Green Tomato Extract Prevents Bone Loss in Ovariectomized Rats, a Model of Osteoporosis

Round 1

Reviewer 1 Report

In terms of formal requirements, the concept of the article is compliant. The title of the paper corresponds to the issues it addresses.   The authors have employed a method which complies with the criteria to be met by scientific papers. After minor corrections, the article may be approved for publication.  In order to increase the value of its content, I suggest the following:

1. Please determine the exact units where the authors were given the permission to conduct their studies.
2. The choice of the sample group, the research procedure and the analysis were correct, but no timescale was provided for the study.
3. Please provide more complete information on project (study) funding.
4. I am not sure whether the study is up to date as no time frame has been specified for it. Please provide this information.

5.The authors should present the limitations of study. State strengths and weaknesses of the study.

6.It does not make a bad impression to mention some of the latest publications in international literature. No reference was made to any recent literature (2020)

Author Response

Dear Reviewer,

Thank you for your comment.

Here we provide you the answers to your comments point by point:

1. This study was fully supported by Korea Food Research Institute. 
2. We had explained the duration of the study in Material and Methods, Animal Works part. We supplemented GTE into rats diet for 12 weeks. 
3. We had added details about the funding in the last part of the manuscript.
4. As stated in Point 2, we had explained the timeline of the study in Material and Methods.
5. We had addressed this suggestion in the last paragraph of the Discussion. 
6. We had updated References as suggested. 

Reviewer 2 Report

Herein the authors investigated the preventive effects of green tomatoes extract  on bone loss in an overectomized rat model. This is an interesting study. However, English language of the manuscript is substandard as it contains several typos and syntax errors.

Comments:

Provide histology imaging of key organs such as liver and kidney.

Perform western blotting experiments on the key proteins investigated in this study.

Author Response

Dear Reviewer,

Thank you for your review.

Here we provide the answer to your comments. 

1. Provide histology imaging of key organs such as liver and kidney.

Answer: The main hypothesis of this study is whether green tomatoes extract (GTE) with high tomatidine content could prevent post-menopausal osteoporosis in OVX rats, thus we only showed relevant data to prove this hypothesis. We are not sure about relevancy of histology imaging of other organs such as liver and kidney. However, if the reviewer were curious about the toxicity of GTE and its impact on key organs, we had showed serum AST and ALT analysis result as a reference in Figure 2. If the histology of key organs were mentioned based on the curiousity of GTE impact on the metabolism status of OVX rats, we added serum Adiponectin/Leptin analysis result in Figure 2.

 

2. Perform western blotting experiments on the key proteins investigated in this study.

Answer: Protein extraction in mineralized tissue such as tibia bone could result in demineralization of the tissue, further resulted in less precise immunoblotting analysis (Cleland, 2015). Also, referring to most of the osteoporosis related studies, changes mRNA level alone is enough to prove the efficacy of the treatment (Moriwaki et al., 2014; Elbahnasawy et al., 2019)

 

Ref.:

Cleland, Timothy P. and Deepak Vasishth. 2015. Bone protein extraction without demineralization utilizing principles from hydroxyapatite chromatography. Anal Biochem.

Elbahnasawy, Amr S.,  E. R. Valeeva, Eman M. El-Sayed, and I. I. Rakhimov. 2019. The Impact of Thyme and Rosemary on Prevention of Osteoporosis in Rats. Journal of Nutrition and Metabolism.

Moriwaki, Sawako, Keiko Suzuki, Masashi Muramatsu, Atsushi Nomura, Fumihide Inoue, Takeshi Into, Yuji Yoshiko, and Shumpei Niida. 2014. Delphinidin, One of the Major Anthocyanidins, Prevents Bone Loss through the Inhibition of Excessive Osteoclastogenesis in Osteoporosis Model Mice. PLos One.

Reviewer 3 Report

Authors results suggest that green tomato extract supplementation prevents severe post-menopausal bone loss by maintaining the regulation of bone homeostasis in OVX rats.

The work is well written and organized, but I suggest, if possible, to improve the introduction with a stronger rationale regarding the use of tomatidine and green tomatoes extract in post-menopausal bone loss.

Moreover I suggest to specify the variety of tomato choosed for chromatographic tomatidine identification.

Author Response

Dear Reviewer,

Thank you for your review.

Here we provide the answers to your comments.

(1) The work is well written and organized, but I suggest, if possible, to improve the introduction with a stronger rationale regarding the use of tomatidine and green tomatoes extract in post-menopausal bone loss.

Answered: We had addressed this issue in the paragraph 4 of Introduction.

(2) Moreover I suggest to specify the variety of tomato choosed for chromatographic tomatidine identification.

Answered: We had addressed this issue in the Materials and Methods: Preparation of Samples.

Reviewer 4 Report

Post-menopausal osteoporosis is usually cured by estrogen-replacement, but this therapy is controversial owing to health-related risks. Nirmala et al. proposed a study focused on green tomato extract (GTE) to contrast osteoporosis bone loss. They investigated both histomorphometrical parameters and gene expression to evaluate the GTE action on bone formation and resorption. The paper is very interesting, well-structured and with very interesting results.

 

Despite the accurate description, some aspects have to be revised:

• The authors indicated that they extracted total RNA from the tibia. Which part of the tibia did they take for RNA extraction? Why the author decided to measure mRNA and not also protein expression? Protein expression could complete the investigation.
• In the paragraph 3.2, the authors showed that white adipose tissue weight decreased in GTE treated rats. The authors should describe better from which part of the rat body they took the white adipose tissue.
• Figure 2C shows that E2 group displayed no significant difference but histogram seems to indicate that there is significative difference. Isn’t it?
• In the paragraph 3.3, histomorphometrical results should be explained more extensively.
• Figure 6E is not well-related to the other parts of the figure and maybe it is not in the right position. Why did not the authors propose it as graphical abstract or as a single image?
• In the discussion section, the authors affirmed that GTE supplementation significantly upregulated Bmp2 and its downstream genes and that Bmp2 might be responsible for the improvement of bone mass in GTE supplemented OVX rats. Anyway, these assertions are dubious. Indeed, results showed that bmp2 is upregulated only in the GH group, whereas all the other osteoblastic, except ALP, are upregulated in both GL and GH groups. How do the authors explain this? They should implement this part of the discussion.

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

Thank you for your advices and comments.

Here we provide the answers to your review.

• The authors indicated that they extracted total RNA from the tibia. Which part of the tibia did they take for RNA extraction? Why the author decided to measure mRNA and not also protein expression? Protein expression could complete the investigation.

Answer: The whole tibia tissue was used in the extraction. Protein extraction in mineralized tissue such as tibia bone could result in demineralization of the tissue, further resulted in less precise immunoblotting analysis (Cleland, 2015). Also, referring to most of the osteoporosis related studies, changes mRNA level alone is enough to prove the efficacy of the treatment (Moriwaki et al., 2014; Elbahnasawy et al., 2019)

Ref.:

Cleland, Timothy P. and Deepak Vasishth. 2015. Bone protein extraction without demineralization utilizing principles from hydroxyapatite chromatography. Anal Biochem.

Elbahnasawy, Amr S.,  E. R. Valeeva, Eman M. El-Sayed, and I. I. Rakhimov. 2019. The Impact of Thyme and Rosemary on Prevention of Osteoporosis in Rats. Journal of Nutrition and Metabolism.

Moriwaki, Sawako, Keiko Suzuki, Masashi Muramatsu, Atsushi Nomura, Fumihide Inoue, Takeshi Into, Yuji Yoshiko, and Shumpei Niida. 2014. Delphinidin, One of the Major Anthocyanidins, Prevents Bone Loss through the Inhibition of Excessive Osteoclastogenesis in Osteoporosis Model Mice. PLos One.

• In the paragraph 3.2, the authors showed that white adipose tissue weight decreased in GTE treated rats. The authors should describe better from which part of the rat body they took the white adipose tissue.

Answer: We had addressed the suggested issue in Table 3.

• Figure 2C shows that E2 group displayed no significant difference but histogram seems to indicate that there is significative difference. Isn’t it?

Answer: We admitted that there was an error in the statistical analysis labelling and corrected it.

• In the paragraph 3.3, histomorphometrical results should be explained more extensively.

Answer: We improved the explanation as suggested.

• Figure 6E is not well-related to the other parts of the figure and maybe it is not in the right position. Why did not the authors propose it as graphical abstract or as a single image?

Answer: As suggested, we separated the mentioned figure into a single image (Figure 7).

• In the discussion section, the authors affirmed that GTE supplementation significantly upregulated Bmp2 and its downstream genes and that Bmp2 might be responsible for the improvement of bone mass in GTE supplemented OVX rats. Anyway, these assertions are dubious. Indeed, results showed that bmp2 is upregulated only in the GH group, whereas all the other osteoblastic, except ALP, are upregulated in both GL and GH groups. How do the authors explain this? They should implement this part of the discussion.

Answer: Despite that we could only proved the statistical significancy of BMP2 mRNA expression upregulation in GH, we did observed minor (±0.2 folds) upregulation in GL group. The expression of BMP2 downstream genes, Runx2 and Smads, were exhibited in the same pattern where GH effect is bigger than GL. Although there were some exceptions seen in Osterix and ALP expressions, the upregulation pattern of both groups on the rest of the downstream signaling genes are also linear with this (Figure 5). Most importantly, these changes were relevant with the phenotype of OVX rats (Figure 3). As suggested, we added explanation about this issue in the discussion (Paragraph 3). 

 

Round 2

Reviewer 2 Report

The authors failed to revise the manuscript as suggested by the reviewer.

Author Response

Dear Reviewer,

 

We are sorry for not able to fulfill your review in Round 1.

However, as we stated in the answer in Round 1, we are unable to perform immunoblotting using tibial bone. Apart from your previously pointed issues, if there are additional parameters you want us to add into our manuscript please let us know.

 

Thank you.