectively.ConclusionsEAS of ST36-GB34 produces a cumulative analgesic effect in neuropathic pain rats, which is frequency-dependent and probably mediated by hippocampal M1 mAChR and β2 nAChR proteins.