Chronic pain, a prevalent and debilitating condition, significantly impacts public health. The International Association for the Study of Pain defines it as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Chronic pain is often accompanied by hyperalgesia and allodynia, affecting physical functioning and quality of life. A strong correlation exists between chronic pain and emotional disorders like anxiety and depression, influencing pain perception and treatment outcomes. Current therapies, such as opioids and NSAIDs, often lack efficacy in addressing the functional and affective components of chronic pain and are associated with adverse effects. The inflammatory process significantly increases mitochondrial reactive oxygen species (ROS), leading to inflammasome activation, particularly NLRP3. NLRP3 inflammasome, composed of NLRP3, ASC, and pro-caspase-1, triggers the release of pro-inflammatory cytokines (IL-1β, IL-18), contributing to inflammatory diseases and depressive-like behaviors. The Nrf2 transcription factor plays a vital role in regulating antioxidant and anti-inflammatory genes. Its activation protects against chronic pain by inducing the expression of antioxidant enzymes such as HO-1, NQO1, GSTM1, and SOD-1. Dimethyl fumarate (DMF), an FDA-approved drug for multiple sclerosis, activates Nrf2, demonstrating analgesic and neuroprotective properties in various pain models. Recent studies synthesized novel DMF derivatives (1m, 1a, 1b) with enhanced HO-1 induction capabilities. However, their antinociceptive, anxiolytic, and antidepressant effects during inflammatory pain remain unexplored. This study aimed to evaluate the effects of DMF, 1m, 1a, and 1b on CFA-induced inflammatory pain, functional deficits, and associated emotional disorders in mice, examining their impact on NLRP3 inflammasome and antioxidant pathways.

The literature review extensively discussed the limitations of current treatments for chronic inflammatory pain, highlighting the need for novel therapies that address both the sensory and emotional aspects of the condition. The review detailed the roles of oxidative stress, NLRP3 inflammasome activation, and the Nrf2 antioxidant pathway in the pathogenesis of inflammatory pain and associated mood disorders. Existing evidence supporting the analgesic and anti-inflammatory effects of DMF and other Nrf2 activators (like sulforaphane) was presented, along with the rationale for investigating the novel HO-1 inducers. The synthesis and in vitro characterization of the novel DMF derivatives were described, emphasizing their potential to be more potent HO-1 inducers compared to DMF itself.

This study used male C57BL/6 mice, subplantarly injected with CFA to induce inflammatory pain. Groups received oral administrations of DMF, 1m, 1a, 1b, or vehicle for four consecutive days starting at day 13 post-CFA injection. Mechanical allodynia was assessed using von Frey filaments, thermal hyperalgesia with the plantar test, and grip strength using a grip strength meter. Anxiety-like behaviors were evaluated using the elevated plus maze and open field tests, while depressive-like behaviors were assessed using the tail suspension and forced swimming tests. Western blotting was performed to analyze the protein expression of NLRP3, Nrf2, HO-1, SOD-1, NQO1, and GSTM1 in paw tissue and amygdala. Statistical analysis involved three-way repeated measures ANOVA and one-way ANOVA with post-hoc Tukey tests. All procedures adhered to ethical guidelines and were approved by the local Animal Use and Care Committee.

Treatment with DMF, 1m, 1a, and 1b significantly inhibited CFA-induced mechanical allodynia and thermal hyperalgesia, and reversed grip strength deficits. 1m showed the most rapid and potent effects. All treatments alleviated anxiety- and depressive-like behaviors associated with inflammatory pain. Western blot analysis revealed that all treatments normalized the upregulation of NLRP3 inflammasome in both paw and amygdala. The treatments significantly enhanced Nrf2 expression in the paw. HO-1 levels were significantly increased in the paw, with 1b showing a greater increase than DMF. SOD-1 expression was also increased in the paw after treatment. No significant changes in NQO1 or GSTM1 expression were observed in the paw. In the amygdala, HO-1 expression was significantly increased by all treatments, while NLRP3 levels were normalized. No significant changes in Nrf2, SOD-1, NQO1, or GSTM1 expression were observed in the amygdala.

The findings demonstrate the significant analgesic and mood-modulating effects of the novel HO-1 inducers and DMF in a model of inflammatory pain. The rapid and potent effects of 1m suggest that it might be a superior therapeutic agent compared to DMF. The observed effects are likely mediated by the suppression of NLRP3 inflammasome activation, reducing inflammation and subsequent pain signaling. Furthermore, the activation of the Nrf2/HO-1/SOD-1 antioxidant pathway in the paw and HO-1 in the amygdala likely contributes to the analgesic and anxiolytic/antidepressant effects. The normalization of NLRP3 in the amygdala suggests that the mood-modulating effects might be, at least in part, due to the modulation of inflammation in brain regions involved in emotional processing. The differential effects on specific antioxidant enzymes (HO-1 and SOD-1) highlights the complexity of the Nrf2-mediated response to inflammatory pain.

This study demonstrates the analgesic and anxiolytic/antidepressant effects of three novel HO-1 inducers (1m, 1a, and 1b) and DMF in mice with CFA-induced inflammatory pain. These effects are likely linked to the suppression of NLRP3 inflammasome and activation of the Nrf2/HO-1/SOD-1 antioxidant pathway. 1m shows the most promise as a potential therapeutic agent for inflammatory pain and associated emotional disorders. Further research is needed to confirm these findings in larger samples, investigate sex differences, and explore the clinical translation of these promising compounds.

This study used only male mice, limiting the generalizability of findings to females. The study focused on a specific inflammatory pain model (CFA-induced), and the results may not be directly applicable to other pain conditions. More research is required to determine the long-term effects of these treatments and to investigate the optimal dosage and treatment duration for clinical applications. Finally, the mechanisms underlying the anxiolytic and antidepressant effects need to be further investigated.