Down‐regulation of M 6 A demethylase ALKBH5 facilitates morphine tolerance via SGK1 in mice

Background and Purpose The development of drug tolerance following prolonged use of opioids, such as morphine, limits their analgesic efficacy and clinical utility. Epigenetic modifications, particularly N 6 ‐methyladenosine (m 6 A), have garnered increasing research interest. AlkB homologue 5 (ALKBH5), a key m 6 A demethylase, has been implicated in cancers and neurological disorders. However, its role in morphine tolerance remains unclear. Experimental Approach A mouse model of morphine tolerance was established via repeated subcutaneous morphine administration. The m 6 A methylation levels and related enzymes expression in the spinal cord were assessed. Functional validation was conducted via adeno‐associated virus (AAV)‐mediated ALKBH5 overexpression in the spinal dorsal horn. Next‐generation sequencing and GO/KEGG pathway analysis were performed to identify potential downstream target genes, followed by validation via pharmacological inhibition of SGK1. Key Results Morphine‐tolerant mice exhibited significantly elevated spinal m 6 A methylation, mainly because of reduced ALKBH5 expression in superficial dorsal horn neurons. ALKBH5 overexpression attenuated the development of morphine tolerance and partly restored opioid analgesic efficacy, reducing spinal m 6 A methylation levels and SGK1 expression. Transcriptomic analysis and subsequent validation identified SGK1‐associated immune signalling pathways as a pivotal mediator of morphine tolerance. Conclusions and Implications Decreased expression of ALKBH5 contributes to elevated spinal m 6 A methylation and SGK1 expression, promoting morphine tolerance. Targeted overexpression of ALKBH5 mitigates morphine tolerance, most likely through modulation of SGK1‐associated immune signalling pathways. These findings suggest that the ALKBH5‐m 6 A‐SGK1 axis may participate in the epigenetic modulation of morphine tolerance and provides a promising molecular target for mitigating morphine tolerance.