Abstract

Background Neuropathic pain (NP) is frequently accompanied by anxiety and depression, and current treatments do not adequately address this comorbidity. The anterior cingulate cortex (ACC) plays a central role in sensory and emotional processing. However, the molecular pathways that connect these functions remain unclear. G protein-coupled receptor 35 (GPR35), an orphan receptor enriched in neurons, has been implicated in neuroinflammation and pain signaling. However, its specific involvement in NP and associated affective disturbances has not been fully elucidated. Methods Peripheral blood GPR35 expression was measured in human patients with NP and healthy controls. In mice, chronic constriction injury (CCI) was used to induce NP. Lentiviral knockdown or overexpression of GPR35 was performed in ACC cells. Behavioral assays were used to assess mechanical and thermal sensitivity, locomotor and anxiety metrics, cognitive performance, and depression-related behaviors. Molecular analyses included western blotting, RT-qPCR, immunofluorescence, RNA sequencing, and co-immunoprecipitation. Additional Nr4a1 knockdown and L-kynurenine (L-Kyna, a GPR35 agonist) administration were used to validate pathway involvement. Results Patients with NP had higher circulating GPR35 levels, which positively correlated with pain intensity. CCI induced a time-dependent increase in GPR35 expression in the ACC of mice, accompanied by hypersensitivity and emotional disturbances. GPR35 knockdown in the ACC worsens mechanical and thermal hypersensitivity, impairs cognition, increases depression-related behaviors, and amplifies microglial activation and pro-inflammatory cytokine production. GPR35 overexpression reversed these effects by reducing hypersensitivity, improving affective behaviors, and restoring the inflammatory balance. Transcriptomic and biochemical analyses identified Nr4a1 as a key downstream effector of GPR35, and Nr4a1 knockdown eliminated the protective effects of GPR35 overex