Pediococcus acidilactici CCFM6432 alleviates anhedonia in major depression through immune-inflammatory modulation: An extended trial analysis

Abstract

BACKGROUND

Anhedonia in major depressive disorder remains therapeutically challenging. Building on our prior randomized evidence of clinical benefit with Pediococcus acidilactici CCFM6432, we further investigated candidate peripheral immune and central reward correlates of treatment response.

AIM

To evaluate whether add-on CCFM6432 alleviates anhedonia through immune-inflammatory modulation and changes in reward-related electrophysiological measures.

METHODS

Adults with major depressive disorder and anhedonia received standard antidepressant therapy plus CCFM6432 or plus placebo for 30 days. Assessments comprised Hamilton Depression Scale; Temporal Experience of Pleasure Scale (total, anticipatory, consummatory); event-related potentials indexing reward anticipation and feedback - stimulus-preceding negativity and feedback-related negativity; and a 13-marker peripheral panel (immune-inflammatory, neurotrophic, neurotransmitter-related). Of 92 screened, 71 were randomized; 55 completed (CCFM6432 group: n = 27; Placebo group: n = 28).

RESULTS

CCFM6432 produced greater reductions in lipopolysaccharide, C-reactive protein, and interleukin-6 vs placebo (P < 0.05). Decreases in these markers were aligned with improvements in the Temporal Experience of Pleasure Scale (total and anticipatory subscales) and with increased stimulus-preceding negativity amplitude, although the latter association attenuated after adjustment for depressive and anxiety symptom changes. No between-group differences were observed for neurotrophic or neurotransmitter-related measures, and feedback-related negativity showed no treatment-related effects.

CONCLUSION

These findings provide preliminary clinical support that immune-inflammatory modulation may contribute to the anhedonia-relieving effects of CCFM6432, particularly in reward anticipation. Larger multicenter studies with multimodal endpoints are warranted to confirm these results and elucidate mechanistic pathways.

Key Words: Anhedonia; Major depressive disorder; Probiotics; Inflammatory markers; Event-related potentials

Core Tip: This study investigated whether Pediococcus acidilactici CCFM6432 alleviates anhedonia in major depressive disorder through immune-inflammatory modulation and reward-related electrophysiological changes. The probiotic significantly reduced inflammatory markers (lipopolysaccharide, C-reactive protein, and interleukin-6) and enhanced reward anticipation, as reflected by increased stimulus-preceding negativity amplitude and higher Temporal Experience of Pleasure Scale scores. These results suggest that CCFM6432 may improve anhedonia via an immune-inflammatory pathway. Large-scale, multicenter studies integrating multimodal biomarkers are warranted to validate and extend these findings.

INTRODUCTION

Major depressive disorder (MDD) is a highly prevalent and disabling psychiatric condition, with core symptoms including depressed mood and anhedonia[1]. Anhedonia is commonly defined as a marked reduction in the ability to experience pleasure or motivation, corresponding to consummatory (CON) and anticipatory (ANT) dimensions, respectively[2,3]. Anhedonia is highly prevalent in MDD, affecting approximately 67%-70% of patients, as reported in recent epidemiological studies[3-5]. This symptom is not only widespread but also frequently persists as a residual feature, exerting detrimental effects on treatment response, social functioning, and prognosis[6-8]. However, conventional antidepressant therapies show limited efficacy in ameliorating anhedonia and are closely associated with treatment resistance, unfavorable outcomes, and an elevated risk of suicide[9,10]. Thus, anhedonia may involve relatively independent pathophysiological mechanisms and should be prioritized as a therapeutic target.

In recent years, the inflammation hypothesis has gained traction as a key explanation for anhedonia. A substantial body of research indicates that elevated levels of peripheral inflammatory markers [e.g., interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)] are strongly associated with the severity of anhedonia[11-15]. Experimental studies further suggest that exogenous inflammatory stimuli [e.g., lipopolysaccharide (LPS) or cytokine administration] can induce depression-like behaviors and anhedonic phenotypes in both animals and healthy humans[13,16]. In clinical studies, heightened inflammatory responses have been closely linked to the onset, progression, and poor antidepressant response of anhedonia[12,17-20]. Moreover, alterations in brain-derived neurotrophic factor (BDNF), its precursors and receptors, as well as metabolic disturbances in neurotransmitter-related pathways [e.g., tryptophan (Trp), dopamine (DA)], have also been implicated in reward dysfunction[12,15,21-24]. Nevertheless, which category of molecular markers across these distinct pathways is most closely related to improvements in anhedonia remains insufficiently validated.

The gut-brain axis, recognized as an important pathway for immune regulation and the pathogenesis of mood disorders[25,26], has attracted increasing attention. Evidence suggests that probiotics may ameliorate depression-related symptoms by improving intestinal barrier integrity, reducing serum LPS levels, and downregulating pro-inflammatory signaling[27,28]. Our previous randomized controlled trial (RCT) demonstrated that adjunctive administration of Pediococcus acidilactici CCFM6432 significantly improved anhedonia in MDD patients, particularly ANT anhedonia, accompanied by enhanced stimulus-preceding negativity (SPN) amplitudes[29]. Complementary animal experiments further revealed that CCFM6432 could enhance the abundance of short-chain fatty acid-producing bacteria, repair intestinal barrier function, and suppress central pro-inflammatory signaling pathways, thereby improving depression-like behaviors[30]. Collectively, these findings suggest that the beneficial effects of CCFM6432 on anhedonia may be closely mediated by gut-brain axis-related immune and inflammatory regulation.

Against this background, the present study conducted an extended analysis based on our prior RCT. We systematically assessed peripheral inflammatory markers [IL-6, IL-1β, TNF-α, CRP, LPS, toll-like receptor-4 (TLR-4), nuclear factor kappaB (NF-κB), nucleotide-binding oligomerization domain 2 (NOD-2)], neurotrophic factors and related proteins [BDNF, Pro-BDNF, tropomyosin related kinase B (TrkB)], as well as neurotransmitter-related indicators (Trp, DA), and integrated these biomarkers with subjective scales [Temporal Experience of Pleasure Scale (TEPS)] and objective electrophysiological indices [SPN, feedback related negativity (FRN)] to evaluate their associations with improvements in anhedonia. The novelty of this study lies in three aspects: (1) For the first time within the RCT framework, multiple categories of peripheral molecular markers were simultaneously measured to comprehensively assess the potential pathways of CCFM6432; (2) By comparing results across pathways, we identified that inflammatory factors were most closely associated with improvements in anhedonia; and (3) By integrating clinical symptomatology, electrophysiological measures, and peripheral blood molecular markers, we established a multimodal chain of evidence, strengthening the proposed role of gut-brain axis-mediated inflammatory mechanisms in anhedonia and providing new clinical and translational insights for precision interventions in MDD.

MATERIALS AND METHODS

Study design

This study was an extended analysis of our previous randomized, double-blind, placebo-controlled clinical trial[29]. In that trial, we reported the beneficial effects of CCFM6432 on anhedonia symptoms and event-related potentials in patients with MDD. The present study further evaluated hematological indicators and their associations with improvements in anhedonia in the same cohort. The trial was conducted from March 2023 to June 2024 at the Affiliated Mental Health Center of Jiangnan University (Wuxi Mental Health Center). The study protocol was approved by the Ethics Committee of the Affiliated Mental Health Center of Jiangnan University (No. WXMHCIRB2023 LLky030) and registered in the Chinese Clinical Trial Registry (No. ChiCTR2400093687). Written informed consent was obtained from all participants.

Participants

In the previous RCT[29], 71 patients completed baseline assessments and were randomly allocated to the CCFM6432 group (n = 36) or the placebo group (n = 35). During follow-up, 16 patients dropped out due to medication adjustments, modified electroconvulsive therapy, antibiotic use, or poor compliance, resulting in incomplete clinical and electroencephalography (EEG) data. Ultimately, 55 patients (27 in the CCFM6432 group and 28 in the placebo group) completed follow-up and provided full clinical and EEG datasets. Although blood samples were collected from all enrolled participants at baseline, laboratory assays of hematological biomarkers were only performed for those who completed follow-up with complete datasets (n = 55) due to funding constraints in this extension analysis. The full recruitment and allocation procedure has been detailed in the original report[29] and is not repeated here.

Interventions

In the original RCT, participants were randomly assigned in a 1:1 ratio to two groups. The CCFM6432 group received standard antidepressant treatment plus daily supplementation with CCFM6432 (≥ 10⁹ colony-forming units/sachet; developed by the School of Food Science and Technology, Jiangnan University, and manufactured by the Yangzhou Institute of Food Biotechnology, Jiangnan University) for 30 consecutive days. The placebo group received standard antidepressant treatment plus a maltodextrin-based placebo identical to CCFM6432 in appearance, taste, color, and weight. No changes were made to the participants’ prescribed antidepressant regimens during the intervention. Treatment adherence was monitored and recorded by nurses (for inpatients) or family members (for outpatients). This study analyzed data from the 55 participants who completed follow-up in the RCT.

Clinical and EEG assessments

Depressive symptoms were assessed using the 17-item Hamilton Depression Rating Scale (HAMD-17), anxiety was assessed with the Hamilton Anxiety Rating Scale (HAMA), and anhedonia was evaluated with the TEPS, which includes ANT and CON subscales. EEG recordings during the “Doors Guessing Task” were used to extract SPN and FRN components, reflecting reward anticipation and feedback processing, respectively. Detailed procedures have been described in our previous report[29].

Hematological measurements

Peripheral venous blood (5 mL) was collected from each participant after an overnight fast at baseline and after 30 days of intervention. Samples were processed immediately by centrifugation at 3000 rpm for 10 minutes at 4 °C; sera were aliquoted and stored at -80 °C until analysis. To avoid batch effects, all samples were tested in a single batch at a third-party laboratory (Nanjing Lapuda Biotechnology Co., Ltd., Nanjing, China). Serum biomarkers were quantified using sandwich Enzyme-linked immunosorbent assay (ELISA) kits (brand: LAPUDA^®; manufacturer: Nanjing Lapuda Biotechnology Co., Ltd., Nanjing, China; catalog and lot numbers listed in Supplementary Table 1) according to the manufacturer’s instructions. We assayed 13 prespecified indicators: Inflammatory factors (IL-6, IL-1β, TNF-α, CRP, LPS, TLR-4, NF-κB, NOD-2); neurotrophic factors and related proteins (BDNF, Pro-BDNF, TrkB); and neurotransmitter-related markers (DA, Trp). These markers were selected based on prior studies, covering three levels - inflammation, neurotrophic regulation, and reward pathways - with the aim of exploring the potential mechanisms of CCFM6432 in a multidimensional framework.

Statistical analysis

All analyses were performed using SPSS version 27.0 (IBM Corp., Armonk, NY, United States). The normality of continuous variables was assessed with the Shapiro-Wilk test. For normally distributed data, independent-samples t tests were used for between-group comparisons; for non-normally distributed data, the Mann-Whitney U test was applied. Categorical variables were analyzed using the χ² test. For hematological indicators, analysis of covariance (ANCOVA) was adopted as the primary analytical approach, with baseline values entered as covariates and follow-up values as dependent variables; partial eta squared (ηp²) was reported as the effect size. Change scores (Δ = baseline - follow-up) were calculated and compared between groups as supplementary analyses. To examine concurrent changes in clinical and biological measures, Pearson or partial correlation analyses were conducted using Δ values. Partial correlations were applied to control for changes in depressive and anxiety symptom severity (ΔHAMD and ΔHAMA), thereby accounting for potential confounding by overall symptom improvement. This approach allowed us to explore associations between reductions in inflammatory markers (CRP, IL-6, LPS) and improvements in anhedonia (TEPS total and subscale scores) as well as EEG indices (SPN and FRN). A two-tailed P value < 0.05 was considered statistically significant.

RESULTS

In the initial screening of the previous RCT, 92 patients were assessed, of whom 10 were excluded for not meeting the inclusion criteria, and 11 declined participation. The remaining 71 patients completed baseline assessments and were randomly allocated to the CCFM6432 group (n = 36) or the placebo group (n = 35). During follow-up, 16 patients dropped out due to medication adjustments, receipt of modified electroconvulsive therapy, antibiotic use, or poor compliance. Ultimately, 55 patients (27 in the CCFM6432 group and 28 in the placebo group) completed follow-up and were included in the present analysis. At baseline, there were no significant between-group differences in demographic or clinical characteristics, including sex, age, body mass index, years of education, illness duration, number of episodes, or antidepressant dosage (all P > 0.05), indicating comparability between groups. The complete baseline characteristics were reported in the previous RCT[29].

With respect to hematological indicators, ANCOVA revealed that after 30 days of intervention, peripheral blood levels of LPS, CRP, and IL-6 were significantly reduced in the CCFM6432 group, with greater decreases than in the placebo group (LPS: F = 6.705, P = 0.012, ηp² = 0.114; IL-6: F = 8.672, P = 0.005, ηp² = 0.143; CRP: F = 11.162, P = 0.002, ηp² = 0.177). No significant between-group differences were observed for the other inflammatory markers (IL-1β, TNF-α, TLR-4, NF-κB, NOD-2). Similarly, no significant group differences were detected for neurotrophic factors and related proteins (BDNF, pro-BDNF, TrkB) or neurotransmitter-related markers (DA, Trp) (Table 1). As a supplementary exploratory sensitivity analysis, we also compared unadjusted change scores. Consistent with the ANCOVA results, LPS showed a significantly greater reduction in the CCFM6432 group, whereas CRP and IL-6 did not reach significance under this less statistically efficient approach (Figure 1). Importantly, the direction of effects remained consistent across both analytical strategies, indicating that the inflammatory findings are robust in their overall pattern despite differences in statistical power.

Figure 1 Comparison of concentration changes in blood biomarker levels between the two groups. This figure summarizes concentration changes in blood biomarker levels between the two groups. ^bP < 0.05 indicates a significant between-group difference (independent-samples t-test). Concentration change = baseline value - follow-up value. BDNF: Brain-derived neurotrophic factor; Pro-BDNF: Pro-brain-derived neurotrophic factor; TrkB: Tropomyosin-related kinase B; DA: Dopamine; Trp: Tryptophan; IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; TLR-4: Toll-like receptor 4; NF-κB: Nuclear factor kappa B; NOD-2: Nucleotide-binding oligomerization domain-containing protein 2; LPS: Lipopolysaccharide; CRP: C-reactive protein; IL-6: Interleukin-6.

Table 1 Blood biomarker levels at baseline and follow-up for the two groups, mean ± SD.

Variable	CCFM6432 baseline	CCFM6432 follow-up	Placebo baseline	Placebo follow-up	F value	P value	ηp2
BDNF (ng/mL)	7.82 ± 3.48	10.53 ± 2.45	7.12 ± 3.49	10.87 ± 3.25	0.163	0.688	0.003
Pro-BDNF (pg/mL)	70.39 ± 22.98	51.56 ± 19.68	69.34 ± 24.66	57.70 ± 27.10	0.900	0.347	0.017
TrkB (ng/mL)	0.42 ± 0.27	0.67 ± 0.24	0.43 ± 0.19	0.66 ± 0.22	0.015	0.902	0.000
DA (pg/mL)	258.55 ± 72.95	221.03 ± 113.67	271.09 ± 86.52	261.96 ± 92.65	1.829	0.182	0.034
Trp (μg/mL)	12.99 ± 3.20	17.56 ± 5.46	12.17 ± 4.47	17.34 ± 8.07	0.000	0.999	0.000
IL-1β (pg/mL)	345.27 ± 177.58	169.06 ± 92.36	339.67 ± 138.30	150.20 ± 75.87	0.658	0.421	0.012
TNF-α (pg/mL)	289.41 ± 97.85	196.58 ± 65.27	327.21 ± 123.55	214.90 ± 115.88	0.404	0.528	0.008
TLR-4 (ng/mL)	5.44 ± 2.31	2.77 ± 1.28	6.03 ± 3.35	3.25 ± 1.71	1.019	0.317	0.019
NF-κB (ng/mL)	2.18 ± 0.75	1.31 ± 0.67	2.47 ± 0.85	1.25 ± 0.88	0.058	0.810	0.001
NOD-2 (ng/mL)	1.68 ± 0.62	0.93 ± 0.45	1.65 ± 0.52	0.90 ± 0.33	0.121	0.730	0.002
LPS (ng/mL)	74.05 ± 30.69	27.89 ± 18.15	68.67 ± 21.99	44.11 ± 27.44	6.705	0.012a	0.114
IL-6 (pg/mL)	148.66 ± 55.29	67.18 ± 18.32	144.03 ± 47.59	80.02 ± 23.09	8.672	0.005a	0.143
CRP (ng/mL)	597.79 ± 314.75	160.08 ± 42.55	543.70 ± 277.00	187.12 ± 47.45	11.162	0.002a	0.177

^aP values indicates a statistically significant difference between groups.

P values represent group differences at day 30 assessed by analysis of covariance (ANCOVA) adjusted for baseline values. All values are presented as mean ± SD. BDNF: Brain-derived neurotrophic factor; CRP: C-reactive protein; DA: Dopamine; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; LPS: Lipopolysaccharide; NF-κB: Nuclear factor kappa B; NOD-2: Nucleotide-binding oligomerization domain-containing protein 2; Pro-BDNF: Pro-brain-derived neurotrophic factor; TLR-4: Toll-like receptor 4; TNF-α: Tumor necrosis factor-alpha; TrkB: Tropomyosin-related kinase B; Trp: Tryptophan.

At baseline, correlation analyses showed that TEPS total scores and ANT subscale scores in MDD patients were negatively correlated with CRP levels (r approximately -0.29 to -0.33), indicating a close relationship between inflammatory status and anhedonia symptoms (Figure 2A). In post-intervention correlation analyses (calculated as baseline minus follow-up values), improvements in TEPS total and ANT subscale scores in the CCFM6432 group were significantly negatively correlated with reductions in CRP and IL-6 (r = -0.48 to -0.52, P < 0.05), meaning that greater decreases in inflammatory markers were associated with greater improvements in anhedonia. Importantly, these correlations remained significant after controlling for changes in depressive and anxiety symptoms (ΔHAMD and ΔHAMA) (r = -0.43 to -0.51, P < 0.05), suggesting that the effect was independent of overall mood improvement. In contrast, although the placebo group showed a similar negative trend, the correlations did not reach statistical significance (Figure 2B-E).

Figure 2 Correlation analyses of baseline and change values. This figure summarizes the Pearson and partial correlation analyses conducted on baseline variables and change values in both the CCFM6432 and placebo groups. Change value = baseline value - follow-up value. A: Pearson correlation analysis of baseline variables in the two groups. ^cP < 0.05; ^dP < 0.01; ^eP < 0.001; ^fP < 0.0001; B: Pearson correlation analysis of change values in the CCFM6432 group. ^gP < 0.05; ^hP < 0.01; ⁱP < 0.001; ^jP < 0.0001; C: Partial correlation analysis of change values in the CCFM6432 group controlling for changes in Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale. ^gP < 0.05; ^hP < 0.01; ⁱP < 0.001; ^jP < 0.0001; D: Pearson correlation analysis of change values in the placebo group. ^kP < 0.05; ^lP < 0.01; ^mP < 0.001; ⁿP < 0.0001; E: Partial correlation analysis of change values in the placebo group controlling for changes in Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale. ^kP < 0.05; ^lP < 0.01; ^mP < 0.001; ⁿP < 0.0001. TEPS: Temporal Experience of Pleasure Scale; ANT: Anticipatory; CON: Consummatory; CRP: C-reactive protein; IL-6: Interleukin-6; LPS: Lipopolysaccharide; SPN: Stimulus-preceding negativity; FRN: Feedback-related negativity.

Regarding EEG indices, the primary focus was the relationship between SPN and hematological markers. In the CCFM6432 group, the reduction in LPS was significantly positively correlated with improvements in SPN amplitude (r = 0.39, P < 0.05), indicating that greater reductions in LPS were associated with enhanced reward anticipation–related neural activity. Although this correlation attenuated and was no longer statistically significant after adjusting for changes in depressive and anxiety symptoms (r = 0.235), the directional trend remained. No such relationship was observed in the placebo group. No significant associations were found between FRN and inflammatory markers. Taken together, the CCFM6432 intervention significantly reduced peripheral blood levels of LPS, CRP, and IL-6 in patients with MDD. Furthermore, reductions in inflammatory markers were closely associated with improvements in anhedonia - particularly ANT pleasure - as well as with enhanced SPN amplitude. These findings suggest that among the multiple pathways assessed, inflammatory markers showed the strongest associations with improvements in anhedonia, supporting a potentially central role of inflammatory mechanisms.

DISCUSSION

As an extension of our previous RCT, this study is the first to simultaneously examine inflammatory factors, neurotrophic factors, and neurotransmitter-related markers in order to explore the peripheral molecular correlates underlying improvements in anhedonia following CCFM6432 intervention. Our results demonstrated that, although multiple pathways were assessed, only reductions in inflammatory markers (LPS, CRP, IL-6) were consistently associated with improvements in anhedonia. In addition, decreases in LPS were aligned with enhanced SPN amplitudes. These findings suggest that inflammatory pathways may play a key role in the alleviation of anhedonia by CCFM6432, providing new clinical evidence that probiotics may exert therapeutic effects primarily through immune-inflammatory regulation. Given the exploratory design and limited sample size, these findings should be considered preliminary and require replication in larger studies before firm conclusions can be drawn.

Previous research has shown that exogenous inflammatory stimuli (e.g., LPS or cytokine administration) can induce depression-like behaviors and anhedonic experiences in animals and healthy individuals[13,16]. In human studies, elevated inflammatory levels in patients with MDD have been closely linked to the severity of anhedonia[12,14,17,18]. Importantly, a recent RCT in MDD patients demonstrated that an acute LPS challenge induces rapid worsening of anhedonia, particularly among individuals with elevated baseline CRP levels[31]. This provides clinical evidence supporting a potential causal link between systemic inflammation and anhedonic symptoms in depression, complementing findings from animal and endotoxin-challenge studies. Mechanistically, inflammation-induced dopaminergic inhibition, kynurenine pathway activation, and reductions in BDNF signaling may converge to disrupt reward circuitry and synaptic plasticity[12,15,23]. Consistent with this framework, our findings demonstrate that CCFM6432 significantly reduced CRP and IL-6 levels, and these reductions were associated with improvements in ANT anhedonia, suggesting that immune-inflammatory modulation may represent a key pathway through which this probiotic exerts its therapeutic effects. This aligns with accumulating evidence indicating that anti-inflammatory interventions can improve depressive symptoms, including anhedonia[32].

Notably, the association between inflammation reduction and improvements in ANT pleasure was more pronounced than that for CON pleasure. This aligns with recent findings that elevated inflammation is more strongly related to ANT deficits than to CON[11,12,17]. For example, patients with higher CRP or IL-6 levels are more likely to show impairments in ANT pleasure rather than CON pleasure[17]. This may be attributable to the greater impact of inflammation on neural circuits involved in reward anticipation, such as the anterior cingulate cortex and ventral striatum, compared to circuits subserving immediate hedonic responses[12,17]. Thus, our study highlights the importance of differentiating anhedonia subtypes in future clinical and mechanistic research to identify specific pathological substrates and treatment targets.

This study also demonstrated that reductions in LPS were associated with enhanced SPN amplitudes, suggesting a potential cross-level association between peripheral inflammation and central reward processing. SPN, an event-related potential component reflecting reward anticipation, is consistently reported to be blunted in patients with MDD[33] and correlates with symptom severity. While our findings align with a “peripheral inflammation-central reward processing” framework, they remain correlational and cannot establish causality. Nevertheless, these results indicate that peripheral inflammatory markers may serve as external correlates of reward-related neural dysfunction, underscoring the need for future longitudinal and mechanistic studies. Moreover, associations between inflammatory improvements and reward anticipation (SPN and TEPS-ANT) were more prominent than those with CON reward processing (FRN and TEPS-CON). This pattern is consistent with evidence that inflammation preferentially disrupts motivational aspects of reward processing[12]. Future studies with larger samples and paradigms optimized for both anticipation and consummation will be essential to delineate subtype-specific neural and immune mechanisms.

Meanwhile, some indicators (e.g., IL-1β, TNF-α, BDNF, TrkB, DA, and Trp) did not show significant changes after intervention. This suggests that the effects of CCFM6432 may be primarily concentrated on specific aspects of immune-inflammatory responses (CRP, IL-6, LPS), rather than generalized inflammatory changes or the neurotransmitter/neurotrophic factors measured here. Previous animal studies have demonstrated that CCFM6432 downregulated hippocampal expression of TLR-4, NF-κB, TNF-α, and IL-1β, suggesting central anti-inflammatory effects[30]. The discrepancy between those findings and our results may be due to differences in study populations (humans vs mice) and biological samples (peripheral blood vs central tissue). In addition, methodological differences and the concomitant use of antidepressant medications in our clinical sample may have contributed. Thus, our findings should be interpreted with caution and require further validation using cerebrospinal fluid assays, neuroimaging, and multi-omics approaches.

From a clinical perspective, this study has several important implications. First, baseline analyses confirmed that inflammatory levels were closely correlated with the severity of anhedonia, suggesting that peripheral inflammatory markers may serve as potential biomarkers. Second, the improvements in anhedonia with CCFM6432 were partly independent of changes in depressive and anxiety symptoms, underscoring the necessity of targeting anhedonia as a distinct therapeutic goal. Third, by integrating hematological indicators and EEG measures, this study provides empirical support for cross-level mechanisms linking peripheral inflammation, central neural activity, and clinical symptoms.

Several limitations should be acknowledged. First, the sample size was modest and derived from a single center, which may limit generalizability and constrain the feasibility of advanced multivariate modeling. Second, hematological biomarkers were collected only at baseline and day 30, limiting the ability to characterize inflammatory trajectories; future studies with denser sampling and longitudinal modeling are warranted. Third, this extension analysis was not originally powered for biomarker outcomes; therefore, multiple comparison correction was not applied, and these findings should be interpreted as preliminary and hypothesis-generating. Fourth, peripheral inflammatory markers cannot fully index central neuroimmune processes (e.g., functional magnetic resonance imaging), and EEG-biomarker associations were based solely on scalp recordings. Future multimodal investigations incorporating neuroimaging, cerebrospinal fluid cytokines, and advanced EEG analyses (e.g., source localization, topographical characterization) are needed to more definitively evaluate the inflammation-reward circuitry. Fifth, the study did not collect fecal or metabolomic samples due to the constraints of the original trial protocol; inclusion of microbiome profiling and gut-derived metabolites (e.g., short-chain fatty acids, Trp-kynurenine markers) will be essential to elucidate gut-brain pathway involvement. Sixth, hematological assays were unavailable for dropouts; although baseline demographic and clinical characteristics were comparable according to data from the same cohort, attrition bias cannot be entirely excluded. Finally, while randomization helped balance antidepressant use between groups, concomitant medication effects cannot be fully ruled out; stratified or multi-arm designs (probiotic alone vs antidepressant alone vs combination therapy) will help isolate treatment-specific mechanisms in future work.

Taken together, this study demonstrates that Pediococcus acidilactici CCFM6432 effectively alleviates anhedonia in patients with MDD by downregulating peripheral inflammatory markers, particularly LPS, CRP, and IL-6. The alignment between decreases in inflammation and enhancements in SPN suggests that gut-brain axis inflammation regulation may represent a key mechanism. These findings not only provide new evidence for the pathophysiology of anhedonia but also highlight probiotics as a potential clinical strategy for precision interventions, warranting replication and extension in larger, multicenter, and multimodal studies.

CONCLUSION

As an extended analysis of a previous RCT, this study demonstrated that adjunctive administration of Pediococcus acidilactici CCFM6432 significantly reduced peripheral inflammatory markers (LPS, CRP, IL-6) in patients with MDD, and these reductions were closely associated with improvements in anhedonia, particularly ANT anhedonia. The observed link between decreases in inflammation and enhancements in SPN suggests that gut–brain axis inflammation regulation may represent a key mechanism. These findings provide both clinical and mechanistic support for probiotic interventions in anhedonia, while emphasizing the need for future validation in larger, multicenter, and multimodal studies.

ACKNOWLEDGEMENTS

We sincerely thank all participants who generously contributed their time and effort to this study.