Protective Effect of Vitamin K2 (MK-7) on Acute Lung Injury Induced by Lipopolysaccharide in Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the manuscript entitled: Protective effect of Vitamin K2 (MK7) on acute lung injury induced by lipopolysaccharide in mice. The manuscript touches on an important topic. In general, it is very interesting, but requires some corrections.

1.     All abbreviations should be explained e.g. line 58 – ROS. Please check the entire manuscript carefully and enter abbreviations when they are used for the first time.

2.     Methods: catalog numbers of ELISA kits, immunochemical antibodies, primers or probes for qPCR and Western Blot antibodies must be provided. This information’s are necessary to verify how the research being conducted and the possibility of repeating it in the future. If qPCR primers were specially designed, sequences should be provided. Moreover, antibody dilutions must also be marked.

3.     The manuscript is methodologically correct and everything is described clearly.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors In the present study, Yang and colleagues investigated the protective potential of vitamin K2 (VK2) against LPS-induced acute lung injury in mice, focusing on the ability of VK2 to modulate key processes associated with ALI pathogenesis, including inflammation, apoptosis, cell-cell interaction, oxidative stress, calcium homeostasis, and autophagy. The primary method used to obtain the data described was the assessment of protein levels in mouse lung tissue by Western blot. It is my understanding that this paper is a follow-up to a study by this author team previously published in PLOS One (Wang, Yulian, et al. "Vitamin K2 (MK-7) attenuates LPS-induced acute lung injury via inhibiting inflammation, apoptosis, and ferroptosis." Plos one 18.11 (2023): e0294763). It is noteworthy that the authors do not cite this article in the present manuscript for unclear reasons. This paper is well done, describes new and interesting results, and may be published in CIMB after some revisions.   Major comments: 1. For all results describing Western blot data, you should indicate not only the presence of up- or down-regulation by the action of LPS or VK2, but also how many times the expression levels changed. Please correct this throughout the manuscript. 2. In the Discussion, please provide a detailed comparison of the results described in this paper with your results previously described in PLOS One. Do they agree? Based on the data from these two papers, what are the possible ALI-associated molecular targets of VK2? 3. Please increase the scale and resolution of all figures in the manuscript. 4. Section 3.1, Figure 1D - please provide a hypothesis as to why dexamethasone caused animal death? How is this consistent with data in the literature? Has this been described previously by other authors in experiments with ALI mice?   Minor comments: 1. Missing spaces  between text and references should be filled in throughout the manuscript. 2. line 58 - Change Ca+2 to Ca2+ (2+ as degree). 3. section 2.2 - Please indicate the method by which VK2 was administered to the mice? By oral gavage? Please specify how VK2 was dissolved (was DMSO used before dissolving in oil) and what volumes were administered to the mice? 4. line 256 - compared to. Please correct. 5. line 301 - VK2 does not upregulate but activates the PI3K/AKT pathway. Please correct. 6. line 331 - Please state the date of ethical approval.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors took into account all my comments from the previous round of review and successfully corrected most of the deficiencies.

Unfortunately, the authors did not always give the correct fold change values when comparing protein expression levels. A striking example: lines 233 and 234 - the authors made a mistake in reporting 0.75-fold and 1-fold, because 0.75-fold in its classical sense indicates a 25% suppression of PINK1 expression by VK2 compared to the LPS group, and 1-fold indicates that the expression level of Mfn2 is not changed by VK2 compared to the LPS group, which is not true (Figure 3E, G). Dear authors, when you compare 2 groups with each other (e.g. the effect of VK2 versus LPS), you should normalize (divide) the protein level in the VK2 group by the level of this protein in the LPS group, instead of subtracting these values from each other.

Another example: line 248 - the authors write that the expression of LC3II and Beclin1 was significantly increased in the LPS group by about 0.5-fold and 0.25-fold, respectively, compared to the control. However, this is not a true statement, in fact, based on Fig. 4B, C, LPS increased LC3II and Beclin1 levels by 1.5-fold and 1.25-fold, respectively, compared with control. Dear authors, please check all fold change values reported in the article and correct them if necessary.

After these minor corrections, the manuscript can be successfully published in CIMB.

Author Response

Thank you to the reviewer for pointing out the key error to us. We apologize for such an error. We have modified all fold change values when comparing protein expression levels.