The Combination of Lactobacillus reuteri RC-14^® and Lactobacillus rhamnosus GR-1^® Induces Anxiolytic-like and Antidepressant-like Effects via Estrogenic Receptors in Ovariectomized Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is a well designed experimental study in ovariectomiezed rats, aiming to investigate whether [in clinical practice] probiotics administration would be given in compination with, or finally to replace estrogen therapy in menopause woman.

I appreciate the phrase "anti-depressant-like effects" since the probiotics they used are not yet recognized as psychobiotics. However, why don't they use a psychobiotic formula? I would like a comment, in discussion section.

Introduction: I would like a paragraph on the effects of estrogen deficiency as well as the role of estrogen replacement therapy on mood status; in other words the 1st paragraph to become longer accompanied by related bibliography. 

Introduction, last paragraph: I would like an explanation why the authors decide to use these two probiotic strains, in this dose, the duration of treatment; and why these two as a combo treatment. Do we know the anti-depressant action of each one seperately? are there any previous studies on these on anxiety/depression? I have no objection on the treatment, but I want to know why?

Good job in general! congratulations!

Author Response

Dear reviewer,

The authors appreciate each of your comments; addressing your requests undoubtedly improves the quality of our manuscript. Below you will find the comments and our response.

It is a well-designed experimental study in ovariectomiezed rats, aiming to investigate whether [in clinical practice] probiotics administration would be given in combination with, or finally to replace estrogen therapy in menopause woman.

Comments 1: I appreciate the phrase "anti-depressant-like effects" since the probiotics they used are not yet recognized as psychobiotics. However, why don't they use a psychobiotic formula? I would like a comment, in discussion section.

Response 1: We thank the reviewer for this insightful observation regarding the use of established psychobiotic formulas versus the specific probiotic strains employed in our study. We have added a paragraph in the Discussion section (lines 364-386) addressing this important methodological consideration and providing justification for our strain selection. Briefly, while we acknowledge that established psychobiotic formulas exist, we selected Lactobacillus reuteri RC-14® and Lactobacillus rhamnosus GR-1® based on: (1) preliminary evidence suggesting their potential psychobiotic properties, (2) their well-established safety profile and current commercial availability as a widely consumed product for women's health, (3) the specific research question addressing their potential interaction with estrogen receptors in the context of menopause-related affective symptoms, which has not been previously explored with established psychobiotic formulas, and (4) the translational relevance of studying a product already in use by consumers. We agree this represents an exploratory approach and have clarified this limitation in the revised manuscript.

Comments 2: Introduction: I would like a paragraph on the effects of estrogen deficiency as well as the role of estrogen replacement therapy on mood status; in other words the 1st paragraph to become longer accompanied by related bibliography. 

Response 2: We sincerely appreciate the reviewer's valuable suggestion to expand the first paragraph of the Introduction to provide a more comprehensive overview of estrogen deficiency effects and the role of estrogen replacement therapy on mood status. We have substantially revised and expanded this paragraph (lines 51-67) to include:

Neurobiological mechanisms by which estrogen deficiency disrupts mood regulation, including effects on serotonergic neurotransmission, brain-derived neurotrophic factor (BDNF) expression, and hypothalamic-pituitary-adrenal (HPA) axis function.

Comprehensive information on hormone replacement therapy (HRT), including its therapeutic efficacy in treating mood disturbances, specific mechanisms of action (modulation of serotonin receptor density, enhancement of noradrenergic function, activation of estrogen receptors in limbic regions, and reduction of neuroinflammation), and associated risks with prolonged use.

Comments 3: Introduction, last paragraph: I would like an explanation why the authors decide to use these two probiotic strains, in this dose, the duration of treatment; and why these two as a combo treatment. Do we know the anti-depressant action of each one seperately? are there any previous studies on these on anxiety/depression? I have no objection on the treatment, but I want to know why?

Response 3: Dear reviewer,

The selection of Lactobacillus reuteri RC-14® and Lactobacillus rhamnosus GR-1® as a combination was based on their commercial availability as Vavig®, a product already consumed worldwide for women's urogenital health [62]. Previous studies have demonstrated that L. reuteri reduces anxiety-like behaviors through IL-6 suppression [12], while L. rhamnosus alleviates depression-related symptoms by modulating serotonin and BDNF pathways [13,41]. The 28-day treatment duration was selected to ensure sufficient time for microbiota modulation and behavioral manifestation [63], while the dose (4.9×10⁹ CFU total) was chosen based on anti-inflammatory efficacy demonstrated in previous research [24] and manufacturer recommendations for optimal probiotic colonization.

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study provides coherent behavioral evidence that Lactobacillus reuteri RC‑14® and Lactobacillus rhamnosus GR‑1® alone and in combination with 17β‑estradiol reduce anxiety- and depression-like behaviors in ovariectomized rats without affecting locomotion, and that these effects are sensitive to tamoxifen pretreatment. The manuscript is suitable in scope for Nutrients but, in its current form, is not suitable for publication.

Specific comments:

• The central claim is that effects are mediated via ERβ, yet there are no direct measures of ER expression or signaling in relevant brain regions or peripheral tissues.​ Tamoxifen is used as an ER antagonist, but it is a SERM with complex, tissue-specific partial agonist/antagonist actions and non‑ER-mediated effects, including neuropsychiatric effects on its own. Inferring specific ERβ involvement solely from systemic tamoxifen reversal is not convincing.
• The probiotic-tamoxifen interaction is interpreted as evidence that probiotics act through estrogen receptors, but alternative explanations (e.g., tamoxifen-induced shifts in microbiota, HPA axis, or inflammation independent of ERβ) are not considered.​
• The authors repeatedly describe the combination of probiotics and estradiol as “synergistic,” especially for FST latency, but the statistical treatment does not formally test interaction. Describing effects as “synergy” implies a formal demonstration that combined treatment exceeds the sum of individual effects, which is not shown.
• The study is framed as modulation of the microbiota-gut-brain axis, but neither gut microbiota composition nor probiotic colonization/survival are assessed. Either add basic microbiota data (e.g., qPCR for the administered strains or 16S overview) or significantly tone down claims about microbiota mediation, focusing instead on behavioral pharmacology with probiotics as bioactive agents.
• While EPM and FST are widely used, their interpretation as models of human anxiety and depression is debated. Acknowledge the limitations of EPM and FST as models of complex human syndromes and soften language around “therapeutic strategy” and “antidepressant-like” effects. Emphasize that data provide preclinical evidence of changes in specific anxiety‑ and despair-related behaviors.
• The conclusion suggests that the probiotic mixture could be an alternative to steroidal estrogen therapy and may allow estrogen dose reduction, yet this goes beyond the presented data. Reframe the translational section to emphasize that findings are hypothesis-generating, not sufficient to propose probiotics as an “alternative” to estrogen therapy. Stress the need for long-term, multi-endpoint animal studies and carefully designed clinical trials before inferring clinical utility.

Author Response

Dear reviewer,

The authors appreciate each of your comments; addressing your requests undoubtedly improves the quality of our manuscript. Below you will find the comments and our response.

The study provides coherent behavioral evidence that Lactobacillus reuteri RC‑14® and Lactobacillus rhamnosus GR‑1® alone and in combination with 17β‑estradiol reduce anxiety- and depression-like behaviors in ovariectomized rats without affecting locomotion, and that these effects are sensitive to tamoxifen pretreatment. The manuscript is suitable in scope for Nutrients but, in its current form, is not suitable for publication.

Specific comments:

Comments 1: The central claim is that effects are mediated via ERβ, yet there are no direct measures of ER expression or signaling in relevant brain regions or peripheral tissues. ​ Tamoxifen is used as an ER antagonist, but it is a SERM with complex, tissue-specific partial agonist/antagonist actions and non‑ER-mediated effects, including neuropsychiatric effects on its own. Inferring specific ERβ involvement solely from systemic tamoxifen reversal is not convincing.

Response 1: This work has a limitation, and future studies could provide more direct evidence. However, some studies highlight the difficulties in studying ERβ because of nonspecific antibodies and the difficulty in distinguishing the functions of ERα and ERβ when they are co-expressed (Song et al., 2022). Song, D., He, H., Indukuri, R., Huang, Z., Stepanauskaite, L., Sinha, I., ... & Williams, C. (2022). ERα and ERβ homodimers in the same cellular context regulate distinct transcriptomes and functions. Frontiers in endocrinology, 13, 930227.

Tamoxifen acts as a selective estrogen receptor modulator, meaning its effects are not uniformly agonistic or antagonistic but are tissue-specific (Komm and Mirkin, 2013). This complexity suggests that systemic tamoxifen effects may result from a combination of varying ERα and ERβ activities in different tissues, rather than a specific action mediated by ERβ alone. The co-expression of ERα and ERβ in many tissues, including the brain, may mediate these effects. Although ERα and ERβ can have distinct roles and may respond differently to pharmacological agents such as tamoxifen – which sometimes acts as an ERβ agonist under conditions where it inhibits ERα (McDonnell and Wardell, 2010)—systemic administration of a drug that interacts with both subtypes makes it difficult to isolate the contribution of one without more specific interventions. The different proportions and activities of ERα and ERβ can significantly influence the response to tamoxifen (Madeira et al., 2013). To clarify the role of ERβ, future studies using highly selective ERβ agonists or antagonists are needed.

Komm, B. S., & Mirkin, S. (2013). Evolution of the tissue selective estrogen complex (TSEC). Journal of Cellular Physiology, 228(7), 1423-1427.

McDonnell, D. P., & Wardell, S. E. (2010). The molecular mechanisms underlying the pharmacological actions of ER modulators: implications for new drug discovery in breast cancer. Current opinion in pharmacology, 10(6), 620-628.

Madeira, M., Mattar, A., Logullo, Â. F., Soares, F. A., & Gebrim, L. H. (2013). Estrogen receptor alpha/beta ratio and estrogen receptor beta as predictors of endocrine therapy responsiveness–a randomized neoadjuvant trial comparison between anastrozole and tamoxifen for the treatment of postmenopausal breast cancer. BMC cancer, 13(1), 425.

His recommendation was taken into account and modified in the manuscript.

Comments 2: The probiotic-tamoxifen interaction is interpreted as evidence that probiotics act through estrogen receptors, but alternative explanations (e.g., tamoxifen-induced shifts in microbiota, HPA axis, or inflammation independent of ERβ) are not considered. ​

Response 2: We thank the reviewer for this insightful comment regarding the interpretation of probiotic-tamoxifen interactions and the need to consider alternative mechanisms beyond direct estrogen receptor modulation. This is indeed an important limitation of our study that deserves careful consideration.

We acknowledge that our interpretation attributing the tamoxifen-induced blockade of probiotic effects primarily to estrogen receptor antagonism may be overly simplistic. As the reviewer correctly points out, tamoxifen could interfere with probiotic effects through several ERβ-independent pathways, including: (1) tamoxifen-induced alterations in gut microbiota composition that could counteract probiotic colonization or metabolic activity, (2) modulation of the HPA axis independently of classic estrogen receptor mechanisms, (3) effects on inflammatory pathways that are not mediated through ERβ signaling, or (4) interference with other neurotransmitter systems (e.g., serotonergic, GABAergic) through non-genomic mechanisms.

To address this valuable critique, we have added a paragraph in the Discussion section (lines 341-355) that explicitly acknowledges these alternative interpretations and discusses the limitations of using tamoxifen as a selective tool to infer mechanism of action. We now emphasize that while our data suggest estrogen receptor involvement, they do not definitively prove that this is the exclusive or even primary mechanism. We have also added that future mechanistic studies using more selective ERβ antagonists (such as PHTPP), direct measurement of estrogen receptor expression in relevant brain regions, assessment of gut microbiota composition following tamoxifen administration, and evaluation of inflammatory markers and HPA axis function would be necessary to dissect the relative contributions of ERβ-dependent versus ERβ-independent mechanisms.

We believe this revision substantially strengthens the manuscript by providing a more nuanced and scientifically rigorous interpretation of our findings.

Comments 3: The authors repeatedly describe the combination of probiotics and estradiol as “synergistic,” especially for FST latency, but the statistical treatment does not formally test interaction. Describing effects as “synergy” implies a formal demonstration that combined treatment exceeds the sum of individual effects, which is not shown.

Response 3: We appreciate your valuable feedback, and the authors have therefore modified that signature in the manuscript.

Comments 4: The study is framed as modulation of the microbiota-gut-brain axis, but neither gut microbiota composition nor probiotic colonization/survival are assessed. Either add basic microbiota data (e.g., qPCR for the administered strains or 16S overview) or significantly tone down claims about microbiota mediation, focusing instead on behavioral pharmacology with probiotics as bioactive agents.

Response 4: We sincerely thank the reviewer for this critical and constructive observation. We fully agree that our study does not provide direct evidence of microbiota composition changes, probiotic colonization, or survival in the gut, and therefore, we cannot make definitive claims about microbiota-gut-brain axis modulation as a primary mechanism. This is indeed a significant limitation of our experimental design.

In response to this valid critique, we have carefully revised the manuscript to significantly tone down all claims regarding microbiota mediation. Specifically, we have:

Revised the Abstract (lines 40-46) to remove or moderate language suggesting direct microbiota modulation and reframed our findings within a behavioral pharmacology framework, describing probiotics as bioactive agents with anxiolytic and antidepressant-like properties.

Modified the Introduction (lines 73-77) to present the microbiota-gut-brain axis as contextual background rather than the primary focus of our investigation, and clarified that our study examines the behavioral effects of probiotic administration rather than microbiota-mediated mechanisms per se.

Substantially revised the Discussion (lines 417-431) to acknowledge explicitly that we did not assess gut microbiota composition, probiotic colonization, or strain survival, remove speculative statements about microbiota changes as the mechanism of action, reframe our interpretation to focus on probiotics as pharmacologically active compounds that produce behavioral effects potentially through multiple pathways (direct metabolite production, immune modulation, neuroendocrine signaling) that may or may not require sustained alterations in microbiota composition and emphasize that future studies with microbiome sequencing, metabolomic profiling, and assessment of probiotic colonization are necessary to determine whether the observed effects are mediated through microbiota composition changes or through other mechanisms

Revised the Conclusions (lines 433-447) to focus on the behavioral and pharmacological findings rather than microbiota-mediated mechanisms.

Comments 5: While EPM and FST are widely used, their interpretation as models of human anxiety and depression is debated. Acknowledge the limitations of EPM and FST as models of complex human syndromes and soften language around “therapeutic strategy” and “antidepressant-like” effects. Emphasize that data provide preclinical evidence of changes in specific anxiety‑ and despair-related behaviors.

Response 5: We greatly appreciate the reviewer's important reminder regarding the limitations of behavioral models and the appropriate interpretation of preclinical findings. We fully agree that the EPM and FST, while widely validated and extensively used in neuropharmacology, have inherent limitations as models of complex human psychiatric syndromes such as anxiety and depression. These tests assess specific behavioral dimensions (e.g., approach-avoidance conflict in EPM, behavioral despair in FST) rather than capturing the full spectrum of human psychopathology.In response to this thoughtful critique, we have revised the manuscript throughout to:

Softened language regarding "therapeutic strategy," "anxiolytic effects," and "antidepressant effects" by consistently using more precise terminology such as "anxiolytic-like effects," "antidepressant-like effects," and "potential therapeutic strategy" or "promising preclinical approach."

Added explicit acknowledgment in the Discussion section (lines 296-307) of the limitations of EPM and FST as models of human anxiety and depression, emphasizing that these tests assess specific behavioral dimensions rather than replicating the complexity of clinical psychiatric disorders.

Reframed our conclusions to emphasize that our data provide preclinical evidence of changes in specific anxiety-related behaviors (approach-avoidance conflict, exploratory behavior) and despair-related behaviors (active coping strategies, behavioral resignation) rather than claiming to model human anxiety and depression disorders per se.

Modified the Abstract and Conclusions to reflect this more cautious interpretation, clarifying that translation to clinical efficacy requires controlled human trials and that our findings represent an initial preclinical step rather than direct evidence of therapeutic utility in menopausal women.

We believe these revisions provide a more scientifically nuanced and appropriately cautious interpretation of our behavioral findings, acknowledging both the value and limitations of these preclinical models.

Comments 6: The conclusion suggests that the probiotic mixture could be an alternative to steroidal estrogen therapy and may allow estrogen dose reduction, yet this goes beyond the presented data. Reframe the translational section to emphasize that findings are hypothesis-generating, not sufficient to propose probiotics as an “alternative” to estrogen therapy. Stress the need for long-term, multi-endpoint animal studies and carefully designed clinical trials before inferring clinical utility.

Response 6: We sincerely thank the reviewer for this critically important observation. We fully acknowledge that our conclusion overstated the translational implications of our findings. The suggestion that probiotics could serve as an alternative to estrogen therapy or allow dose reduction indeed goes beyond what our preclinical data can support. Our study provides only preliminary behavioral evidence in an animal model and lacks the comprehensive data (long-term safety, dose-response relationships, clinical endpoints, microbiota analysis, mechanistic confirmation) necessary to make such therapeutic recommendations.

In response to this essential critique, we have substantially revised the manuscript to:

Reframed the Abstract conclusion (lines 40-46) to emphasize that our findings are hypothesis-generating and represent preliminary preclinical evidence requiring extensive validation before any clinical recommendations can be made. In addition, the wording in the introduction was modified (lines 100-106).

Completely revised the paragraph of the Discussion (lines 374-377) and (426-431)

Modified the Conclusions section (lines 433-447) to present findings as raising interesting possibilities for future investigation rather than proposing clinical applications.

Removed or softened all language throughout the manuscript suggesting that probiotics could replace or reduce estrogen therapy, replacing it with cautious, hypothesis-generating framing.

We believe these revisions provide a scientifically appropriate and responsible interpretation of our findings, clearly distinguishing between preliminary preclinical observations and the extensive evidence base required for clinical recommendations.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your prompt response to all of my comments 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have addressed all of the reviewer's comments and clarified all of the concerns. I have no further comments.