Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study utilized macroscopic measurements, biochemical assays, histology, and multicolor flow cytometry to investigate the therapeutic differences between free cells and microencapsulated synbiotics of Levilactobacillus brevis LBH1070 in popliteal lymph node samples from healthy and arthritic mice in a complete Freund’s adjuvant (CFA)-induced model. The study focused on quantifying the local antioxidant effects and targeted immunomodulation of infiltrating CD3⁺/CD4⁺ T lymphocytes and associated pro-inflammatory cytokines. Additionally, the study examined the comparative efficacy against allopathic phenylbutazone and analyzed the physiological constraints of the murine gastrointestinal tract on synbiotic delivery.

 

However, several points need clarification and improvement before publication. These pertain to experimental design, methodology, statistical analysis, data interpretation, and manuscript presentation. Some conclusions are over-interpreted, and certain methods require more detailed justification or technical clarity.

 

1. Line 15. The author's third affiliation number is incorrectly labeled (it was labeled as 2). Please correct this formatting.

 

2. While the Introduction discusses the complex autoimmune mechanisms of human rheumatoid arthritis (RA) at length, the Complete Freund's Adjuvant (CFA) model utilized here primarily drives localized, mycobacterial antigen-induced chronic inflammation. The Discussion should briefly address the inherent limitations of using the CFA model to mimic a systemic human autoimmune disease.

 

3. Line 147: The study exclusively uses male CD-1 mice. Given that human RA epidemiological data shows a pronounced female predominanc, particularly in young-onset cohorts. What was the scientific rationale behind selecting an all-male cohort? A brief justification should be added to the Methods or Discussion.

 

4. Line 175. The authors stated that only one lymph node (n=1 per group) was used for histological evaluation. A sample size of n=1 is insufficient to reflect biological variation. Histological analysis should be repeated, using at least n=3 to n=5 lymph nodes per group, and a quantitative histological score should be provided. Please explain the reason for using only one lymph node for histological assessment.

 

5. Line 190. The absolute weight data missing on day 28. Based on the reported mouse weight (25±5 g) and relative PLN ratios (0.03%-0.14%, Line 307), a single popliteal lymph nodeweighs only 7.5-35 mg. Homogenizing this minute tissue in 2 mL PBS introduces a 57 to 260-fold dilution, which theoretically places MDA and reactive carbonylconcentrations far below the limit of detection for conventional TBARS and DNPH assays. Please clarify if "2 ml" is a typographical error or if tissues were pooled. In addition, please clearly present the absolute weight recorded on day 28 in the results.

 

6. Lines 195 and 204: The subsection numbering "2.3.2.2" is duplicated for both Lipid Peroxidation and Protein Oxidation.

 

7. Lines 202、 There is an inconsistency in units between the text and figures(using µM/g vs. µmol/g). Please standardize this unit formatting throughout the manuscript.

 

8. Lines 234-247. The antibody list excludes anti-CD3, yet Section 3.4 presents data explicitly gating on total C'D3+ T cells. Without indicating the use of an anti-CD3 antibody, it is unclear how these T-cellpopulations were definitively identified. Please update the methods with thecomplete antibody cocktail and include the flow cytometry gating strategydiagram to clarify this immunophenotyping step.

 

9. Page 10. The visual charts/panels for Figures 5 and 6 appear to be missing from the current draft, though their captions are present. Please ensure all figures are correctly embedded.

 

10. Please review the text for erroneous hyphenations and word breakages occurring mid-line, for example, "de-crease"(Line 315).

 

11. Lines 375-383. The percentage reductions stated in this section do not mathematically align with the reported absolute values. For example, a decrease from 362 to 244 µmol MDA/g is described as a 59% reduction, whereas the actual math yields 32.6%. Similarly, a drop from 992 to 586 µmol CR/g is a 40.9% decrease, not 82%. Please carefully re-verify and correct all calculation baselines throughout Section 3.3.

 

12. Lines 471-473. The mean values reported for IL-1β-producing T cells and TNF-α-producing cells in the arthritic control group are identical (both listed as 27.41). Since it is unusual for two completely different cytokine-producing subsets to yield the exact same mean value, please re-check the raw dataset or look for a potential error during drafting.

 

13. Given that this study evaluates both a probiotic and a synbiotic formulation, the lack of gut microbiota profiling (such as 16S rRNA sequencing) leaves a significant gap. The hypothesis that the synbiotic performed poorly due to accelerated gastrointestinal transit and failed release remains speculative without factual fecal or intestinal microbiota data. If microbiota profiling was not performed, this limitation should be transparently addressed in the Discussion.

 

14. A core finding is that the microencapsulated synbiotic showed lower efficacy than the free cells, which is attributed to rapid gastrointestinal transit and incomplete matrix breakdown in mice. However, without accompanying in vitro release kinetic data at varying pH levels (simulating gastric vs. intestinal fluids), it is difficult to determine if the issue is premature gastric degradation or poor intestinal release. Please provide in vitro release curves of the microcapsules at different pH levels, otherwise the rationale for the design of this synbiotic formulation will be seriously questioned.

 

15. Line 583-585. The text consistently cites "Amdekar et al., 2013," but the corresponding entry in the reference list (Reference #4) indicates the publication year as 2012. Please cross-check and reconcile this citation year inconsistency.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Arthritis affects one in every hundred people worldwide (according to the WHO data). This joint disease is accompanied by excruciating pain, and treatment typically involves medications with a wide range of side effects. The ongoing development of effective methods and medications based on probiotic bacteria for the treatment of arthritis is highly relevant and in demand. The study “Anti-inflammatory potential of Levilactobacillus brevis LBH1070 and its synbiotic in a murine model of experimental arthritis”, submitted for publication in Microorganisms, examines the effects of probiotics in a mouse model of arthritis induced by administration of complete Freund's adjuvant.

               The work is well thought out and rationally planned. The Introduction provides a comprehensive understanding of the key events in the pathogenesis of the disease, which is based on inflammation. Information on the use of bacteria in the treatment of arthritis and their characteristics is provided. In the Introduction, the authors justify the use of all "players" in experiments studying the effects of probiotics and synbiotics; provide descriptions of their effects, and for the anti-inflammatory drug phenylbutazone, provide information on the use and side effects.

The Materials and Methods section is clearly written and contains all the necessary information about the microorganisms used, their encapsulation, reagents, and mice. Experiments on mice were conducted in accordance with the recommendations and standards of the National and International committees for the care and use of laboratory animals. The number of animals used (7 per group) was adequate for the experimental objectives. The experimental model of arthritis is described clearly and in detail. It is worth noting the careful selection of the parameters for evaluating the impact results, which made it possible to obtain convincing results.

The authors conducted a wide set of studies aimed at investigating the effects of used "medications" on the key components of arthritis pathogenesis. These studies examined histologic pictures, oxidative stress, lipid peroxidation, protein oxidation, superoxide dismutase activity, glutathione peroxidase activity, and immunomodulatory activity. The Immunomodulatory activity study included an analysis of changes in a wide range of cytokines inside and outside the cells of the popliteal lymph node. The description of experiments allows them to be reproduced by other researchers.

The "Results" section shows all visible lesions on the affected paw in mice and their changes after treatment with the "drugs." Visual differences are demonstrated in a photograph of the paw swelling and confirmed by measurement data. The anti-edema effect of free-form probiotics is impressive, with an effect similar to that of the potent drug phenylbutazone. The equally striking changes in the size of the lymph nodes due to edema and treatment presented by the authors confirm the anti-edema effect of probiotics.

               Microphotographs confirm the "macro" data. Unfortunately, not all images are of high quality; I recommend replacing the blurry images (Fig. 4A - x10; 4C - all three images, 4E - x10) with images of acceptable quality.

Data on the influence of probiotics and synbiotics on the parameters of oxidative stress, lipid peroxidation, protein oxidation, as well as on the activity of superoxide dismutase, glutathione peroxidase and immunomodulatory activity, are presented in full and well-illustrated, the necessary statistical processing has been performed.

The Discussion section is well written, drawing on current published data. The authors outline possible mechanisms for the effects of Levilactobacillus brevis LBH1070. For example, a significant reduction in the number of CD3⁺ T cells and CD3⁺/CD4⁺ T cells producing IL-1β and TNF-α mediates the probiotic's anti-inflammatory effect in an experimental model of CFA-induced arthritis. Interestingly, the effect of the bacteria outperforms that of the potent anti-inflammatory drug phenylbutazone. The probiotic significantly reduced oxidative stress level, decreasing lipid and protein oxidation. This resulted in the preservation of membrane integrity in the lymphoid tissue of the popliteal fossa.

In my opinion, the lack of a progressive effect of the synbiotic formula compared to the free strain in biological reactions, at least in the mouse model used, deserves special attention. This indicates the need for a thorough analysis of the effects of encapsulated forms of bacteria in animal host.  

Overall, the results presented in the manuscript “Anti-inflammatory potential of Levilactobacillus brevis LBH1070 and its synbiotic in a murine model of experimental arthritis”, demonstrate the potential of L. brevis LBH1070 as a viable strain with promising probiotic properties. The strain convincingly demonstrated anti-inflammatory, antioxidant, and immunomodulatory effects in mice, suggesting the potential for these effects in humans.

I have carefully read the manuscript and have not found any flaws that would prevent its publication in its current form. However, I would like to see all the high-quality micrographs in this article, so I ask the authors to make this change. I think that the authors have preserved tissue blocks and will easily make new sections and new photographs.

Author Response

Please see the attachment

Author Response File: Author Response.pdf