scholarly journals Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation

2021 ◽  
Author(s):  
Yan-Yan Wu ◽  
Hai-Long Zhang ◽  
Xiaomin Lu ◽  
Han Du ◽  
Yong-Chang Li ◽  
...  
Keyword(s):  
Dorsal Horn ◽  
Spinal Dorsal Horn ◽  
Visceral Pain ◽  
Cpg Island ◽  
Dna Demethylation ◽  
Luciferase Assay ◽  
Unknown Etiology ◽  
Physical Interaction ◽  
Long Term Effects ◽  
Spinal Dorsal

AbstractIrritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1–TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.

scholarly journals Involvement of Brn3a-positive spinal dorsal horn neurons in the transmission of visceral pain in inflammatory bowel disease model mice

2021 ◽  
Author(s):  
Kazuhiko Nishida ◽  
Shinji Matsumura ◽  
Takuya Kobayashi
Keyword(s):  
Inflammatory Bowel Disease ◽  
Dorsal Horn ◽  
Bowel Disease ◽  
Spinal Dorsal Horn ◽  
Visceral Pain ◽  
Dorsal Horn Neurons ◽  
Spinal Dorsal ◽  
Pain Transmission ◽  
Inflammatory Bowel ◽  
Spinal Dorsal Horn Neurons

Spinal dorsal horn plays crucial roles in the transmission and processing of somatosensory information. Although spinal neural circuits which process several distinct types of cutaneous sensation have been extensively studied, those responsible for visceral pain transmission remain poorly understood. In the present study, we analyzed the dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) model mice to characterize the spinal dorsal horn neurons involved in visceral pain transmission. DSS-treated mice exhibited increased abdominal licking behavior, suggestive of experiencing visceral pain. Immunostaining of c-fos, a marker indicating neuronal activity, demonstrated that numerous c-fos-positive cells were found bilaterally in the lumbosacral spinal dorsal horn, and their distribution was particularly abundant in the shallow dorsal horn. Neurochemical characterization of these neurons revealed that the percentage of the POU transcription factor Brn3a-positive neurons among the c-fos-positive neurons in the shallow dorsal horn was 30-40% in DSS-treated mice, which was significantly higher than that in the somatic pain model mice. We further demonstrated by neuronal tracing that within the shallow dorsal horn, Brn3a-positive neurons are represented more highly in spino-solitary projection neurons than in spino-parabrachial projection ones. These results raised the possibility that Brn3a-positive spinal dorsal horn neurons make a large contribution to visceral pain transmission, and part of which was mediated through spino-solitary pathway.

scholarly journals Upregulation of Spinal ASIC1 and NKCC1 Expression Contributes to Chronic Visceral Pain in Rats

2021 ◽  
Vol 13 ◽  
Author(s):  
Yong-Chang Li ◽  
Yuan-Qing Tian ◽  
Yan-Yan Wu ◽  
Yu-Cheng Xu ◽  
Ping-An Zhang ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Signaling Pathway ◽  
Dorsal Horn ◽  
Spinal Dorsal Horn ◽  
Visceral Pain ◽  
Intrathecal Injection ◽  
Adult Rats ◽  
Sodium Potassium ◽  
Spinal Dorsal ◽  
Whole Cell Patch Clamp

Aims: To determine whether acid-sensing ion channel 1 (ASIC1)–sodium-potassium-chloride cotransporter 1 (NKCC1) signaling pathway participates in chronic visceral pain of adult rats with neonatal maternal deprivation (NMD).Methods: Chronic visceral pain was detected by colorectal distension (CRD). Western blotting and Immunofluorescence were performed to detect the expression and location of ASIC1 and NKCC1. Whole-cell patch-clamp recordings were performed to record spinal synaptic transmission.Results: The excitatory synaptic transmission was enhanced and the inhibitory synaptic transmission was weakened in the spinal dorsal horn of NMD rats. ASIC1 and NKCC1 protein expression in the spinal dorsal horn was significantly up-regulated in NMD rats. Incubation of Amiloride reduced the amplitude of mEPSCs. Incubation of Bumetanide (BMT) increased the amplitude of mIPSCs. Intrathecal injection of ASIC1 or NKCC1 inhibitors reversed the threshold of CRD in NMD rats. Also, Amiloride treatment significantly reversed the expression of NKCC1 in the spinal dorsal horn of NMD rats.Conclusion: Our data suggest that the ASIC1-NKCC1 signaling pathway is involved in chronic visceral pain in NMD rats.

scholarly journals Targeting spinal TRAF6 expression attenuates chronic visceral pain in adult rats with neonatal colonic inflammation

2020 ◽  
Vol 16 ◽  
pp. 174480692091805 ◽  
Author(s):  
Rui-Xia Weng ◽  
Wei Chen ◽  
Jia-Ni Tang ◽  
Qian Sun ◽  
Meng Li ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Irritable Bowel Syndrome ◽  
Dorsal Horn ◽  
Spinal Dorsal Horn ◽  
Visceral Pain ◽  
Intrathecal Injection ◽  
Visceral Hypersensitivity ◽  
Colonic Inflammation ◽  
Spinal Dorsal ◽  
Irritable Bowel

Background Irritable bowel syndrome is one of the most common gastrointestinal disorders. It is featured by abdominal pain in conjunction with altered bowel habits. However, the pathophysiology of the syndrome remains largely unknown. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be involved in neuropathic pain. The aim of this study was to investigate roles and mechanisms of TRAF6 in the chronic visceral hypersensitivity. Methods Visceral hypersensitivity was induced by neonatal colonic inflammation and was identified by colorectal distention. The protein level, RNA level, and cellular distribution of TRAF6 and its related molecules were detected with Western blot, quantitative polymerase chain reaction, and immunofluorescence. In vitro spinal cord slice recording technique was performed to determine the synaptic transmission activities. Results Neonatal colonic inflammation rats displayed visceral hypersensitivity at the age of six weeks. The expression of TRAF6 was obviously upregulated in spinal cord dorsal horn of neonatal colonic inflammation rats at the age of six weeks. Immunofluorescence study showed that TRAF6 was dominantly expressed in spinal astrocytes. Intrathecal injection of TRAF6 small interfering RNA (siRNA) significantly reduced the amplitude of spontaneous excitatory postsynaptic currents at the spinal dorsal horn level. Furthermore, knockdown of TRAF6 led to a significant downregulation of cystathionine β synthetase expression in the spinal dorsal horn of neonatal colonic inflammation rats. Importantly, intrathecal injection of TRAF6 siRNA remarkably alleviated visceral hypersensitivity of neonatal colonic inflammation rats. Conclusions Our results suggested that the upregulation of TRAF6 contributed to visceral pain hypersensitivity, which is likely mediated by regulating cystathionine β synthetase expression in the spinal dorsal horn. Our findings suggest that TRAF6 might act as a potential target for the treatment of chronic visceral pain in irritable bowel syndrome patients.

Attenuation of Remifentanil-Induced Hyperalgesia by Betulinic Acid Associates with Inhibiting Oxidative Stress and Inflammation in Spinal Dorsal Horn

Pharmacology ◽  
2018 ◽  
Vol 102 (5-6) ◽  
pp. 300-306 ◽  
Author(s):  
Chang-Cheng Lv ◽  
Man-Li Xia ◽  
Shu-Juan Shu ◽  
Fei Chen ◽  
Li-Shan Jiang
Keyword(s):  
Oxidative Stress ◽  
Dorsal Horn ◽  
Betulinic Acid ◽  
Spinal Dorsal Horn ◽  
Manganese Superoxide Dismutase ◽  
Visceral Pain ◽  
Sprague Dawley ◽  
Spinal Dorsal ◽  
Pre Treatment ◽  
Factor Α

Remifentanil-induced hyperalgesia (RIH) is known to be associated with oxidative stress and inflammation. Betulinic acid (BA) was reported to reduce visceral pain owing to its anti-oxidative and anti-inflammatory potential. Here, we ­explored whether BA can attenuate RIH through inhibiting oxidative stress and inflammation in spinal dorsal horn. Sprague-Dawley rats were randomly divided into 4 groups: Control, Incision, RIH, and RIH pre-treated with BA. After pretreated with BA (25 mg/kg, i.g.) for 7 days, rats were subcutaneously infused with remifentanil (40 μg/kg) for 30 min during right plantar incision surgery to induce RIH. The paw withdrawal mechanical threshold (PWMT), paw withdrawal thermal latency (PWTL), spinal oxidative stress and inflammatory mediators were determined. Intraoperative remifentanil infusion induced postoperative hyperalgesia, as evidenced by the significant decrease in PWMT and PWTL (p < 0.01), and the significant increase in oxidative stress and inflammation evidenced by up-regulations of malondialdehyde, 3-nitrotyrosine, interleukin-1β and tumour necrosis factor-α (p < 0.01) in spinal dorsal horn and matrix metalloproteinase-9 (MMP-9) activity (p < 0.01) in dorsal root ganglion, as well as a decrease in manganese superoxide ­dismutase activity (p < 0.01) compared with control and ­incision groups. All these results mentioned above were markedly reversed by pre-treatment with BA (p < 0.01) compared with RIH group. These findings demonstrated that BA can effectively attenuate RIH, which associates with potentially inhibiting oxidative stress and subsequently down-regulating MMP-9-related pro-inflammatory cyokines in spinal dorsal horn.

scholarly journals Blockade of Erythropoietin-Producing Human Hepatocellular Carcinoma Receptor B1 in Spinal Dorsal Horn Alleviates Visceral Pain in Rats

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Chen-Li Sun ◽  
Cheng-Wen Li ◽  
Nong He ◽  
Yuan-Zhang Tang ◽  
Xiu-Liang Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dorsal Horn ◽  
Spinal Dorsal Horn ◽  
Visceral Pain ◽  
Intrathecal Administration ◽  
Human Hepatocellular Carcinoma ◽  
Control Group ◽  
Visceral Hyperalgesia ◽  
Spinal Dorsal ◽  
Phosphorylated Form

Objective. This experiment was designed to determine whether erythropoietin-producing human hepatocellular carcinoma (Eph) receptors were involved in the development of visceral pain. Methods. Adult male Sprague-Dawley rats were randomly divided into three groups receiving different treatments (n = 16 per group): intracolonic vehicle (control group), intracolonic 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) (TNBS group), and intracolonic TNBS and intrathecal EphB1 receptor blocking reagent (TNBS + EphB2-Fc group). Visceral hyperalgesia was evaluated with quantification of visceral pain threshold induced by colorectal distention. The spinal expressions of EphB1 and ephrinB2 and levels of their phosphorylated forms (p-EphB1 and p-ephrinB2) were assessed by Western blotting and immunohistochemistry. Results. The TNBS-treated rats developed significant visceral hyperalgesia. The spinal expressions of EphB1, p-EphB1, ephrinB2, and p-ephrinB2 were significantly increased in the TNBS group compared with the control group, but visceral hyperalgesia and elevation of spinal EphB1 and p-EphB1 expressions were evidently alleviated by intrathecal administration of EphB2-Fc in the TNBS + EphB2-Fc group. The number of EphB1- and p-EphB1-immunopositive cells, the average optical (AO) value of EphB1, and its phosphorylated form in the spinal dorsal horn were significantly increased in the TNBS group than in the control group, but they were obviously reduced by intrathecal administration of EphB2-Fc. There were no significant differences in the number of ephrinB2- and p-ephrinB2-immunopositive cells and the AO value of ephrinB2 and its phosphorylated form between the TNBS and TNBS + EphB2-Fc groups. Conclusion. EphB1 receptors in the spinal dorsal horn play a pivotal role in the development of visceral pain and may be considered as a potential target for the treatment of visceral pain.

scholarly journals Regulation of Peripheral Clock to Oscillation of Substance P Contributes to Circadian Inflammatory Pain

2012 ◽  
Vol 117 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Jing Zhang ◽  
Huili Li ◽  
Huajing Teng ◽  
Ting Zhang ◽  
Yonglun Luo ◽  
...  
Keyword(s):  
Substance P ◽  
Dorsal Root Ganglion ◽  
Dorsal Horn ◽  
Inflammatory Pain ◽  
Dorsal Root ◽  
Spinal Dorsal Horn ◽  
Luciferase Assay ◽  
Nociceptive Response ◽  
Spinal Level ◽  
Spinal Dorsal

Background The daily fluctuations of many physiologic and behavioral parameters are differentially influenced by either central or peripheral clocks in mammals. Since substance P (SP) oscillates in some brain tissues and plays an indispensable role in modulating inflammatory pain at the spinal level, we speculated that SP mediates circadian nociception transmission at the spinal level. Methods In the present study behavioral observation, real-time polymerase chain reaction, luciferase assay, chromatin immunoprecipitation, and immunohistochemistry stain methods were used to investigate the role of SP in the spinal circadian nociception transmission and its regulation mechanism. Results Our results showed that under transcriptional regulation of BMAL1:CLOCK heterodimers, SP's coding gene Tac1 expression oscillates in dorsal root ganglion (n = 36), but not in the spinal dorsal horn. Further, the expression of SP cycled in the spinal dorsal horn, and this rhythmicity was potentially determined by circadian expression of Tac1 in dorsal root ganglion. Furthermore, the variation of SP expression induced by formalin was fluctuated in a similar rhythm to behavioral nociceptive response induced by formalin (n = 48); and the nociceptive behavioral circadian rhythm could be abolished through blockade of the SP-Neurokinin 1 receptor pathway (n = 70). Lastly, the variations of spinal SP expression and behavioral nociceptive response were in step, and both were changed by the deletion mutation of clock gene. Conclusions We conclude that spinal SP probably plays a pivotal role in modulating circadian inflammatory pain and suggest that peripheral circadian-regulated signaling is potentially an essential pathway for circadian nociceptive transmission.

scholarly journals Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Qi An ◽  
Chenyan Sun ◽  
Ruidi Li ◽  
Shuhui Chen ◽  
Xinpei Gu ◽  
...  
Keyword(s):  
Neuropathic Pain ◽  
Dorsal Horn ◽  
Nerve Injury ◽  
Spinal Dorsal Horn ◽  
Microglial Activation ◽  
Microglial Cells ◽  
Gene Promoters ◽  
Spinal Dorsal ◽  
Related Peptide ◽  
Calcitonin Gene

Abstract Background Calcitonin gene-related peptide (CGRP) as a mediator of microglial activation at the transcriptional level may facilitate nociceptive signaling. Trimethylation of H3 lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that regulates inflammatory-related gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K27me3 in microglial activation after nerve injury, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. Methods Microglial cells (BV2) were treated with CGRP and differentially enrichments of H3K27me3 on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on microglial activation and EZH2/H3K27me3 signaling in CCI-induced neuropathic pain. Results Overexpressions of EZH2 and H3K27me3 were confirmed in spinal microglia of CCI rats by immunofluorescence. CGRP treatment induced the increased of H3K27me3 expression in the spinal dorsal horn and cultured microglial cells (BV2) through EZH2. ChIP-seq data indicated that CGRP significantly altered H3K27me3 enrichments on gene promoters in microglia following CGRP treatment, including 173 gaining H3K27me3 and 75 losing this mark, which mostly enriched in regulation of cell growth, phagosome, and inflammation. qRT-PCR verified expressions of representative candidate genes (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) and real-time cell analysis (RTCA) verified microglial proliferation. Additionally, CGRP treatment and CCI increased expressions of ITGAM, ADAM10, MCP-1, and CX3CR1, key mediators of microglial activation in spinal dorsal horn and cultured microglial cells. Such increased effects induced by CCI were suppressed by CGRP antagonist and EZH2 inhibitor, which were concurrently associated with the attenuated mechanical and thermal hyperalgesia in CCI rats. Conclusion Our findings highly indicate that CGRP is implicated in the genesis of neuropathic pain through regulating microglial activation via EZH2-mediated H3K27me3 in the spinal dorsal horn.

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