Quantitative Distribution of Theobromine in Mouse Brain Regions and Its Mitigation of Nicotine Withdrawal-Induced Anxiety and Depression through Neuroinflammation Suppression

Nicotine withdrawal is often accompanied by anxiety and depression-like behaviors, which are closely linked to neuroinflammation. Theobromine (TB), a bioactive compound found in cocoa, has demonstrated potential anti-inflammatory and neuroprotective properties. This study aimed to quantify the distribution of TB in various tissues-particularly brain regions involved in emotional regulation-and evaluate its efficacy in alleviating nicotine withdrawal-induced emotional disturbances in mice. We first developed a high-efficiency liquid chromatography-tandem mass spectrometry quantitative method for TB and found that its highest concentration was in the blood, followed by the liver, with the lowest levels in the brain. Within the various brain regions, the nucleus accumbens (NAc) exhibited the highest concentration of TB (41.64 ± 17.14 μg/g). Behavioral experiments revealed that TB significantly reduced anxiety and depression-like behaviors induced by nicotine withdrawal. Molecular analyses showed that TB markedly decreased the expression of proinflammatory cytokines (C-C motif chemokine ligand 4, C-C motif chemokine ligand 5, cyclooxygenase-2, toll-like receptor 4, and inducible nitric oxide synthase) in the NAc and lowered levels of interleukin-2, interleukin-6, and tumor necrosis factor-α in both the NAc and serum. In summary, by optimizing liquid chromatography-tandem mass spectrometry technology, we discovered for the first time, to our knowledge, that TB is distributed across nearly all brain regions, with the highest concentration in the NAc. This provides a pharmacological basis for its neuroprotective and mood-regulating effects. TB effectively mitigates anxiety and depression-like behaviors associated with nicotine withdrawal in mice, likely through its anti-inflammatory actions in the NAc and systemic circulation. This study offers compelling evidence for TB as a natural anti-inflammatory and mood-regulating agent, laying the groundwork for developing novel adjunctive treatments for nicotine withdrawal. SIGNIFICANCE STATEMENT: To the authors' knowledge, this study provides the first comprehensive mapping of theobromine (TB) distribution in the brain, specifically focusing on its interactions with common withdrawal and addiction-related pathways, identifying its highest concentration in the nucleus accumbens-a key region for emotional regulation. Demonstrating that TB effectively alleviates nicotine withdrawal-induced anxiety and depression-like behaviors by suppressing neuroinflammation, the findings of this study support the development of TB-based therapies, presenting a novel strategy to aid individuals in overcoming nicotine dependence.