Policosanol Stimulates Osteoblast Differentiation via Adenosine Monophosphate-Activated Protein Kinase-Mediated Expression of Insulin-Induced Genes 1 and 2

Round 1

Reviewer 1 Report (Previous Reviewer 2)

As my questionable comments, the authors are fully described in the revised manuscript.

The quality of English is good.

Author Response

Thank you for your review of the revised manuscript.

Reviewer 2 Report (Previous Reviewer 1)

1.    Abstract: Line 25-27, conclusion: what do the results indicate or suggest? I suggest that the authors add such an indication as well as the implications of their findings into the conclusion. For example, “Taken together, these results show that……via AMPK-mediated INSIGs expression, indicating or suggesting……”.

2.    Introduction: Paragraph 2, line 40-48, there is 1 sentence lacking to wrap up this whole paragraph. I suggest that the authors add 1 sentence at the end of this paragraph. For example, “However, how policosanol affects bone formation is unclear.”

3.    Introduction: Line 43-47, “The major function of policosanol……and inhibits hepatic lipid accumulation”, I do not understand why the authors introduce the major function of policosanol, and the effect of policosanol on cardiovascular diseases. These sentences are misleading. This study is about osteogenesis. If the major function of policosanol is to increase high-density lipoprotein cholesterol, why do the authors then investigate the effect of policosanol on osteogenesis? The authors can introduce the different functions of policosanol, but they should explain the importance of the effect of policosanol on osteogenesis, or at least explain the relation between the major (other) function(s) of policosanol and the effect of policosanol on osteogenesis.

4.    Introduction: Line 49-55, “The adenosine monophosphate-activated protein kinase……increases osteoblast differentiation”, the authors introduced the function of AMPK in different cell types. However, only 1 sentence addresses bone research. I suggest the authors to elaborate more about the function of AMPK in bone cells, especially osteoblasts.

5.    Introduction: Line 65, “policosanol inhibits PI-induced VSMC……”, please provide first the full names of “PI” and “VSMC”, and thereafter use the abbreviations.

6.    Materials and Methods: The descriptions of relative ALP activity and relative mineralization quantification are lacking. Please provide this information in the materials and methods section.

7.    Results: The description of the results is unclear. Please elaborate more on the findings. For example, line 176-177, “As shown in Figure 1a, policosanol induced the mRNA expression of osteogenic markers such as Dlx5 and Runx2”, this sentence does not describe what is actually shown in figure 1a. Dlx5 gene expression is increased by policosanol at day 1 (about 4-fold), day 2 (about 4-fold) and day 4 (about 4-fold). Runx2 gene expression is increased by policosanol at day 1 (about 2-fold), and day 4 (about 7-fold), but not at day 2 (about 2-fold). Runx2 gene expression is similar at day 1 and day 2. However, there is a significant difference at day 1 (2 *, p<0.01), but not at day 2. Please double check the statistical analysis. In addition, I am wondering whether there are any significant differences between day 1 (Runx2) and day 4 (Runx2), and between day 2 (Runx2) and day 4 (Runx2) in figure 1a?

8.    Results: Line 177- 178, “Furthermore, policosanol increased Dlx5 and Runx2 protein levels (Figure 1b)”, this sentence does not adequately describe what is shown in figure 1b. In figure 1b, Runx2 protein expression is increased by policosanol at day 2 and 4, but not at day 6 and 8. Please double check the statistical analysis between day 0 (Runx2) and day 8 (Runx2) in figure 1b. Line 178-180, “Moreover, under osteogenic conditions, policosanol showed additional effects on ALP activity and mineralization”, please describe clearly the results of ALP activity and mineralization as shown in figure 1c and 1d. What are “Rel. ALP activity” and “Rel. mineralization” in figure 1c and 1d?

The quality of English language should be improved.

Author Response

1. Abstract: Line 25-27, conclusion: what do the results indicate or suggest? I suggest that the authors add such an indication as well as the implications of their findings into the conclusion. For example, “Taken together, these results show that……via AMPK-mediated INSIGs expression, indicating or suggesting……”.

 

Thank you very much for your suggestion. In response to your suggestion, we have additionally written an implication to our findings.

 

2. Introduction: Paragraph 2, line 40-48, there is 1 sentence lacking to wrap up this whole paragraph. I suggest that the authors add 1 sentence at the end of this paragraph. For example, “However, how policosanol affects bone formation is unclear.”

 

Thank you for your suggestion. As you mentioned, we added that sentence.

 

3. Introduction: Line 43-47, “The major function of policosanol……and inhibits hepatic lipid accumulation”, I do not understand why the authors introduce the major function of policosanol, and the effect of policosanol on cardiovascular diseases. These sentences are misleading. This study is about osteogenesis. If the major function of policosanol is to increase high-density lipoprotein cholesterol, why do the authors then investigate the effect of policosanol on osteogenesis? The authors can introduce the different functions of policosanol, but they should explain the importance of the effect of policosanol on osteogenesis, or at least explain the relation between the major (other) function(s) of policosanol and the effect of policosanol on osteogenesis.

 

Thank you very much for your thoughtful comments. The intention of sentence you mentioned was written with the intention of introducing the basic function of policosanol. And the meaning of policosanol's main function, cholesterol level control, on bone metabolism is written in the discussion section of the manuscript. If you look at paragraph 5 of the discussion, "High cholesterol levels reduce the proliferation and differentiation of osteoblasts, thereby inhibiting bone formation and increasing the risk of osteoporosis." I think it contains the meaning of controlling the level of cholesterol in the bone metabolism process. Additionally, since few studies have been conducted on the effects of policosanol on bone formation, we wanted to conduct this study.

 

4. Introduction: Line 49-55, “The adenosine monophosphate-activated protein kinase……increases osteoblast differentiation”, the authors introduced the function of AMPK in different cell types. However, only 1 sentence addresses bone research. I suggest the authors to elaborate more about the function of AMPK in bone cells, especially osteoblasts.

 

Thank you for your meaningful suggestion. As you mentioned, we have additionally written an explanation of what role AMPK plays in osteoblast differentiation.

 

5. Introduction: Line 65, “policosanol inhibits PI-induced VSMC……”, please provide first the full names of “PI” and “VSMC”, and thereafter use the abbreviations.

 

Thank you for your comment. As you mentioned, we have provided the full name of the abbreviation.

 

6. Materials and Methods: The descriptions of relative ALP activity and relative mineralization quantification are lacking. Please provide this information in the materials and methods section.

 

Thanks for the thoughtful advice. As mentioned, we have additionally written the contents related to the relative quantification of ALP activity and mineralization in the Materials and Methods section.

 

7. Results: The description of the results is unclear. Please elaborate more on the findings. For example, line 176-177, “As shown in Figure 1a, policosanol induced the mRNA expression of osteogenic markers such as Dlx5 and Runx2”, this sentence does not describe what is actually shown in figure 1a. Dlx5 gene expression is increased by policosanol at day 1 (about 4-fold), day 2 (about 4-fold) and day 4 (about 4-fold). Runx2 gene expression is increased by policosanol at day 1 (about 2-fold), and day 4 (about 7-fold), but not at day 2 (about 2-fold). Runx2 gene expression is similar at day 1 and day 2. However, there is a significant difference at day 1 (2 *, p<0.01), but not at day 2. Please double check the statistical analysis. In addition, I am wondering whether there are any significant differences between day 1 (Runx2) and day 4 (Runx2), and between day 2 (Runx2) and day 4 (Runx2) in figure 1a?

 

Thanks for the very meaningful suggestions. As you mentioned, the description of the figure has been rewritten in more detail. In addition, errors in the statistical processing process were identified, and errors were corrected by performing statistical processing again. Lastly, we checked whether there was a significant difference between the values of day 1 and 2 and the value of day 4 in Runx2 gene expression, which was the part you were curious about, and as a result, it was confirmed that there was a significant difference. The contents of checking the significant difference are shown in the figure below.

 

 

 

 

##: compared with day 1

$$: compared with day 2

 

 

8. Results: Line 177- 178, “Furthermore, policosanol increased Dlx5 and Runx2 protein levels (Figure 1b)”, this sentence does not adequately describe what is shown in figure 1b. In figure 1b, Runx2 protein expression is increased by policosanol at day 2 and 4, but not at day 6 and 8. Please double check the statistical analysis between day 0 (Runx2) and day 8 (Runx2) in figure 1b. Line 178-180, “Moreover, under osteogenic conditions, policosanol showed additional effects on ALP activity and mineralization”, please describe clearly the results of ALP activity and mineralization as shown in figure 1c and 1d. What are “Rel. ALP activity” and “Rel. mineralization” in figure 1c and 1d?

 

Thanks for the thoughtful advice. As you mentioned, similar to the previous answer, I edited and wrote the manuscript in detail to provide a more detailed explanation of the figure. In the case of statistical processing, it was used only to indicate an increase in protein level by policosanol treatment, but as you mentioned, a statistically significant difference was additionally displayed even in a decrease in protein level. It made me rethink about representing data. Thank you again.

 

In addition to the above information, a picture file is attached, so a word file is attached.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (Previous Reviewer 1)

The authors did good job.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript by Kim et al. addresses the effects of policosanol on osteoblast differentiation via AMPK-mediated expression of INSIG1 and 2 in vitro and in vivo. The authors investigated osteogenic gene expression, ALP activity, extracellular mineralization, AMPK, and INSIGs expression in MC3T3-E1 pre-osteoblasts treated with policosanol. Furthermore, the authors discussed the expression of INSIGs regulated by AMPK using  silencing and transfection methodology. In vivo experiments demonstrated that policosanol can induce zebrafish fin regeneration. This manuscript is interesting, and well structured. However, the authors need to improve their manuscript at several points, as well as the English language written. Specific issues need clarification and should be addressed, which are indicated below.

1.    Abstract: Conclusion does not answer the aim or hypothesis of this study. I suggest the authors to rephrase the aim or hypothesis of this study.

2.    Introduction: I suggest to change the sentence (“In addition,……osteoblast differentiation)” (line 52-54) into “In addition, plant-derived ……osteoblast differentiation, such as policosanol.”

3.    Introduction: Please rephrase this sentence in line 56-58 (“It contains……carbon atoms in cosanol”). It is difficult to follow.

4.    Introduction: The main function of policosanol is to increase the level of high-density lipoprotein cholesterol and decrease the level of low-density lipoprotein cholesterol. In addition, the function of policosanol reduces blood pressure and inhibits fat production (line 58-63). However, in the abstract, the authors only introduced the additional function of policosanol, not the main function of policosanol. I suggest the authors to rephrase these contents.

5.    Discussion: How do AMPK and INSIs regulate osteoblast differentiation in vascular smooth muscle cells and MC3T3-E1 cells? What is the difference between the 2 cell types treated with policosanol?

6.    Discussion: In the whole section of discussion, the authors introduced some knowledges based on literature and their own work. However, the authors did not explain clearly why and how the effect of policosanol on osteoblast differentiation via AMPK-mediated expression of INSIGs based on their findings in this study. For example, line 323-329 (“In this study,……osteoblast differentiation.”), the authors only descripted the results, did not explain the reason.

7.    The authors already published several similar work about the effect of policosanol on osteoblast differentiation via AMPK-mediated expression of INSIGs. Please make it clear what is the novelty in this study.

8.    Conclusion: I suggest the authors to combine the 2 paragraphs into 1 paragraph.

Reviewer 2 Report

The manuscript is improved and well-constructed as commented.