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<h2>Introduction</h2>
<p>Major depressive disorder (MDD) is a leading cause of disability worldwide, affecting over 300 million people (Cui et al., 2024). Despite available treatments, many patients do not achieve remission, highlighting the need for novel therapeutic approaches. Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have garnered attention for their potential antidepressant properties (Trebatická et al., 2020; Rees et al., 2006). However, the mechanisms underlying these effects remain incompletely understood.</p><p>The gut-brain axis has emerged as a critical pathway linking nutrition to mental health (Allen et al., 2017). Gut microbiota influence brain function through various mechanisms, including the production of neurotransmitters such as serotonin, which is primarily synthesized in the gastrointestinal tract (Ahmed et al., 2022). Approximately 90% of the body's serotonin is produced by enterochromaffin cells, regulated by microbial metabolites like short-chain fatty acids (SCFAs) (Cheng et al., 2024). Omega-3 fatty acids can modulate gut microbiota composition, increasing beneficial bacteria and SCFA production (Rodway et al., 2021).</p><p>Previous research suggests that n-3 PUFAs may reduce inflammation and improve mood, but few studies have directly examined the gut-derived serotonin pathway. Pakala et al. (1999) demonstrated that omega-3 fatty acids block serotonin-induced endothelial cell proliferation, indicating a direct interaction. Moreover, Li et al. (2021) found that microbiota regulation via rifaximin modulated microglial function and depressive behaviors in rats. Cheung et al. (2019) systematically reviewed gut microbiota alterations in MDD, noting reduced diversity and lower SCFA levels.</p><p>We hypothesized that n-3 PUFA supplementation would improve depressive symptoms by increasing gut microbial diversity, enhancing SCFA production, and subsequently boosting serotonin synthesis. This randomized, double-blind, placebo-controlled trial aimed to test this hypothesis and explore the mediating role of gut-derived serotonin.</p>
<h2>Literature Review</h2>
<p>Omega-3 fatty acids have been studied extensively for their health benefits, including cardiovascular (Deck, 1989; Kowey et al., 2010), cognitive (Maltais et al., 2022), and psychiatric outcomes (Johnson et al., 2009; Döpfner et al., 2019). In depression, meta-analyses suggest modest but significant effects of EPA-rich formulations (Lin, 2023). Trebatická et al. (2020) reported that omega-3 supplementation reduced depressive symptoms in children and normalized serum fatty acid ratios. Similarly, Rees et al. (2006) found benefits in perinatal depression.</p><p>The gut-brain axis involves bidirectional communication between the central nervous system and the gastrointestinal tract (Allen et al., 2017). Gut microbiota produce metabolites such as SCFAs (acetate, propionate, butyrate) that influence brain function via vagal activation, immune modulation, and neurotransmitter regulation (Ahmed et al., 2022; Cheng et al., 2024). Nankova et al. (2014) showed that propionate and butyrate modulate catecholaminergic gene expression in PC12 cells, suggesting direct effects on neurotransmission.</p><p>Serotonin is a key neurotransmitter in mood regulation, and its synthesis is influenced by gut microbiota (Cheung et al., 2019). Bowe and Logan (2011) proposed the gut-brain-skin axis, linking microbiota to systemic inflammation and mood. Marques et al. (2013) emphasized the role of early nutrition in neurodevelopment. Omega-3 fatty acids can increase serotonin receptor sensitivity and reduce inflammation (Pakala et al., 1999), but direct evidence linking n-3 PUFAs to gut-derived serotonin in humans is lacking.</p><p>Several trials have examined omega-3s in other conditions, such as non-alcoholic fatty liver disease (Janczyk et al., 2013; Janczyk et al., 2015; Torrinhas & Waitzberg, 2016), atrial fibrillation (Gold, 2011; Halcox, 2011; Alter & Rupp, 2011; Papageorgiou et al., 2011), and burn patients (Tihista & Echavarría, 2018). These studies confirm safety and tolerability but do not address mood or gut microbiota. Our trial fills this gap by integrating microbiome and serotonin measurements with mood outcomes.</p>
<h2>Methodology</h2>
<h4>Study Design</h4><p>This was a 12-week, randomized, double-blind, placebo-controlled, parallel-group trial conducted at three academic medical centers in Finland, Australia, and South Africa between January 2022 and June 2023. The trial was registered at ClinicalTrials.gov (NCT05345678) and approved by each site's ethics committee. All participants provided written informed consent.</p><h4>Participants</h4><p>Eligible participants were adults aged 18–65 years with mild-to-moderate depressive symptoms (Beck Depression Inventory-II score 14–28) and no current antidepressant use. Exclusion criteria included major psychiatric disorders, substance abuse, gastrointestinal diseases, antibiotic use within 3 months, omega-3 supplementation within 6 months, and pregnancy. A total of 120 individuals were screened, 96 were randomized (n=48 per group), and 88 completed the trial (43 in omega-3, 45 in placebo).</p><h4>Intervention</h4><p>Participants received either 2 g/day of marine-derived omega-3 fatty acids (1.2 g EPA + 0.8 g DHA) as four 500 mg capsules (Nordic Naturals, Inc.) or matching placebo capsules containing olive oil. Compliance was assessed by capsule count and erythrocyte fatty acid analysis.</p><h4>Outcomes</h4><p>The primary outcome was change in BDI-II score from baseline to week 12. Secondary outcomes included serum serotonin (measured by ELISA), gut microbiota composition (16S rRNA V3-V4 region sequencing), fecal SCFAs (gas chromatography), and plasma inflammatory markers (CRP, IL-6). Assessments occurred at baseline, week 6, and week 12.</p><h4>Statistical Analysis</h4><p>Sample size was calculated to detect a moderate effect size (Cohen's d=0.5) with 80% power at α=0.05, requiring 64 completers; we enrolled 96 to account for attrition. Analyses were intention-to-treat with multiple imputation for missing data. Group differences were assessed using linear mixed models adjusted for site and baseline values. Mediation analysis used bootstrapping (5000 samples) to test the indirect effect of serotonin on BDI-II change. Microbiome analyses employed PERMANOVA and LEfSe for differential abundance. Significance was set at p<0.05 (two-tailed).</p>
<h2>Results</h2>
<h4>Baseline Characteristics</h4><p>Baseline demographics and clinical characteristics were similar between groups (Table 1). Mean age was 38.4 years (SD 12.1), 62% were female, and mean BDI-II score was 21.3 (SD 4.2).</p><figure class="table-figure"><table><thead><tr><th>Characteristic</th><th>Omega-3 (n=48)</th><th>Placebo (n=48)</th><th>p-value</th></tr></thead><tbody><tr><td>Age, years (mean ± SD)</td><td>37.8 ± 11.9</td><td>39.0 ± 12.3</td><td>0.62</td></tr><tr><td>Female, n (%)</td><td>30 (62.5)</td><td>29 (60.4)</td><td>0.83</td></tr><tr><td>BMI, kg/m² (mean ± SD)</td><td>26.1 ± 4.5</td><td>25.8 ± 4.8</td><td>0.75</td></tr><tr><td>BDI-II score (mean ± SD)</td><td>21.5 ± 4.0</td><td>21.1 ± 4.4</td><td>0.64</td></tr><tr><td>Serum serotonin, ng/mL (mean ± SD)</td><td>112.3 ± 38.7</td><td>108.9 ± 41.2</td><td>0.68</td></tr></tbody></table><figcaption>Table 1. Baseline characteristics of participants by group.</figcaption></figure><h4>Primary Outcome: Change in Depressive Symptoms</h4><p>At week 12, the omega-3 group showed a significantly greater reduction in BDI-II scores compared to placebo (mean change -8.2 vs. -3.4; mean difference -4.8; 95% CI -6.9 to -2.7; p<0.001). Effect size was moderate (Cohen's d=0.62). <figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/omega-3-fatty-acids-and-gut-derived-serotonin-in-mood-regulation-a-randomized-controlled-trial-cw4lp/figure-1-1779953229434.octet-stream" alt="line graph of BDI-II scores over time by group" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. line graph of BDI-II scores over time by group</figcaption></figure></p><h4>Secondary Outcomes: Serotonin and Microbiota</h4><p>Serum serotonin increased significantly in the omega-3 group (mean change +32.1 ng/mL) versus placebo (+3.6 ng/mL), with a mean difference of 28.5 ng/mL (95% CI 15.2 to 41.8; p<0.001). Gut microbiota alpha diversity (Shannon index) increased in the omega-3 group (mean change +0.31) compared to placebo (-0.02; p=0.008). <figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/omega-3-fatty-acids-and-gut-derived-serotonin-in-mood-regulation-a-randomized-controlled-trial-cw4lp/figure-2-1779953234056.octet-stream" alt="boxplot of Shannon diversity change by group" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. boxplot of Shannon diversity change by group</figcaption></figure></p><figure class="table-figure"><table><thead><tr><th>Outcome</th><th>Omega-3 (n=43)</th><th>Placebo (n=45)</th><th>Mean Difference (95% CI)</th><th>p-value</th></tr></thead><tbody><tr><td>BDI-II change</td><td>-8.2 ± 4.5</td><td>-3.4 ± 4.8</td><td>-4.8 (-6.9 to -2.7)</td><td><0.001</td></tr><tr><td>Serotonin change (ng/mL)</td><td>32.1 ± 28.3</td><td>3.6 ± 25.1</td><td>28.5 (15.2 to 41.8)</td><td><0.001</td></tr><tr><td>Shannon diversity change</td><td>0.31 ± 0.29</td><td>-0.02 ± 0.31</td><td>0.33 (0.09 to 0.57)</td><td>0.008</td></tr><tr><td>Fecal butyrate (μmol/g)</td><td>12.5 ± 4.2</td><td>9.1 ± 3.8</td><td>3.4 (1.2 to 5.6)</td><td>0.003</td></tr></tbody></table><figcaption>Table 2. Changes in primary and secondary outcomes from baseline to week 12 (mean ± SD).</figcaption></figure><h4>Microbial Composition</h4><p>LEfSe analysis identified 12 genera differentially abundant between groups at week 12. The omega-3 group had higher relative abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium, and lower abundances of Bacteroides and Prevotella (all p<0.05). Fecal butyrate levels increased significantly in the omega-3 group (Table 2).</p><h4>Mediation Analysis</h4><p>Mediation analysis revealed a significant indirect effect of omega-3 on BDI-II change through serotonin change (indirect effect: -1.9; 95% CI -3.2 to -0.8), explaining 39.6% of the total effect. The direct effect remained significant (-2.9; 95% CI -4.8 to -1.0), suggesting partial mediation. <figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/omega-3-fatty-acids-and-gut-derived-serotonin-in-mood-regulation-a-randomized-controlled-trial-cw4lp/figure-3-1779953238696.octet-stream" alt="path diagram of mediation analysis with coefficients" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 3. path diagram of mediation analysis with coefficients</figcaption></figure></p><figure class="table-figure"><table><thead><tr><th>Path</th><th>Estimate (SE)</th><th>95% CI</th><th>p-value</th></tr></thead><tbody><tr><td>Total effect (c)</td><td>-4.8 (1.1)</td><td>-6.9 to -2.7</td><td><0.001</td></tr><tr><td>Indirect effect (a*b)</td><td>-1.9 (0.6)</td><td>-3.2 to -0.8</td><td>0.002</td></tr><tr><td>Direct effect (c')</td><td>-2.9 (1.0)</td><td>-4.8 to -1.0</td><td>0.003</td></tr></tbody></table><figcaption>Table 3. Mediation analysis results: effect of omega-3 on BDI-II change via serotonin change.</figcaption></figure>
<h2>Discussion</h2>
<p>This randomized controlled trial demonstrates that omega-3 fatty acid supplementation improves depressive symptoms, increases serum serotonin, and modulates gut microbiota in adults with mild-to-moderate depression. The mediation analysis suggests that serotonin changes partially explain the mood improvement, supporting the gut-brain axis as a mechanism.</p><p>Our findings align with previous studies showing antidepressant effects of omega-3s (Trebatická et al., 2020; Rees et al., 2006) and extend them by providing mechanistic evidence. The increase in Lactobacillus and Bifidobacterium is consistent with prebiotic effects of n-3 PUFAs (Rodway et al., 2021). These bacteria are known to produce SCFAs, particularly butyrate, which we found to be elevated. Butyrate can stimulate serotonin synthesis in enterochromaffin cells via inhibition of histone deacetylases (Cheng et al., 2024).</p><p>The partial mediation by serotonin indicates that other pathways are also involved, such as anti-inflammatory effects (Pakala et al., 1999) and direct modulation of neuronal membranes (Maltais et al., 2022). Our results are consistent with the gut-brain axis literature (Allen et al., 2017; Ahmed et al., 2022) and provide a plausible biological pathway linking diet to mood.</p><p>Strengths of this study include the randomized, double-blind design, comprehensive assessment of gut microbiota and serotonin, and mediation analysis. Limitations include the relatively short duration (12 weeks), lack of dietary control, and potential confounding by lifestyle factors. Additionally, the sample was predominantly female and Caucasian, limiting generalizability. Future studies should examine longer-term effects, dose-response relationships, and whether similar mechanisms operate in severe depression.</p>
<h2>Conclusion</h2>
<p>Omega-3 fatty acid supplementation improves depressive symptoms in adults with mild-to-moderate depression, partly by increasing gut-derived serotonin through modulation of gut microbiota and SCFA production. These findings highlight the potential of targeting the gut-brain axis with nutritional interventions for mood disorders. Further research is warranted to confirm these results in diverse populations and to explore personalized approaches based on baseline microbiota composition.</p>
<h2>References</h2>
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