Beneficial Effects of Probiotics on Liver Injury Caused by Chronic Alcohol Consumption

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article is interesting, but it should indicate and emphasize the novelty more. Below is information on what should be corrected:

-Line 72, the word "in vitro" should be in italics

-Line 38, the word "LPS", is missing an abbreviation description

-Lines 44-52 no literature 

-The introduction includes abbreviations that should be described so that every reader knows the full name. Only then can the abbreviation be used.

-Methodologies lack citations. The authors relied on already known research and modified it.

-The discussion is described theoretically and correctly, but there is also no information on how much higher the concentration of e.g. "TNF-α, IL-6, VEGF, TGF-β1" is in the presented study and in other studies. What concentration is appropriate.

Author Response

Dear reviewer:

Thank you for reviewing the manuscript and giving us your valuable comments. The answers to your questions are as follows.

Q: -Line 72, the word "in vitro" should be in italics

A: This error has been corrected.

Q: -Line 38, the word "LPS", is missing an abbreviation description

A: The full spelling of “LPS” has been added.

Q: -Lines 44-52 no literature

A: The references have been added.

Q: -The introduction includes abbreviations that should be described so that every reader knows the full name. Only then can the abbreviation be used.

A: The full spelling has been added.

Q: -Methodologies lack citations. The authors relied on already known research and modified it.

A: The references have been added.

Q: -The discussion is described theoretically and correctly, but there is also no information on how much higher the concentration of e.g. "TNF-α, IL-6, VEGF, TGF-β1" is in the presented study and in other studies. What concentration is appropriate.

A: Considering the differences between humans and animals, there is no way to have a reference threshold defined like the human body check. In animal experiments the effective impact is mainly demonstrated by comparing significant changes between groups.

Thank you and best regards.

Yours sincerely,

Hengxian Qu

Reviewer 2 Report

Comments and Suggestions for Authors

Your manuscript was easy to read and presents an interesting, coherent piece of research. It can, however, be improved, especially regarding the description of methods (please see detailed comments below).

Detailed comments:

Line 11 - fermentum should be in italics.

Line 22 - I would suggest using a less assertive sentence formulation regarding the fragment "... so that liver inflammation was relieved", e.g., "indicating relieve of liver inflammation".

Line 38 - LPS - please write in full; it's the first time you mention this in your text.

Line 56 - L. rhamnosus - please give the full name of this species - it's the first time you mention it in your text. If this is a commercial strain and you want to keep the name it is known by in the market, you could write Lactobacillus (Lactiplantibacillus) plantarum.

Line 80 - 81 - Please replace "the bacterial body" with "the resulting pellet".

Line 84 - The sentence "Dispense according to the daily requirement and store at -80 °C", as well as the next sentence, do not make gramatical sense in this context. They need rephrasing. I would suggest something along the lines of "Bacterial cultures were stored a -80 °C and defrosted at 30 °C, in a water bath, prior to use".

Line 110 - How were the animals sacrificed? This is an important information. The sacrifice method used can change your results and it certainly has ethical implications. Therefore, it is important to give this information, to allow both reviewers and readers to have a critical view of your work.

Lines 134 - 139 - Please use the infinite form of the verbs in this fragment of the text. E.g., "1 µl was added" instead of "we added 1 µ", or "it was washed once with PBS" instead of "we wash it once with PBS". Also, please pay attention to verbal tenses - it should be "washed" and not "wash".

Line 136 - Type of PBS used (pH)?

Lines 145 - 231 - Throughout the entire Results section, whenever you metion "significant differences", "significantly lower", "significantly higher", etc., please be consistent in giving the P value, or at least the significance level (e.g., P<0.05). This information is missing in a few instances.

 

Comments on the Quality of English Language

Some parts of the Material and Methods section need rewritting, as they are not gramatically correct. Please refer to the detailed comments to locate these parts.

Author Response

Dear reviewer:

Thank you for reviewing the manuscript and giving us your valuable comments. The answers to your questions are as follows.

Line 11 - fermentum should be in italics.

A: This error had been corrected.

Line 22 - I would suggest using a less assertive sentence formulation regarding the fragment "... so that liver inflammation was relieved", e.g., "indicating relieve of liver inflammation".

A: Changes have been made with reference to your comments.

Line 38 - LPS - please write in full; it's the first time you mention this in your text.

A: The full spelling of “LPS” has been added.

Line 56 - L. rhamnosus - please give the full name of this species - it's the first time you mention it in your text. If this is a commercial strain and you want to keep the name it is known by in the market, you could write Lactobacillus (Lactiplantibacillus) plantarum.

A: Changes have been made with reference to your comments.

Line 80 - 81 - Please replace "the bacterial body" with "the resulting pellet".

A: Changes have been made with reference to your comments.

Line 84 - The sentence "Dispense according to the daily requirement and store at -80 °C", as well as the next sentence, do not make gramatical sense in this context. They need rephrasing. I would suggest something along the lines of "Bacterial cultures were stored a -80 °C and defrosted at 30 °C, in a water bath, prior to use".

A: Changes have been made with reference to your comments.

Line 110 - How were the animals sacrificed? This is an important information. The sacrifice method used can change your results and it certainly has ethical implications. Therefore, it is important to give this information, to allow both reviewers and readers to have a critical view of your work.

A: Animal handling has been added. Information related to animal ethics has also been added.

Lines 134 - 139 - Please use the infinite form of the verbs in this fragment of the text. E.g., "1 µl was added" instead of "we added 1 µ", or "it was washed once with PBS" instead of "we wash it once with PBS". Also, please pay attention to verbal tenses - it should be "washed" and not "wash".

A: Changes have been made with reference to your comments.

Line 136 - Type of PBS used (pH)?

A: The pH of PBS has been added.

Lines 145 - 231 - Throughout the entire Results section, whenever you metion "significant differences", "significantly lower", "significantly higher", etc., please be consistent in giving the P value, or at least the significance level (e.g., P<0.05). This information is missing in a few instances.

A: The significance level (e.g., P<0.05) has been added.

Thank you and best regards.

Yours sincerely,

Hengxian Qu

Reviewer 3 Report

Comments and Suggestions for Authors

In this research article, the authors investigate the protective effects of lactobacilli supplementation against ethanol-induced liver damage, in vivo. Two lactobacilli previously isolated from long-lived volunteers that have shown antioxidant capacity in vitro were utilized in this study. The authors concluded that both lactobacilli may have protective effects against ethanol-induced damage due to their anti-inflammatory and antioxidant activity. Major modifications should be made to the manuscript, to be recommended for publication.

Lines 72-74: the aims and the objectives of this manuscript should be clearly stated.

Line 77: isolated instead of “separated”.

Lines 78-80: this sentence should be revised for clarity.

Lines 80, 123: rpm instead of r/min, although g would be more appropriate.

Lines 84-85: these sentences should be revised. This is not a protocol.

Line 94, Table 1: why were methionine and choline bitartrate selected as drugs in this experimental design? To the best of my knowledge, these are used as supplements and not as drugs to treat ethanol-induced damage of the liver. I strongly advise the authors to include an approved drug for this condition. The following claims in the manuscript about the performance of the two strains compared to the “drugs” should be revised accordingly.

Have the authors evaluated the performance of other lactobacillus strains in their experimental model?

Lines 86-103: an ethics statement should be added to the text, as well as the ethics approval number for the study.

Line 108: Please include the manufacturer.

Lines 124-125: this sentence should be revised. This is not a protocol.

Lines 127-132, 134-136: please include the manufacturer of the kit used. Also, the concentration of the reagents must be included in the text; not the volume used.

Lines 146-157: all this data is descriptive and presented qualitatively, is there any quantitative measure of the animal behavior, physiology, appearance, stool quality etc during the trials?

Lines 159-170, Figure 1A: using EM for pathological examination is uncommon. Histological changes are more appropriately examined using appropriate stains in tissue blocks. There is no annotation of the structures shown in Figure 1A, and thus it is difficult for readers with no background in EM to understand what they are seeing.

Author Response

Dear reviewer:

Thank you for reviewing the manuscript and giving us your valuable comments. The answers to your questions are as follows.

Lines 72-74: the aims and the objectives of this manuscript should be clearly stated.

A: The aims and objectives of this study have been modified .

Line 77: isolated instead of “separated”.

A: The "isolated" has been changed to "separated".

Lines 78-80: this sentence should be revised for clarity.

A: This sentence has been revised.

Lines 80, 123: rpm instead of r/min, although g would be more appropriate.

A: The " r/min "has been changed to “rpm”.

Lines 84-85: these sentences should be revised. This is not a protocol.

A: This section has been modified.

Line 94, Table 1: why were methionine and choline bitartrate selected as drugs in this experimental design? To the best of my knowledge, these are used as supplements and not as drugs to treat ethanol-induced damage of the liver. I strongly advise the authors to include an approved drug for this condition. The following claims in the manuscript about the performance of the two strains compared to the “drugs” should be revised accordingly.

Have the authors evaluated the performance of other lactobacillus strains in their experimental model?

A: The drug used in this study is a drug approved in China called dongbaogantai. The main ingredients in the drug are methionine and choline bitartrate. It is one of the more commonly used liver medications.

We have also evaluated the performance of other lactobacillus strains in rat models such as high-fat diet and immunocompromised.

Lines 86-103: an ethics statement should be added to the text, as well as the ethics approval number for the study.

A: The ethics statement and the ethics approval number have been added at the end of the article.

Line 108: Please include the manufacturer.

A: The manufacturer has been added.

Lines 124-125: this sentence should be revised. This is not a protocol.

A: This part of the treatment is mainly for the subsequent tests of 2.6 and 2.7.

Lines 127-132, 134-136: please include the manufacturer of the kit used. Also, the concentration of the reagents must be included in the text; not the volume used.

A: This section has been supplemented with references.

Lines 146-157: all this data is descriptive and presented qualitatively, is there any quantitative measure of the animal behavior, physiology, appearance, stool quality etc during the trials?

A: This section focuses on a description of the state of the rat. Your comments are a very important reference and will be noted in subsequent studies that focus on quantitative documentation.

Lines 159-170, Figure 1A: using EM for pathological examination is uncommon. Histological changes are more appropriately examined using appropriate stains in tissue blocks. There is no annotation of the structures shown in Figure 1A, and thus it is difficult for readers with no background in EM to understand what they are seeing.

A: Your suggestion is very meaningful for our subsequent experiments, and we will carefully consider the way of staining subsequently. In this study, the labeling of individual points was not carried out because it was a global observation and comparison.

Thank you and best regards.

Yours sincerely,

Hengxian Qu

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been revised according to comments.

Author Response

Dear reviewer

Thank you for your work and for taking time out of your busy schedule to review this article!

I wish you a wonderful day!

Yours sincerely,

Hengxian Qu

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for their responses. However, the resubmitted manuscript cannot be recommended for publication as the authors did not respond satisfactorily to several major comments:

1. Lines 152-163: The behavioral analysis is only qualitative with no systematic, quantitative measures provided. No figures, photos, or any other evidence for these results is provided.

2. EM is not appropriate to determine the physiology in tissues. Most importantly, the structures are not annotated. Figure 1A does not hold scientific validity or rigor.

3. Have the authors evaluated the performance of other lactobacillus strains in their experimental model? The authors did not provide information on other bacterial strains tested.

Other minor points:

Lines 83-85: this wording is not clear. What is the “bacterial fluid of the experimental strain”?

Lines 94, Table 1: is this the scientific name of the drug? Also, using a drug is different from using two purified compounds. The text must be revised, the correct treatments must be included in the text, as well as the reason for the selection of these two compounds. The reason why these two strains were selected from all strains isolated from the volunteers should be included in the text.

Lines 114-115: are these in-house kits? If not, please include the LOT number. The manufacturers of all reagents must be added to the text.

Line 140: the concentration of reagents should be included in the text and not the volume used.

Author Response

Dear reviewer

Thank you for your work and for taking time out of your busy schedule to review this article!

I have made further changes to the article as you suggested and your suggestions have made it even better. Detailed answers are provided below.

 

Q: 1. Lines 152-163: The behavioral analysis is only qualitative with no systematic, quantitative measures provided. No figures, photos, or any other evidence for these results is provided.

A: We did neglect in the conduct of the experiment to record some of the photos, etc. of the animals' behavior that you mentioned. However, we also believe that the most important thing in behavioral science is the objective description of the behavior after drinking and the recording of the mortality rate. These post-drinking behaviors cannot be properly quantified.

Q: 2. EM is not appropriate to determine the physiology in tissues. Most importantly, the structures are not annotated. Figure 1A does not hold scientific validity or rigor.

A: As you pointed out, it is true that EM is not the most appropriate way to look at histopathology, but there are some studies in the literature where this method has been used. We have annotated the structures with reference to your suggestion.

Q: 3. Have the authors evaluated the performance of other lactobacillus strains in their experimental model? The authors did not provide information on other bacterial strains tested.

A: I apologize for misunderstanding you in my earlier comments. We have looked at the probiotic properties of other strains in other studies. However, in this study, only these two strains were explored for their mitigating effects on alcoholic liver injury in an animal model. In in vitro studies, we have compared the antioxidant and other abilities of these two strains with other strains, and these two strains specifically have better in vitro properties. So these two strains were selected for the study.

Q: Lines 83-85: this wording is not clear. What is the “bacterial fluid of the experimental strain”?

A: This sentence has been modified.

Q: Lines 94, Table 1: is this the scientific name of the drug? Also, using a drug is different from using two purified compounds. The text must be revised, the correct treatments must be included in the text, as well as the reason for the selection of these two compounds. The reason why these two strains were selected from all strains isolated from the volunteers should be included in the text.

A: This is the Chinese name for the drug that contains these two compounds. This is one of the more widely used drugs in China. Further information on the drug has been added in this paper.

In addition, Line71-73 describes the reasons for the selection of these two strains.

Q: Lines 114-115: are these in-house kits? If not, please include the LOT number. The manufacturers of all reagents must be added to the text.

A: This is a case of lack of clarity in the previous presentation and the manufacturer of the kit has been restated.

Q: Line 140: the concentration of reagents should be included in the text and not the volume used.

A: The kit was purchased here to perform labeling and staining, and the concentration used was not indicated in the kit. Specific information on the kits has been added in this article.

 

I hope that the above response and modifications will satisfy you. If you have any questions, I look forward to your suggestions and responses!

Thanks again for your work! I wish you a wonderful day!

Yours sincerely,

Hengxian Qu