BMAL1 in Anterior Cingulate Cortex Pyramidal Neurons Relieves Pulpitis‐Induced Nocturnal Pain

ABSTRACT Aim Nocturnal exacerbation of pulpitis‐induced pain severely disrupts patients' sleep and quality of life, increasing emergency dental care burden. However, the exact mechanisms underlying nocturnal pain remain poorly understood, limiting the development of targeted analgesic strategies. Here, we explored the circadian characteristics of pulpitis‐induced pain in mice and the role of clock genes in its nocturnal exacerbation. Methodology An experimental pulpitis model was established via pulp exposure, with stability confirmed by Micro‐computed tomography and haematoxylin and eosin staining. Von Frey test was used to investigate the circadian characteristics of pulpitis‐induced pain. Immunofluorescence (IF) staining, in vivo fibre photometry, and stereotaxic injection were combined to evaluate the circadian variation of neuronal activity in the anterior cingulate cortex (ACC), as well as its role in modulating the circadian rhythm of pulpitis‐induced pain. Single‐nuclei RNA sequencing (snRNA‐seq) was performed to screen differentially expressed genes. Quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blot, and IF staining were used to detect the expression changes and neuronal expression profile of brain and muscle ARNT‐like protein 1 (BMAL1). Adeno‐associated virus selectively overexpressed BMAL1 in pyramidal neurons was constructed. Combined with behavioural tests, Western blot, and in vivo fibre photometry, the role and underlying mechanism of BMAL1 in the nocturnal exacerbation of pulpitis‐induced pain were evaluated. Result On Day 3 after pulp exposure, the pain threshold exhibited a distinct circadian rhythm, reaching its minimum during the light phase. Excitotoxic lesion and fibre photometry experiments further indicated that neurons in the ACC are critical for regulating the circadian rhythm of pulpitis‐induced pain. Using snRNA‐seq, qRT‐PCR, Western blot, and IF staining, we screened and validated a significant reduction in BMAL1 expression in the ACC of mice with pulpitis during the light phase, particularly in pyramidal neurons. Furthermore, overexpression of BMAL1 in ACC pyramidal neurons alleviated the nocturnal exacerbation of pulpitis‐induced pain, alongside decreased activation of the Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII) signaling pathway and reduced pyramidal neuronal excitability. Conclusion Our findings elucidate the core role of BMAL1 in contributing to nocturnal pain exacerbation of pulpitis and offer a valuable therapeutic target for the management of nocturnal pain in patients with pulpitis.