Qi-Lu Li, Jia-Jia Wang, Meng-Xing Zhang, Qi-Di Sun, De-Sheng Xu, Fang-Cao Pi, Ming-Ming Peng, Can-Ling Feng, Zi-Han Zhu, Yan Wang, Zi-Rui Li, Juan-Min Li, Yuan-Yuan Li, Yan Dong, Ying Xie, Yun-Da Yao, Hua Zhou
This study provides the first evidence that SIN suppresses P2X2R transcription via upregulation of DNMT1, leading to P2X2R promoter hypermethylation-a previously unrecognized epigenetic mechanism underlying the analgesic action of this natural product. This DNMT1-dependent pathway offers a novel therapeutic target for analgesic strategies focusing on epigenetic regulation.
BACKGROUND: Chronic inflammatory pain lacks effective and non-addictive therapeutic options. Sinomenine (SIN), a non-opioid alkaloid derived from the traditional Chinese medicine Sinomenium acutum, has demonstrated analgesic potential in clinical practice, yet its precise mechanism remains incompletely understood.
PURPOSE: This study aimed to investigate the analgesic mechanism of SIN and to identify a novel epigenetic pathway involved.
METHODS: The effects of SIN were evaluated in complete Freund's adjuvant-induced chronic inflammatory pain models in rats and lipopolysaccharide stimulated BV2 microglial cells using behavioral tests, enzyme-linked immunosorbent assay, Western blot, reverse transcription quantitative PCR, immunohistochemistry, hematoxylin and eosin staining, calcium imaging, and amplicon bisulfite sequencing. Pharmacological interventions with a P2X2 purinergic receptor (P2X2R) agonist BZATP and antagonist A317491, as well as small interfering RNA mediated knockdown of DNA methyltransferase 1 (DNMT1) in BV2 cells, were employed to dissect the pathway.
RESULTS: SIN dose-dependently alleviated pain hypersensitivity and inflammation, accompanied by downregulation of P2X2R expression. SIN inhibited P2X2R-dependentoveractivation of downstream extracellular signal-regulated kinase 1/2 signaling. Amplicon bisulfite sequencing revealed that SIN promoted methylation of the P2X2R gene promoter. Importantly, DNMT1 knockdown in BV2 cells completely abolished the suppressive effect of SIN on P2X2R expression.
CONCLUSION: This study provides the first evidence that SIN suppresses P2X2R transcription via upregulation of DNMT1, leading to P2X2R promoter hypermethylation-a previously unrecognized epigenetic mechanism underlying the analgesic action of this natural product. This DNMT1-dependent pathway offers a novel therapeutic target for analgesic strategies focusing on epigenetic regulation.