Molecular Basis of Surfactin-Induced Macrophage Modulation and Its Implications in Medication-Related Osteonecrosis of the Jaw Pathogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research direction of this manuscript is innovative and worthy of further in-depth study. However, the article has problems such as insufficient research content, inadequate depth, unreasonable data statistics and analysis, and the amount of animal data not conforming to medical statistics. The above problems still need to be improved.

 

1. In the Introduction section, the surface manifestations and effects of MRONJ should be described. Meanwhile, the latest epidemiological data should be added.
2. In the Introduction section, is surfactin applied in the treatment of MRONJ? If there are any relevant reports, they should be described.
3. Has the concentration of "LPS (100 ng/mL)" been evaluated through CCK-8? Or based on the reference reports?
4. The effect of surfactin concentration on J774.1 cells was evaluated by CCK-8. The impact on RAW264.7 Cells has not been evaluated? Why?
5. Why were J774.1 cells and RAW264.7 Cells chosen?
6. In the "Animal Experiment" section, how were the doses above "a mixture of 50 μg LPS 348 and 2.5 mg surfactin" determined?
7. In the "Animal Experiment" section, the number of animals "In the LPS group (n=3) and In the LPS + surfactin group (n=3)" is too small, and its data is not reliable. It does not meet the requirements of medical statistics.
8. A large amount of data statistics in bar charts are inaccurate. Verification, modification and addition are required. For example, Figure 2, Figure 3, Figure 6 and Figure 7. In Figure 6, when comparing LPS (+) with JNK Inhibitor Group II, is it "*" or "**"? In Figure 7, when comparing LPS (+) with the AG-490 group, is it "*" or "**"?

Comments on the Quality of English Language

It can be improved.

Author Response

Reviewer 1

The research direction of this manuscript is innovative and worthy of further in-depth study. However, the article has problems such as insufficient research content, inadequate depth, unreasonable data statistics and analysis, and the amount of animal data not conforming to medical statistics. The above problems still need to be improved.

 

1. In the Introduction section, the surface manifestations and effects of MRONJ should be described. Meanwhile, the latest epidemiological data should be added.

According to the reviewer’s comments, we added the latest epidemiological data and modified the Introduction in the revised manuscript (lines 44-47).

 

2. In the Introduction section, is surfactin applied in the treatment of MRONJ? If there are any relevant reports, they should be described.

Thank you for your suggestions. Although no approved pharmaceuticals currently exist, surfactin possesses various biological activities and holds promise for application in the medical field. However, we did not mention about it. Therefore, we modified the Introduction section in the revised manuscript (lines 77-79).

 

3. Has the concentration of "LPS (100 ng/mL)" been evaluated through CCK-8? Or based on the reference reports?

The concentration of LPS added to the macrophage cell line was determined based on the results of our previous studies. Furthermore, CCK-8 analysis confirmed that 100 ng/mL LPS does not inhibit the proliferation of J774.1 cells. So, we added Supplemental Figure 1 and modified the Discussion section with new reference in the revised manuscript (lines 217-221).

 

4. The effect of surfactin concentration on J774.1 cells was evaluated by CCK-8. The impact on RAW264.7 Cells has not been evaluated? Why?

We have already investigated the effect of surfactin on the proliferation of RAW264.7 cells and found that proliferation inhibition is not induced at concentrations of 50 μg/mL or lower. However, we did not mention about it. Therefore, we modified the Results section in the revised manuscript (lines 115-117).

 

5. Why were J774.1 cells and RAW264.7 Cells chosen?

Given the limited information available on surfactin in the oral innate immune response, this study was conducted as a preliminary investigation. Accordingly, mouse monocyte-macrophage cell lines were used to facilitate future analyses employing genetically modified mouse models. Among the available mouse monocyte/macrophage cell lines, J774.1 and RAW264.7 were selected for this study because they have been demonstrated to respond to LPS derived from periodontopathic bacteria. We have added this description to the Discussion section of the revised manuscript (lines 215-217).

 

6. In the "Animal Experiment" section, how were the doses above "a mixture of 50 μg LPS and 2.5 mg surfactin" determined?

Thank you for your helpful comment. The dosage of LPS and surfactin in animal experiments was determined based on the results of preliminary experiments and in vitro experiments. However, we did not mention about it. So, we added Supplemental Figure 2 and modified the Materials and Methods in the revised manuscript (lines 369-371).

 

7. In the "Animal Experiment" section, the number of animals "In the LPS group (n=3) and In the LPS + surfactin group (n=3)" is too small, and its data is not reliable. It does not meet the requirements of medical statistics.

We appreciate your critical suggestions. As the reviewers pointed out, the animal experiments in this study represent preliminary investigations and are insufficient to provide conclusive evidence of surfactin's ability to control the pathophysiology of MRONJ. Further studies are required, including investigations with increased sample sizes and verification of the long-term pharmacological effects of surfactin. We have added this information and revised the Discussion section in the revised manuscript (lines 294-298).

 

8. A large amount of data statistics in bar charts are inaccurate. Verification, modification and addition are required. For example, Figure 2, Figure 3, Figure 6 and Figure 7. In Figure 6, when comparing LPS (+) with JNK Inhibitor Group II, is it "*" or "**"? In Figure 7, when comparing LPS (+) with the AG-490 group, is it "*" or "**"?

We appreciate your important suggestions. Confidence intervals describe the uncertainty of the estimate and not the data, so the variance of the variable was described by its standard deviation. To improve clarity, we included dot plots in the graph bars of the revised figures (Figure 2, 3, 4A, 6C, 6D, 7C). We also carefully reviewed the results of the statistical analysis.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript demonstrates that surfactin selectively suppresses LPS-induced IL-6 production in macrophages through inhibition of the JNK–c-Jun–AP-1 axis and the JAK/STAT signaling pathway, and that this immunomodulatory effect translates into reduced osteonecrosis in an MRONJ animal model. Together, the findings provide mechanistic and in vivo evidence supporting surfactin as a promising therapeutic candidate for MRONJ. Although the authors discuss the roles of the NF-κB, MAPK, and JAK/STAT signaling pathways in the regulation of IL-6 expression in the Discussion section, these pathways are not adequately introduced in the Introduction or Results sections. Instead, the manuscript proceeds directly to presenting the experimental findings. Providing brief background information on these signaling pathways earlier in the manuscript would help improve clarity and better contextualize the results for readers. In Fig. 5C, it is notable that surfactin inhibited c-Jun phosphorylation only in the presence of LPS, while surfactin alone did not promote c-Jun phosphorylation in the absence of LPS. This observation is interesting and should be explicitly pointed out and discussed in the manuscript.

Author Response

Reviewer 2

This manuscript demonstrates that surfactin selectively suppresses LPS-induced IL-6 production in macrophages through inhibition of the JNK–c-Jun–AP-1 axis and the JAK/STAT signaling pathway, and that this immunomodulatory effect translates into reduced osteonecrosis in an MRONJ animal model. Together, the findings provide mechanistic and in vivo evidence supporting surfactin as a promising therapeutic candidate for MRONJ. Although the authors discuss the roles of the NF-κB, MAPK, and JAK/STAT signaling pathways in the regulation of IL-6 expression in the Discussion section, these pathways are not adequately introduced in the Introduction or Results sections. Instead, the manuscript proceeds directly to presenting the experimental findings. Providing brief background information on these signaling pathways earlier in the manuscript would help improve clarity and better contextualize the results for readers. In Fig. 5C, it is notable that surfactin inhibited c-Jun phosphorylation only in the presence of LPS, while surfactin alone did not promote c-Jun phosphorylation in the absence of LPS. This observation is interesting and should be explicitly pointed out and discussed in the manuscript.

We appreciate your insightful suggestions. Accordingly, we have added a description of the signaling pathways involved in IL-6 expression to the Introduction section of the revised manuscript with some references (lines 66-71). Furthermore, as noted by reviewer, stimulation of surfactin alone activated c-jun phosphorylation. However, we do not mention about it. So, we added new reference and modified Discussion in the revised manuscript (lines 257-262).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Just as the author stated, "the animal experiments in this study represent preliminary investigations and are insufficient to provide conclusive evidence of surfactin's ability to control the pathophysiology of MRONJ" 
Regarding the results obtained for (n=3), is it reliable? Can the author increase the number of animals for verification? 

Author Response

Just as the author stated, "the animal experiments in this study represent preliminary investigations and are insufficient to provide conclusive evidence of surfactin's ability to control the pathophysiology of MRONJ". Regarding the results obtained for (n=3), is it reliable? Can the author increase the number of animals for verification?

 

We appreciate the reviewer’s important feedback. As noted, the sample size was insufficient for statistically robust analyses; therefore, Figure 8 and the Results (lines 198-199) and Discussion (lines 290-292) linessections have been revised. The current study primarily addresses the molecular mechanisms by which surfactin may regulate MRONJ pathology using cell biology–based approaches. Additional in vivo studies with larger sample sizes, including immunohistochemical staining and hard tissue analyses using 3D CT and calcein labeling, are currently underway and will be reported separately.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

It is hoped for more comprehensive and in-depth research.