Proteomic analysis of differential protein expression in neuropathic pain and electroacupuncture treatment models

Acupuncture has long been used for pain relief. Although recent studies have shown that acupuncture can reduce neuropathic pain, the mechanism of this effect is not clear and little information is available regarding proteins that are involved in the development of neuropathic pain and the effects of acupuncture. We have developed an animal model for neuropathic pain using young adult male Sprague-Dawley rats. The model was confirmed by behavioral tests. Electroacupuncture (EA) treatment was applied to Zusanli (ST36) of neuropathic pain model to examine the analgesic effect of EA. The protein expression profile of the hypothalamus in both neuropathic pain and EA treatment models was analyzed using two-dimensional electrophoresis-based proteomics. We detected thirty-six proteins that were differentially expressed in the neuropathic pain model compared with normal rats and that restored to normal expression levels after EA treatment. Twenty-one of these proteins were identified in the MS-FiT database and are involved in a number of biological processes, including inflammation, enzyme metabolism and signal transduction. Potential applications of our results include the identification and characterization of signaling pathways involved in EA treatment and further exploration of the role of selected identified proteins in the animal model.     

Changes in expression of nociceptin/orphanin FQ and its receptor in spinal dorsal horn during electroacupuncture treatment for peripheral inflammatory pain in rats

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain modulation. It was reported that electroacupuncture (EA) had a potent analgesic effect on inflammatory pain by activating various endogenous transmitters such as the opioid peptides. In the present study, we investigated the effect of EA on peripheral inflammatory pain and the expression of N/OFQ and the NOP receptor in the spinal dorsal horn of rats, using a behavioral test, RT-PCR, immunohistochemistry and Western blot analysis techniques. The results showed: (1) EA had an accumulative analgesic effect on chronic inflammatory pain; (2) in the superficial layers of the spinal dorsal horn, the level of mRNA of the precursor protein for N/OFQ (preproN/OFQ, ppN/OFQ) was increased and the N/OFQ immunoreactivity was decreased after peripheral inflammation, and could be significantly increased by EA treatment; (3) both mRNA and protein levels of the NOP receptor in the spinal dorsal horn were significantly increased after chronic inflammatory pain and could be further enhanced by EA treatment. The present data demonstrated that EA could activate the endogenous N/OFQ-NOP receptor system, and this might underlie the effectiveness of EA in the treatment of inflammatory pain.     

Suppressing PKC-dependent membrane P2X3 receptor upregulation in dorsal root ganglia mediated electroacupuncture analgesia in rat painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a common and troublesome diabetes complication. Protein kinase C (PKC)-mediated dorsal root ganglia (DRG) P2X3 receptor upregulation is one important mechanism underlying PDN. Accumulating evidence demonstrated that electroacupuncture (EA) at low frequency could effectively attenuate neuropathic pain. Our previous study showed that 2-Hz EA could relieve pain well in PDN. The study aimed to investigate whether 2-Hz EA relieves pain in PDN through suppressing PKC-mediated DRG P2X3 receptor upregulation. A 7-week feeding of high-fat and high-sugar diet plus a single injection of streptozotocin (STZ) in a dose of 35 mg/kg after a 5-week feeding of the diet successfully induced type 2 PDN in rats as revealed by the elevated body weight, fasting blood glucose, fasting insulin and insulin resistance, and the reduced paw withdrawal threshold (PWT), as well as the destructive ultrastructural change of sciatic nerve. DRG plasma membrane P2X3 receptor level and DRG PKC expression were elevated. Two-hertz EA failed to improve peripheral neuropathy; however, it reduced PWT, DRG plasma membrane P2X3 receptor level, and DRG PKC expression in PDN rats. Intraperitoneal administration of P2X3 receptor agonist αβ-meATP or PKC activator phorbol 12-myristate 13-acetate (PMA) blocked 2-Hz EA analgesia. Furthermore, PMA administration increased DRG plasma membrane P2X3 receptor level in PDN rats subject to 2-Hz EA treatment. These findings together indicated that the analgesic effect of EA in PDN is mediated by suppressing PKC-dependent membrane P2X3 upregulation in DRG. EA at low frequency is a valuable approach for PDN control.     

Analgesic effect of electroacupuncture on chronic neuropathic pain mediated by P2X3 receptors in rat dorsal root ganglion neurons

Adenosine 5'-triphosphate disodium (ATP) gated P2X receptors, especially the subtype P2X(3), play a key role in transmission of pain signals in neuropathic pain, ATP has been documented to play a significant role in the progression of pain signals, suggesting that control of these pathways through electroacupuncture (EA) is potentially an effective treatment for chronic neuropathic pain. EA has been accepted to effectively manage chronic pain by applying the stimulating current to acupoints through acupuncture needles. To determine the significance of EA on neuropathic pain mediated by P2X(3) receptors in the dorsal root ganglion (DRG) neurons, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and the expression of P2X(3) receptors in the DRG neurons was assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). In addition, the currents which were evoked in DRG neurons isolated from rats following chronic constriction injury (CCI) by the P2X(3) receptors agonists i.e. ATP and α,β-methylen-ATP (α,β-meATP) were examined through the experimental use of whole cell patch clamp recording. The present study demonstrates that EA treatment can increase the MWT and TWL values and decrease the expression of P2X(3) receptors in DRG neurons in CCI rats. Simultaneously, EA treatment attenuates the ATP and α,β-meATP evoked currents. EA may be expected to induce an apparent induce analgesic effect by decreasing expression and inhibiting P2X(3) receptors in DRG neurons of CCI rats. There is a similar effect on analgesic effect between rats with contralateral EA and those with ipsilateral EA.     

DDD-028: A potent potential non-opioid,non-cannabinoid analgesic for neuropathic and inflammatory pain

DDD-028 (4), a novel pentacyclic pyridoindolobenzazepine derivative was evaluated in vitro for receptor binding affinity and in vivo for analgesic activity using rodent models of neuropathic and inflammatory pain. DDD-028 does not bind to opioid, cannabinoid, dopamine, or histamine receptors. DDD-028 is very active even at the low oral dose of 1–5 mg/kg in both neuropathic, (spinal nerve ligation and chronic constriction injury) and inflammatory (Complete Freund’s Adjuvant Induced) models of pain. DDD-028 appears to be about 6-fold more potent than pregabalin and indomethacin. Visual observation of all the animals used in these studies indicated that DDD-028 is well tolerated without any sedation. Thus, DDD-028 seems to be a promising candidate for the treatment of neuropathic and inflammatory pain without the possible side effects or abuse potential associated with opioid or cannabinoid activities.     

ARA 290 relieves pathophysiological pain by targeting TRPV1 channel: Integration between immune system and nociception

ARA 290 is an erythropoietin-derived polypeptide that possesses analgesic and tissue protective effect in many diseases such as diabetes and cancer. The analgesic effect of ARA 290 is mediated by its anti-inflammatory and immunomodulatory functions, or more specifically, by targeting the innate repair receptor (IRR) to down-regulate inflammation to alleviate neuropathic pain. However, whether other mechanisms or pathways are involved in ARA 290-mediated analgesic effect remains elusive. In this study, we are particularly interested in whether ARA 290 could directly target peripheral nociceptors by blocking or influencing receptors in pain sensation. Using calcium imaging, cell culture and behavioral tests, we demonstrated that ARA 290 was able to specifically inhibit TRPV1 channel activity, and relieve the mechanical hypersensitivity induced by capsaicin. Our study suggested that ARA 290 could potentially function as a novel antagonist for TRPV1 channel. This finding would not only contribute to the development of new pain treatment using ARA 290, but also help to improve our understanding of the integration between the immune system and the peripheral nervous system.     

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

     

Early Repeated Administration of CXCR4 Antagonist AMD3100 Dose-Dependently Improves Neuropathic Pain in Rats After L5 Spinal Nerve Ligation

AMD3100 is a specific C-X-C chemokine receptor type 4 (CXCR4) antagonist which blocks the interaction between CXCR4 and CXCL12. Multiple lines of evidence suggest that AMD3100 has analgesic effects on many pathological pain states, including peripheral neuropathic pain. However, little is known about the underlying mechanisms. In the current study, we investigated the effect of different doses of AMD3100 on neuropathic pain in rats after L5 spinal nerve ligation. We used naloxone methiodide (NLXM) to further determine whether AMD3100-mediated analgesic effect was opioid-dependent. Behavioral study showed that early repeated administration of AMD3100 (2 and 5 mg/kg, i.p.) dose-dependently alleviates peripheral neuropathic pain. Flow cytometry, immunofluorescence and NLXM experiments showed that AMD3100 alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. Furthermore, we found that pro-inflammatory cytokines were down-regulated by AMD3100 using Enzyme-linked Immunosorbent Assay. Our data indicate that AMD3100 dose-dependently alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. This finding suggests that AMD3100 may be a viable pharmacotherapeutic strategy for the treatment of neuropathic pain.     

Modification of antiallodynic and antinociceptive effects of morphine by peripheral and central action of fluoxetine in a neuropathic mice model

We have previously reported that serotonin concentration was reduced in the brain of mice with neuropathic pain and that it may be related to reduction of morphine analgesic effects. To further prove this pharmacological action, we applied fluoxetine, a selective serotonin reuptake inhibitor, to determine whether it suppressed neuropathic pain and examined how its different administration routes would affect antinociceptive and antiallodynic effects of morphine in diabetic (DM) and sciatic nerve ligation (SL) mice, as models of neuropathic pain. Antiallodynia and antinociceptive effect of drugs were measured by using von Frey filament and tail pinch tests, respectively. Fluoxetine given alone, intracerebroventicularly (i.c.v., 15 microg/mouse) or intraperitoneally (i.p., 5 and 10 mg/kg) did not produce any effect in either model. However, fluoxetine given i.p. enhanced both antiallodynic and antinociceptive effects of morphine. Administration of fluoxetine i.c.v., slightly enhanced only the antiallodynic effect of morphine in SL mice. Ketanserine, a serotonin 2A receptor antagonist (i.p., 1 mg/kg) and naloxone, an opioid receptor antagonist (i.p., 3 mg/kg), blocked the combined antinociceptive effect of fluoxetine and morphine. Our data show that fluoxetine itself lacks antinociceptive properties in the two neuropathy models, but it enhances the analgesic effect of morphine in the periphery and suggests that co-administration of morphine with fluoxetine may have therapeutic potential in treatment of neuropathic pain.     

Inhibition of Spinal Interlukin-33/ST2 Signaling and Downstream ERK and JNK Pathways in Electroacupuncture Analgesia in Formalin Mice

Although acupuncture is widely used to manage pain, it remains highly controversial, largely due to the lack of a clear mechanism for its benefits. Here, we investigated the role of IL-33, a novel interleukin (IL)-1 family member, and its receptor ST2 in the analgesic effects of electroacupuncture (EA) on formalin-induced inflammatory pain. The results showed that 1) EA stimulation of ipsilateral Zusanli (ST 36) and Yanglingquan (GB 34) acupoints for 30 min remarkably suppressed the two phases of formalin-induced spontaneous pain; 2) subcutaneous or intrathecal administration of recombinant IL-33 (rIL-33) significantly inhibited the analgesic effect of EA, whereas the ST2 antibody potentiated EA analgesia in formalin mice; 3) EA treatment decreased the up-regulation of IL-33 and ST2 protein following formalin injection; and 4) the suppression of the formalin-induced expression of spinal phosphorylated ERK and JNK induced by EA treatment was significantly attenuated following subcutaneous rIL-33 delivery, and was further decreased by the ST2 antibody. These data suggest that EA alleviates formalin-induced inflammatory pain, at least partially, by inhibiting of spinal IL-33/ST2 signaling and the downstream ERK and JNK pathways.     

Intrathecal Infusion of Hydrogen-Rich Normal Saline Attenuates Neuropathic Pain via Inhibition of Activation of Spinal Astrocytes and Microglia in Rats

Background

Reactive oxygen and nitrogen species are key molecules that mediate neuropathic pain. Although hydrogen is an established antioxidant, its effect on chronic pain has not been characterized. This study was to investigate the efficacy and mechanisms of hydrogen-rich normal saline induced analgesia.

Methodology/Principal findings

In a rat model of neuropathic pain induced by L5 spinal nerve ligation (L5 SNL), intrathecal injection of hydrogen-rich normal saline relieved L5 SNL-induced mechanical allodynia and thermal hyperalgesia. Importantly, repeated administration of hydrogen-rich normal saline did not lead to tolerance. Preemptive treatment with hydrogen-rich normal saline prevented development of neuropathic pain behavior. Immunofluorochrome analysis revealed that hydrogen-rich normal saline treatment significantly attenuated L5 SNL-induced increase of 8-hydroxyguanosine immunoreactive cells in the ipsilateral spinal dorsal horn. Western blot analysis of SDS/PAGE-fractionated tyrosine-nitrated proteins showed that L5 SNL led to increased expression of tyrosine-nitrated Mn-containing superoxide dismutase (MnSOD) in the spinal cord, and hydrogen-rich normal saline administration reversed the tyrosine-nitrated MnSOD overexpression. We also showed that the analgesic effect of hydrogen-rich normal saline was associated with decreased activation of astrocytes and microglia, attenuated expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord.

Conclusion/Significance

Intrathecal injection of hydrogen-rich normal saline produced analgesic effect in neuropathic rat. Hydrogen-rich normal saline-induced analgesia in neuropathic rats is mediated by reducing the activation of spinal astrocytes and microglia, which is induced by overproduction of hydroxyl and peroxynitrite.     

Modulated expression of genes associated with NO signal transduction contributes to the cholesterol-lowering effect of electro-acupuncture

Electro-acupuncture (EA) at Fenglong acupoint (ST40) can lower the levels of serum cholesterol and triacylglycerols. To study the hepatic genes responsible for the cholesterol-lowering effect of EA, suppression subtractive hybridization combined with the switch mechanism at the 5'-end of RNA template cDNA synthesis and long-distance PCR were employed using hepatic tissues from hypercholesterolemia and EA-treated mice. 68 % of the identified genes are involved in metabolism, immune response, and signal transduction pathways. Real-time PCR and western blot indicate that EA at ST40 induces the expression of nNOS and Mt1, two genes involved in NO signal transduction. EA treatment for hypercholesterolemia thus involves the modulation of several biological pathways and provides a physiological link between NO signal transduction and the cholesterol-lowering effect of EA.     

反复电针对慢性痛的累加治疗作用及其机制研究

本研究从基础和临床两方面观察了反复电针对慢性痛的累加治疗作用,并结合疼痛患者及慢性痛动物模型中几种神经肽的放射免疫测定及相应受体拮抗剂的药理学研究结果,探讨了产生累加效应的可能机制。结果表明,在临床脊髓损伤性痉挛患者,１００Ｈｚ穴位体表电刺激有效地缓解痉挛并有累加效应;在临床慢性痛患者,２／１５Ｈｚ变频ＴＥＮＳ刺激有效地治疗疼痛并具有累加效应。在关节炎模型大鼠,电针刺激能产生明显的镇痛并具有累加效     

Effect of lappaconitine on neuropathic pain mediated by P2X3 receptor in rat dorsal root ganglion

ATP facilitates initiation and transmission of the neuropathic pain at the dorsal root ganglion (DRG) level via the P2X receptors, especially the subtype P2X(3). Lappaconitine (LA) is an active principle isolated from Chinese herbal medicine and possesses analgesic effect. The aim of this study was to investigate the effect of LA on chronic constriction injury (CCI)-induced neuropathic pain mediated by P2X(3) receptor in the DRG neurons. In the presence of CCI and/or LA, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X(3) receptor expression in the DRG neurons was evaluated by immunohistochemistry and Western blotting. Following intrathecal administration of P2X(3) receptor oligonucleotide, the effect of LA on pain thresholds was assessed. Furthermore, the effect of LA on the P2X(3) receptor agonists ATP- and α,β-meATP-induced inward currents (I(ATP) and I(α,β-meATP)) in the acutely dissociated rat DRG neurons was investigated by whole cell patch-clamp. The results included: (1) There showed reduction of pain thresholds, enhancement of I(ATP) and I(α,β-meATP) and up-regulation of P2X(3) receptor expression in rat DRG neurons when neuropathic pain occurred. (2) In the presence of LA, the decreased pain thresholds, the up-regulated P2X(3) receptor expression and the enhanced I(ATP) and I(α,β-meATP) were reversible in the CCI rats. (3) The down-regulated P2X(3) receptor expression with pretreatment of P2X(3) receptor antisense oligonucleotide significantly attenuated the analgesic effect of LA. These results indicate that the analgesic effect of LA involves decrease of expression and sensitization of the P2X(3) receptors of the rat DRG neurons following CCI.     

DGCR5 attenuates neuropathic pain through sponging miR-330-3p and regulating PDCD4 in CCI rat models

Neuropathic pain caused by somatosensory nervous system dysfunction is a serious public health problem. Some long noncoding RNAs (lncRNAs) can participate in physiological processes involved in neuropathic pain. However, the effects of lncRNA DGCR5 in neuropathic pain have not been explored. Therefore, in our current study, we concentrated on the biological roles of DGCR5 in neuropathic pain. Here, it was observed that DGCR5 was significantly decreased in chronic sciatic nerve injury (CCI) rat models. DGCR5 overexpression was able to alleviate neuropathic pain development including mechanical and thermal hyperalgesia. In addition, the current understanding of miR-330-3p function in neuropathic pain remains largely incomplete. Here, we found that miR-330-3p was greatly increased in CCI rats and DGCR5 can modulate miR-330-3p expression negatively. Upregulation of DGCR5 repressed inflammation-correlated biomarkers including interleukin 6 (IL-6), tumor necrosis factor α, and IL-1β in CCI rats by sponging miR-330-3p. The negative correlation between DGCR5 and miR-330-3p was confirmed in our current study. Inhibition of miR-330-3p suppressed neuropathic pain progression by restraining neuroinflammation in vivo. In addition, PDCD4 was predicted as a downstream target of miR-330-3p. Furthermore, PDCD4 was significantly increased in CCI rats and DGCR5 regulated PDCD4 expression through sponging miR-330-3p in CCI rat models. Taken these together, it was implied that DGCR5/miR-330-3p/PDCD4 axis participated in neuropathic pain treatment.     

Metformin relieves neuropathic pain after spinal nerve ligation via autophagy flux stimulation

Neuropathic pain is a well‐known type of chronic pain caused by damage to the nervous system. Autophagy is involved in the development and/or progression of many diseases, including neuropathic pain. Emerging evidence suggests that metformin relieves neuropathic pain in several neuropathic pain models; however, metformin's cellular and molecular mechanism for pain relief remains unknown. In this study, we investigated the therapeutic effects of metformin on pain relief after spinal nerve ligation (SNL) and its underlying mechanism of autophagy regulation. Behavioural analysis, histological assessment, expression of c‐Fos and molecular biological changes, as well as ultrastructural features, were investigated. Our findings showed that the number of autophagosomes and expression of autophagy markers, such as LC3 and beclin1, were increased, while the autophagy substrate protein p62, as well as the ubiquitinated proteins, were accumulated in the ipsilateral spinal cord. However, metformin enhanced the expression of autophagy markers, while it abrogated the abundance of p62 and ubiquitinated proteins. Blockage of autophagy flux by chloroquine partially abolished the apoptosis inhibition and analgesic effects of metformin on SNL. Taken together, these results illustrated that metformin relieved neuropathic pain through autophagy flux stimulation and provided a new direction for metformin drug development to treat neuropathic pain.     

孤啡肽受体对痛觉调控作用的研究进展

孤啡肽受体是继经典的mu阿片受体、kappa阿片受体和delta阿片受体之后发现的又一类新型阿片受体,不仅在结构上具有同上述阿片受体相类似的特征,而且可介导相同或相似的细胞内生物学反应.孤啡肽受体对痛觉反应具有独特的调控模式.一方面,在背根神经节以及脊髓水平,孤啡肽受体主要介导镇痛效应,并且在脊髓水平还与其他阿片受体有协同效应以增强镇痛效果.另一方面,在脊髓上水平,孤啡肽受体往往产生痛敏而拮抗了其他阿片受体的镇痛效应.此外孤啡肽受体对痛觉的调控在不同物种间也表现一定的差异性.这为进一步阐明内源性阿片系统的痛觉调控作用提供一定的理论依据.     

Dynorphinergic system alterations in the corticostriatal circuitry of neuropathic mice support its role in the negative affective component of pain

The dynorphinergic system is involved in pain transmission at spinal level, where dynorphin exerts antinociceptive or pronociceptive effects, based on its opioid or non‐opioid actions. Surprisingly, little evidence is currently available concerning the supraspinal role of the dynorphinergic system in pain conditions. The present study aimed to investigate whether neuropathic pain is accompanied by prodynorphin (Pdyn) and κ‐opioid receptor (Oprk1) gene expression alterations in selected mouse brain areas. To this end, mice were subjected to chronic constriction injury of the right sciatic nerve and neuropathic pain behavioral signs were ascertained after 14 days. At this interval, a marked increase in Pdyn mRNA in the anterior cingulate cortex (ACC) and prefrontal cortex (PFC) was observed. Oprk1 gene expression was increased in the PFC, and decreased in the ACC and nucleus accumbens (NAc). No changes were observed in the other investigated regions. Because of the relationship between dynorphin and the brain‐derived neurotrophic factor, and the role of this neurotrophin in chronic pain‐related neuroplasticity, we investigated brain‐derived neurotrophic factor gene (Bdnf) expression in the areas showing Pdyn or Oprk1 mRNAs changes. Bdnf mRNA levels were increased in both the ACC and PFC, whereas no changes were assessed in the NAc. Present data indicate that the dynorphinergic system undergoes quite selective alterations involving the corticostriatal circuitry during neuropathic pain, suggesting a contribution to the negative affective component of pain. Moreover, parallel increases in Pdyn and Bdnf mRNA at cortical level suggest the occurrence of likely interactions between these systems in neuropathic pain maladaptive neuroplasticity.