Intrathecal amylin reverses morphine tolerance through site-specific DNA methylation of Pdyn and Bdnf promoters in rats.

Chronic use of opioids such as morphine can lead to tolerance, opioid-induced hyperalgesia, and addiction. Amylin has demonstrated analgesic properties and the ability to reverse morphine properties when injected intrathecally in rats. Our previous study correlated these effects with reduced spinal cord Bdnf expression. To further investigate the mechanisms underlying amylin's reversal of morphine tolerance, we analyzed the mRNA expression of Pdyn and DNA methyltransferases (Dnmt3a and Dnmt3b), along with the methylation status of Pdyn and Bdnf exon IV promoters in the lumbar spinal cord of rats. Quantitative RT-PCR showed that morphine elevated Pdyn mRNA levels, an effect reversed by co-administration of amylin (1-60 pmol) between days 6 and 10 post-tolerance induction. The highest amylin dose also significantly upregulated Dnmt3a expression. Methylation-specific PCR (MSP-PCR) revealed unmethylated Pdyn and Bdnf promoters in saline-treated and morphine-only rats, but 20-100% methylation in rats treated with both morphine and amylin, and 100% methylation with amylin alone. Bisulfite sequencing confirmed site-specific methylation at CpG - 197 in the Bdnf promoter and CpG - 110 in the Pdyn promoter. These molecular changes, observed in animals with behaviorally confirmed morphine tolerance and amylin- induced reversal, suggest that amylin reverses morphine tolerance through mechanisms associated with Dnmt3a upregulation and site-specific methylation of Pdyn and Bdnf promoters, revealing a novel epigenetic correlation.