Intraperitoneal 5-Azacytidine Alleviates Nerve Injury-Induced Pain in Rats by Modulating DNA Methylation

Abstract

To investigate the role of DNA methylation in modulating chronic neuropathic pain (NPP), identify possible target genes of DNA methylation involved in this process, and preliminarily con rm the medicinal value of the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-AZA) in NPP by targeting gene methylation. MethodsTwo rat NPP models, chronic constriction injury (CCI) and spinal nerve ligation (SNL), were used. The DNA methylation pro les in the lumbar spinal cord were assayed using an Arraystar Rat RefSeq Promoter Array. The underlying genes with differential methylation were then identi ed and submitted to Gene Ontology and pathway analysis. Methyl-DNA immunoprecipitation quantitative PCR (MeDIP-qPCR) and quantitative reverse transcription-PCR (RT-qPCR) were used to con rm gene methylation and expression.The protective function of 5-AZA in NPP and gene expression were evaluated via behavioral assays and RT-qPCR, respectively. ResultsAnalysis of the DNA methylation patterns in the lumbar spinal cord indicated that 1205 differentially methylated fragments in CCI rats were located within DNA promoter regions, including 638 hypermethylated fragments and 567 hypomethylated fragments. The methylation levels of Grm4, Htr4, Adrb2, Kcnf1, Gad2 and Pparg, which are associated with long-term potentiation (LTP) and glutamatergic synapse pathways, were increased with a corresponding decrease in their mRNA expression, in the spinal cords of CCI rats. Moreover, we found that the intraperitoneal injection of 5-AZA (4 mg/kg) attenuated CCI-or SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, the mRNA expression of hypermethylated genes such as Grm4, Htr4, Adrb2, Kcnf1 and Gad2 was reversed after 5-AZA treatment. ConclusionIncreasing methylation is a novel negative regulatory mechanism of target gene expression in chronic NPP. In rats, the intraperitoneal injection of 5-AZA alleviated spinal nociception, an effect accompanied by the reversed expression of hypermethylated genes. Thus, DNA methylation inhibition represents a promising strategy for protection against chronic NPP following nerve injury. Our study lays a theoretical foundation for 5-AZA to become a clinical targeted drug.1. Introduction hypermethylated gene mRNA expression induced by chronic NPP following CCI-or spinal nerve ligation (SNL). Materials And Methods AnimalsIn this study, male Sprague-Dawley (SD) rats weighing 220-250 g were employed. All animals were kept in a setting with a temperature of 22 ± 1°C, a relative humidity of 50 ± 1%, and a cycle of light and dark of 12 hr. They had access to food and water at all times in their separate cages. All protocols were approved by the ethics committee of the Fujian Cancer Hospital and adhered to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Experimental paradigmBehavioral testing was performed before surgery and at 3, 5, 7, 10 and 14 Days after surgery in each rat. Under deep anesthesia, all the rats were killed and their ipsilateral lum...

Pathology of pain and its implications for therapeutic interventions

“…Patients suffering from chronic pain universally undergo significant changes in DNA methylation states, particularly in promoter regions. 184 , 185 Global methylation data have been used to investigate pain-associated mechanisms with the support of bioinformatic analysis, such as G-protein coupled cholinergic signaling, neuron development and immunomodulation. 184 , 186 , 187 DNA methylation has quick responses to pain.…”

“… 196 , 197 Systemic inhibition of DNMT activity results in alleviation of neuropathic pain. 185 Therefore, it can be concluded that despite manifold targets of DNA methylation, its overall effects are pain hypersensitivity.…”

The Epigenetics of Neuropathic Pain: A Systematic Update

“…The latter agent also reduced CCI-evoked mechanical allodynia and heat hyperalgesia, suggesting that CCI may lead to epigenetic silencing of some putative antinociceptive genes via DNA methylation. In a genome-wide analysis of the spinal cords of rats with CCI, 638 hypermethylated and 567 hypomethylated sites within promoter regions were revealed [13].…”

“…The methylation levels of the genes of the metabotropic glutamate receptor type 4 (mGlu4), serotonin 5-HT 4 receptor, β 2 adrenergic receptor, and K v 5.1 channel were increased in both the CCI and SNL models of neuropathic pain in the rat [13]. Accordingly, the mRNAs of these receptors/channels were downregulated in the spinal cord.…”