Qulian Guo

Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury.

The pathogenesis of neuropathic pain remains largely unknown. Epigenetic mechanisms may play a major role in regulating expression of pro- or antinociceptive genes. DNA methylation is a major epigenetic mechanism in vertebrates, and methyl- CpG-binding protein 2 (MeCP2) is directly involved in methylation-mediated gene silencing. To determine how changes in global DNA methylation and MeCP2 expression occur following chronic constriction injury (CCI) and how repression of DNA methylation affects these changes and attenuates neuropathic pain, we used intrathecal 5-azacytidine, a DNA methyltransferase inhibitor, in CCI rats. Rats received 0.9% saline or 5-azacytidine (10μmol·d(-1)) via spinal injection once daily from day 3 to day 14 after CCI surgery. Global DNA methylation and MeCP2 expression increased in the spinal cord in CCI rats on day 14 after CCI surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine from day 5 to day 14 after CCI surgery. The increases in global DNA methylation and MeCP2 expression in the spinal cord in CCI rats were also significantly inhibited by intrathecal 5-azacytidine. These results demonstrate that increased global DNA methylation and MeCP2 expression in the spinal cord after nerve damage may play an important role in neuropathic pain. 5-azacytidine shows potential for treating neuropathic pain.

Remote ischemic postconditioning protects the brain from global cerebral ischemia/reperfusion injury by up-regulating endothelial nitric oxide synthase through the PI3K/Akt pathway

Remote ischemic postconditioning (RIPoC) attenuates ischemia/reperfusion (I/R) injury in the heart, lung and hind limb. RIPoC performed in the hind limb reduces brain injury following focal cerebral ischemia in rats. Whether RIPoC has a neuroprotective effect with respect to global cerebral I/R injury is, however, unknown, and the mechanism of neuroprotection needs further elucidation. Here we investigated whether RIPoC could reduce global cerebral I/R injury in rats and whether this neuroprotective effect was induced by up-regulating endothelial nitric oxide synthase (eNOS) through the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. Global cerebral ischemia was performed via 8min of four-vessel occlusion. Neuronal density, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. The expression of eNOS, phosphorylated eNOS (Ser1177), Akt and phosphorylated Akt (Ser473) in the CA1 region was measured after reperfusion. RIPoC significantly attenuated delayed neuronal death and reduced the spatial learning and memory deficits associated with global cerebral ischemia. Pre-administration of N(ω)-nitro-l-arginine methyl ester (a nonselective NOS inhibitor) significantly abolished the neuroprotective effect of RIPoC. Moreover, pre-administration of LY294002 (a highly selective inhibitor of PI3K) not only significantly reversed the neuroprotective effect of RIPoC, but also obviously inhibited the up-regulation of eNOS induced by RIPoC. Our findings suggest that RIPoC protects the brain against global cerebral I/R injury and that this neuroprotection is mediated by up-regulating eNOS through the PI3K/Akt pathway.

Curcumin Alleviates Neuropathic Pain by Inhibiting p300/CBP Histone Acetyltransferase Activity-Regulated Expression of BDNF and Cox-2 in a Rat Model

The management of neuropathic pain is still a major challenge because of its unresponsiveness to most common treatments. Curcumin has been reported to play an active role in the treatment of various neurological disorders, such as neuropathic pain. Curcumin has long been recognized as a p300/CREB-binding protein (CBP) inhibitor of histone acetyltransferase (HAT) activity. However, this mechanism has never been investigated for the treatment of neuropathic pain with curcumin. The aim of the present study was to investigate the anti-nociceptive role of curcumin in the chronic constriction injury (CCI) rat model of neuropathic pain. Furthermore, with this model we investigated the effect of curcumin on P300/CBP HAT activity-regulated release of the pro-nociceptive molecules, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (Cox-2). Treatment with 40 and 60 mg/kg body weight curcumin for 7 consecutive days significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia, whereas 20 mg/kg curcumin showed no significant analgesic effect. Chromatin immunoprecipitation analysis revealed that curcumin dose-dependently reduced the recruitment of p300/CBP and acetyl-Histone H3/acetyl-Histone H4 to the promoter of BDNF and Cox-2 genes. A similar dose-dependent decrease of BDNF and Cox-2 in the spinal cord was also observed after curcumin treatment. These results indicated that curcumin exerted a therapeutic role in neuropathic pain by down-regulating p300/CBP HAT activity-mediated gene expression of BDNF and Cox-2.

Effects of intrathecal epigallocatechin gallate, an inhibitor of Toll-like receptor 4, on chronic neuropathic pain in rats.

Numerous studies revealed that spinal inflammation and immune response play an important role in neuropathic pain. In this study, we investigated the effects of intrathecal injection of a Toll-like receptor (TLR4) inhibitor epigallocatechin gallate (EGCG) on neuropathic pain induced by chronic constriction injury of the sciatic nerve (CCI). A total of 120 rats were randomly assigned into 4 groups: sham-operated group, CCI group, CCI plus normal saline group and CCI plus EGCG group. CCI and sham surgeries were performed and both thermal hyperalgesia and mechanical allodynia were tested. Lumbar spinal cord was sampled and the mRNA and protein expressions of TLR4 and High Mobility Group 1 protein (HMGB1) were detected, the contents of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-10 (IL-10) were measured by ELISA, and immunohistochemistry for nuclear factor kappa B (NF-κB) was also carried out. When compared with the sham group, both mechanical and heat pain thresholds were significantly decreased, and the mRNA and protein expressions of TLR4 and HMGB1, the contents of TNF-α, IL-1β and IL-10 in the spinal cords and NF-κB expression in the spinal dorsal horn were markedly increased in CCI rats (P<0.05). After intrathecal injection of EGCG (1mg/kg) once daily from 1day before to 3days after CCI surgery, the expressions of TLR4, NF-κB, HMGB1, TNF-α and IL-1β were markedly decreased while the content of IL-10 in the spinal cord increased significantly accompanied by dramatical improvement of pain behaviors in CCI rats (P<0.05). These results show that the TLR4 signaling pathway plays an important role in the occurrence and development of neuropathic pain, and the therapy targeting TLR4 might be a novel strategy in the treatment of neuropathic pain.

p300 exerts an epigenetic role in chronic neuropathic pain through its acetyltransferase activity in rats following chronic constriction injury (CCI)

BackgroundNeuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646.ResultsUsing the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord.ConclusionsThe results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.

Suppression of MicroRNA-155 Attenuates Neuropathic Pain by Regulating SOCS1 Signalling Pathway

Chronic neuropathic pain is an unfavourable pathological pain characterised by allodynia and hyperalgesia which has brought considerable trouble to people’s physical and mental health, but effective therapeutics are still lacking. MicroRNAs (miRNAs) have been widely studied in the development of neuropathic pain and neuronal inflammation. Among various miRNAs, miR-155 has been widely studied. It is intensively involved in regulating inflammation-associated diseases. However, the role of miR-155 in regulating neuropathic pain development is poorly understood. In the present study, we aimed to investigate whether miR-155 is associated with neuropathic pain and delineate the underlying mechanism. Using a neuropathic pain model of chronic constriction injury (CCI), miR-155 expression levels were markedly increased in the spinal cord. Inhibition of miR-155 significantly attenuated mechanical allodynia, thermal hyperalgesia and proinflammatory cytokine expression. We also demonstrated that miR-155 directly bound with the 3′-untranslated region of the suppressor of cytokine signalling 1 (SOCS1). The expression of SOCS1 significantly decreased in the CCI rat model, but this effect could be reversed by miR-155 inhibition. Furthermore, knockdown of SOCS1 abrogated the inhibitory effects of miR-155 inhibition on neuropathic development and neuronal inflammation. Finally, we demonstrated that inhibition of miR-155 resulted in the suppression of nuclear factor-κB and p38 mitogen-activated protein kinase activation by mediating SOCS1. Our data demonstrate the critical role of miR-155 in regulating neuropathic pain through SOCS1, and suggest that miR-155 may be an important and potential target in preventing neuropathic pain development.

Mechanical hypersensitivity, sympathetic sprouting, and glial activation are attenuated by local injection of corticosteroid near the lumbar ganglion in a rat model of neuropathic pain.

Background and Objectives: Inflammatory responses in the lumbar dorsal root ganglion (DRG) play a key role in pathologic pain states. Systemic administration of a common anti-inflammatory corticosteroid, triamcinolone acetonide (TA), reduces sympathetic sprouting, mechanical pain behavior, spontaneous bursting activity, and cytokine and nerve growth factor production in the DRG. We hypothesized that systemic TA effects are primarily due to local effects on the DRG. Methods: Male Sprague-Dawley rats were divided into 4 groups: SNL (tight ligation and transection of spinal nerves) and normal with and without a single dose of TA injectable suspension slowly injected onto the surface of DRG and surrounding region at the time of SNL or sham surgery. Mechanical threshold was tested on postoperative days 1, 3, 5, and 7. Immunohistochemical staining examined tyrosine hydroxylase and glial fibrillary acidic protein in DRG and CD11B antibody (OX-42) in spinal cord. Results: Local TA treatment attenuated mechanical sensitivity, reduced sympathetic sprouting in the DRG, and decreased satellite glia activation in the DRG and microglia activation in the spinal cord after SNL. Conclusions: A single injection of corticosteroid in the vicinity of the axotomized DRG can mimic many effects of systemic TA, mitigating behavioral and cellular abnormalities induced by spinal nerve ligation. This provides a further rationale for the use of localized steroid injections clinically and provides further support for the idea that localized inflammation at the level of the DRG is an important component of the spinal nerve ligation model, commonly classified as neuropathic pain model.

Intrathecal lentiviral-mediated RNA interference targeting PKCγ attenuates chronic constriction injury-induced neuropathic pain in rats.

In the spinal cord, protein kinase C isoform γ (PKCγ) plays an important role in the development of central pain sensitization. However, there are currently no specific PKCγ inhibitors available. Therefore, the aim of the present study was to assess the role of PKCγ in the modulation of pain using a more selective experimental tool. Although small interfering RNAs have been used to silence genes in neurons, in vivo delivery of RNA interference (RNAi) remains a major challenge, thus limiting its applications. Here we developed a highly efficient method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ for in vivo gene silencing in the spinal cord of rats. This method decreased the expression of PKCγ mRNA and protein, and additionally attenuated chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia for more than 6 weeks. Our study suggests that PKCγ is a potential RNAi target for neuropathic pain. Furthermore, the lentiviral vector delivery strategy holds great promise as a novel approach for the treatment of neuropathic pain and study of PKCγ gene function.

Cyclooxygenase inhibitors suppress the expression of P2X3 receptors in the DRG and attenuate hyperalgesia following chronic constriction injury in rats

Recent evidence suggests that P2X(3) receptors express abundantly in nociceptive sensory neurons and play an important role in neuropathic pain. Upregulation of prostaglandin E2 (PGE2) after nerve injure is involved in the pathogenesis of neuropathic pain. An increase of P2X(3) receptors after chronic constriction injury (CCI) to the sciatic nerve has also been reported, the mechanisms are not known clearly. In this study, we examined the effects of systemic administration of cyclooxygenase (COX) inhibitors on analgesia and the expression of P2X(3) receptors in the dorsal root ganglia (DRG) in CCI rats. Rats received 0.9% saline, the nonselective COX inhibitor ibuprofen (40mgkg(-1)day(-1)) or the selective COX-2 inhibitor celecoxib (30mgkg(-1)day(-1)) by gavage twice daily from 3 to 14 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were markedly attenuated by celecoxib from 5 to 14 days after surgery, and relieved by ibuprofen treatment from 7 to 10 days after surgery. The increase of P2X(3) receptors in the DRG in CCI rats on day 14 after surgery was also significantly inhibited; the effect of ibuprofen was stronger than that of celecoxib. These results demonstrate that up-regulated COX/PGE2 after nerve damage may play an important role in neuropathic pain. They are highly involved in the expression of P2X(3) receptors in the DRG in CCI rats.

Gene knockdown with lentiviral vector-mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats.

Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKCγ isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKCγ short hairpin RNA (LV‐shPKCγ) down‐regulated the expression of the PKCγ gene and reversed MT.