Disruption of Circadian NRF2 Expression and Its Impact on Pain Sensitivity in Diabetes Peripheral Neuropathy

Objective: Diabetic peripheral neuropathy (DN) is a common complication of diabetes, characterized by symptoms that are milder during the day and worsen at night. This study aims to uncover the role of circadian NRF2 expression in dorsal root ganglia (DRG) in regulating pain sensitivity and explores its disruption on neuropathic pain. Method: Male BKS.Cg-Lepr^db/db/J (db/db) type 2 diabetic mice (T2DM) were used as a model for DN. Diurnal pain sensitivity in the mice was evaluated through force withdrawal threshold (FWT) and thermal withdrawal latency (TWL) at Zeitgeber Time (ZT) 2 and ZT14 from 8 to 16 weeks of age. Sciatic nerve conduction velocity (SNCV) and oxidative stress levels in DRG were evaluated. Oxidative stress levels and antioxidant activity were assessed using fluorescent probe staining. NRF2 expression was evaluated through molecular and histological methodologies. The circadian regulatory genes (Clock, Bmal1), inflammatory factors (IL-6 and IL-10), and NRF2 target gene HO-1 were all detected by qRT-PCR. To directly investigate NRF2's role, AAV-mediated intrathecal injection was used to knock down NRF2 in DRG, disrupting its circadian rhythmicity. Results: A significant diurnal variation in neuropathic pain sensitivity was observed in db/db mice, with increased pain sensitivity at ZT2 compared to ZT14. Elevated ROS levels were detected in the DRG of db/db mice, especially at ZT2. In db/+ mice, NRF2 showed diurnal rhythms with higher expression at ZT2, a pattern disrupted in db/db mice, accompanied by elevated ROS levels and inflammation in the DRG. NRF2 knockdown yielded distinct effects: in db/db mice, it further elevated ROS levels at ZT14, impaired antioxidant capacity, and imbalance between pro-inflammatory and anti-inflammatory factors without significantly altering pain sensitivity, whereas in db/+ mice, it reduced pain thresholds and induced diurnal variations in pain sensitivity. Conclusion: The circadian rhythmicity of NRF2 in nondiabetic (db/+) mice is essential for maintaining the balance between anti- and pro-ROS, as well as inflammation, thereby preventing pain exacerbation and diurnal variations. Its disruption increases oxidative stress and inflammation, associated with induced diurnal pain sensitivity. In diabetic (db/db) mice, the loss of NRF2 rhythmicity exacerbates oxidative stress but minimally affects pain sensitivity, indicating a ceiling effect in pain sensitivity. These findings highlight NRF2 rhythmicity as a potential chronotherapeutic target for managing diabetic neuropathy.