Protective Effect of Panicum dichotomiflorum in a Rodent Model of Testosterone-Induced Benign Prostatic Hyperplasia

Round 1

Reviewer 1 Report

The manuscript is interesting and the topic always actual. However, the outcomes should be better described not only using charts but also by few concise phrases. In Discussions it should be mentioned the study limitations as well.

Author Response

Editor Scientia pharmaceutica

January 13, 2024

Dear editor:

We have revised the manuscript entitled “Protective effect of Panicum dichotomiflorum in a rodent model of testosterone-induced benign prostatic hyperplasia” according to the suggestions made by reviewers. We have revised the text as indicated below, where we seek to address all of the reviewers’ comments in detail. I would like give sincere thanks to all reviewers for their time and effort reviewing this manuscript. All of their comments and suggestions were very constructive and critical which resulted in marked improvement of the manuscript. Responses to their inquiries are attached as below.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Hyo-Jung Kwun, D.V.M., Ph.D.

Associate Professor

Department of Veterinary Pathology, College of Veterinary Medicine

Chungnam National University

99 Daehak-ro, Yuseong-gu, Daejeon 305-764

South Korea

Tel: 82-42-821-6755

Fax: 82-42-821-8903

Email: [email protected]

Reviewer Comments:

Reviewer #1

The manuscript is interesting and the topic always actual. However, the outcomes should be better described not only using charts but also by few concise phrases. In Discussions it should be mentioned the study limitations as well.

Answer: We appreciate your comments. We have revised discussion section and added the study limitations in discussion section (page 12, line 395-405).

Reviewer 2 Report

The manuscript describes the action of Panicum dichotomiflorum extract on benign prostate hyperplasia in a clear and comprehensive way and provides new data for the search of potential BPH treatments. Yet there are parts of presented work that must be improved prior to publication.

Introduction:

In introduction section authors describe several previously found components of PD (tricin, luteolin, luteolin-4'-O-β-D-glucopyranoside, luteolin-7-O-β-D-glucopyranoside, lignan, pinoresinol) and their biological action as basis for potential anti-BPH effect of PD. Yet, in section 3.1 authors present only tetrahydroxyflavonoid-C-hexosyl-O-deoxy-hexoside and dichotomin as main PD extract components. The difference between expected and observed components should be addressed in discussion.

Materials and methods:

The number of animals used is not provided. How many animals were assigned to each group and were animal randomized by weight?

As usually in BPH model animals are castrated to reduce the influence of endogenous testosterone, were the rats castrated or not before the start of testosterone administration?

What was used as vehicle for testosterone injections, and for finasteride and PD extract?

As the methanol was used as solvent for PD extract, how did authors ensure that effects described are due caused by PD and not methanol?

At which point of experiment the blood was obtained?

How and when experiment was stopped and how animals were euthanized?

Line 130 (For each prostate, a tissue sample was obtained from the ventral lobe and fixed in 10% formalin) and Line 138 (Isolated prostate were fixed in 10% formalin.) contradict each other – were whole prostate fixed or only samples from ventral lobes?

Line 150 – “Proteins (15 μg each) were separated” - how the proteins were obtained?

Results:
The weight of animals at the end of experiment should be provided as the resulting relative prostate weight can be influenced by substances affecting body mass.

Table 2 – for more clear presentation it may be beneficial to round up absolute prostate weights to 2 decimal places and present relative weight as mg/100g body weight

Also, lines 195-196 state marked increase of both absolute and relative prostate weight, but in Table 2 statistical difference is designated only for relative weight.

Please check the scales for testosterone and dihydrotestosterone in Figure 2 – are you sure its ng and pg per milliliter and not per liter?

Also, it should be noted that testosterone level was lower in finasteride-treated groups compared to BPH control. As finasteride inhibits conversion of testosterone to dihydrotestosterone, lower DHT levels, as also shown in current work, are expected, but as testosterone is provided externally at controlled rate, it is expected for its levels to remain the same or even rise. Can observed BPH inhibition be caused by lower circulating testosterone levels? Please address in Discussion.

Discussion:
As PD in given dose decreased prostate weight in lesser degree than finasteride, it should have other advantages to be considered an alternative drug, such as lower side effects. Some information on PD toxicity should be included.

Comments for author File: Comments.pdf

Language comments
While readable, significant language improvement is required across whole manuscript.

Several examples:

Line 16 – “expression were significantly inhibites following the treatment with PD. Testosterone-induced increases of prostate gland epithelial thickness…” should be “significantly inhibited” and “increase of prostate gland epithelial thickness…”

Line 139 – “Epithelial thickness was evaluatedd from the ventral lobes of prostates as described previously [33]. In briefly, 30 randomly 140 selected regions for each prostate were measured in thickness (μm).” should be “Epithelial thickness (μm) was measured in the ventral lobes of prostate glands as described previously [33]. For each prostate, 30 randomly selected regions were evaluated.”

Line 143 – “Five ventral lobe of prostate of each animal were randomly assigned by incubation overnight with anti-PCNA” – did authors meant five regions?

Line 177 – “We used a software called GraphPad Prism 6 for statistical analyses.” should be “We used GraphPad Prism (Software provider?) software for statistical analysis.”

Author Response

Editor Scientia pharmaceutica

January 13, 2024

Dear editor:

We have revised the manuscript entitled “Protective effect of Panicum dichotomiflorum in a rodent model of testosterone-induced benign prostatic hyperplasia” according to the suggestions made by reviewers. We have revised the text as indicated below, where we seek to address all of the reviewers’ comments in detail. I would like give sincere thanks to all reviewers for their time and effort reviewing this manuscript. All of their comments and suggestions were very constructive and critical which resulted in marked improvement of the manuscript. Responses to their inquiries are attached as below.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Hyo-Jung Kwun, D.V.M., Ph.D.

Associate Professor

Department of Veterinary Pathology, College of Veterinary Medicine

Chungnam National University

99 Daehak-ro, Yuseong-gu, Daejeon 305-764

South Korea

Tel: 82-42-821-6755

Fax: 82-42-821-8903

Email: [email protected]

Reviewer Comments:

Reviewer #2

The manuscript describes the action of Panicum dichotomiflorum extract on benign prostate hyperplasia in a clear and comprehensive way and provides new data for the search of potential BPH treatments. Yet there are parts of presented work that must be improved prior to publication.

Introduction:

1. In introduction section authors describe several previously found components of PD (tricin, luteolin, luteolin-4'-O-β-D-glucopyranoside, luteolin-7-O-β-D-glucopyranoside, lignan, pinoresinol) and their biological action as basis for potential anti-BPH effect of PD. Yet, in section 3.1 authors present only tetrahydroxyflavonoid-C-hexosyl-O-deoxy-hexoside and dichotomin as main PD extract components. The difference between expected and observed components should be addressed in discussion.

Answer: We appreciate your comments. We have added the explanation in discussion (page 12, line 395-405).

Materials and methods:

2. The number of animals used is not provided. How many animals were assigned to each group and were animal randomized by weight?

Answer: We have added the animal number of each group in materials and methods (page 3, line 120).

3. As usually in BPH model animals are castrated to reduce the influence of endogenous testosterone, were the rats castrated or not before the start of testosterone administration? What was used as vehicle for testosterone injections, and for finasteride and PD extract?

Answer: This is very critical point. We appreciate your comments. In our previous studies, we established the BPH rat model (Res Rep Urol. 2022;14:313-326. Evid Based Complement Alternat Med. 2022;2022:7902920. J Ethnopharmacol. 2020;255:112779. J Ethnopharmacol. 2019;233:115-122. Biol Pharm Bull. 2019;42(1):1-9). We administrated testosterone for 4 weeks without castration and relative prostate weight was significantly increased in testosterone-treated rats compared with vehicle-treated normal control rat. In the present study, we used the same animal model to induce BPH.

The vehicle used in this study was corn oil or PBS for testosterone or finasteride/PD extract, respectively. We have added this information in Table 2 (page 6).

4. As the methanol was used as solvent for PD extract, how did authors ensure that effects described are due caused by PD and not methanol? At which point of experiment the blood was obtained? How and when experiment was stopped and how animals were euthanized?

Answer: In the present study, we extracted PD using methanol. The no observed adverse effect level (NOAEL) for methanol was 500 mg/kg /day. In addition, in 90-day oral gavage study of SD rats, the effect of methanol on prostate has not been reported (OECD, 2004, https://www.industrialchemicals.gov.au/sites/default/files/Methanol_Human%20health%20tier%20II%20assessment.pdf). Many studies have reported the protective effect of methanol-extracted natural products against BPH, but the effect was not attributed to methanol origin. (Vet Res Forum. 2023;14:59-64. Evid Based Complement Alternat Med. 2017;2017:9124240. Biosci Biotechnol Biochem. 2009;73(9):1911-4).  

As your suggestion, we have now added the experiment methods for blood collection and necropsy in materials and methods (page 3, line 129-130).

5. Line 130 (For each prostate, a tissue sample was obtained from the ventral lobe and fixed in 10% formalin) and Line 138 (Isolated prostate were fixed in 10% formalin.) contradict each other – were whole prostate fixed or only samples from ventral lobes?

Answer: We apologize for the confusion. Isolated ventral lobes of prostates were divided into two parts for formalin and other analysis (ELISA, Western blot, and RT-qPCR). We have now clarified the description in materials and methods (page 3, line 135-136).

6. Line 150 – “Proteins (15 μg each) were separated” - how the proteins were obtained?

Answer: We have now added the description in materials and methods (page 4, line 155-157).

Results:

7. The weight of animals at the end of experiment should be provided as the resulting relative prostate weight can be influenced by substances affecting body mass.

Answer: Thank you for important question. As your suggestion, the body weight at the end of experiment have been now added in Table 2 (page 6).

8. Table 2 – for more clear presentation it may be beneficial to round up absolute prostate weights to 2 decimal places and present relative weight as mg/100g body weight

Answer: As your suggestion, we have revised Table 2 (page 6).

9. Also, lines 195-196 state marked increase of both absolute and relative prostate weight, but in Table 2 statistical difference is designated only for relative weight.

Answer: We apologize for the confusion. As your comment, we have now added the statistical difference in Table 2 (page 6).

10. Please check the scales for testosterone and dihydrotestosterone in Figure 2 – are you sure its ng and pg per milliliter and not per liter?

Answer: We apologize for the confusion. We have now changed the scale correctly in Figure 2.

11. Also, it should be noted that testosterone level was lower in finasteride-treated groups compared to BPH control. As finasteride inhibits conversion of testosterone to dihydrotestosterone, lower DHT levels, as also shown in current work, are expected, but as testosterone is provided externally at controlled rate, it is expected for its levels to remain the same or even rise. Can observed BPH inhibition be caused by lower circulating testosterone levels? Please address in Discussion.

Answer: In previous studies, finasteride decreased testosterone level of serum (Nutrients. 2017;9(10):1070, Biol Pharm Bull. 2019;42(1):1-9) and we added the description in discussion (page 10, line 325-328).

Discussion

12. As PD in given dose decreased prostate weight in lesser degree than finasteride, it should have other advantages to be considered an alternative drug, such as lower side effects. Some information on PD toxicity should be included.

Answer: This is very critical point. We appreciate your comments. It has been reported that PD has hepatotoxicosis in horses and sheep (J Vet Diagn Invest 2019, 31, 90-93. J Vet Intern Med 2006, 20, 1414-1421). As your suggestion, we have evaluated any toxicity in our experimental setting and have now added the result (page 10, line 299-311).

Language comments : While readable, significant language improvement is required across whole manuscript.

Several examples:

13. Line 16 – “expression were significantly inhibites following the treatment with PD. Testosterone-induced increases of prostate gland epithelial thickness…” should be “significantly inhibited” and “increase of prostate gland epithelial thickness…”

Answer: We apologize for the confusion. We have carefully checked the entire manuscript and all the figures and corrected the typographical errors. Furthermore, we have received English proofreading service (https://sciencemanager.com).

14. Line 139 – “Epithelial thickness was evaluatedd from the ventral lobes of prostates as described previously [33]. In briefly, 30 randomly 140 selected regions for each prostate were measured in thickness (μm).” should be “Epithelial thickness (μm) was measured in the ventral lobes of prostate glands as described previously [33]. For each prostate, 30 randomly selected regions were evaluated.”

Answer: We have corrected the sentence (page 3, line 143-144).

15. Line 143 – “Five ventral lobe of prostate of each animal were randomly assigned by incubation overnight with anti-PCNA” – did authors meant five regions?

Answer: We apologize for the confusion. We have clarified the description (page 4, line 147-148).

16. Line 177 – “We used a software called GraphPad Prism 6 for statistical analyses.” should be “We used GraphPad Prism (Software provider?) software for statistical analysis.”

Answer: We have corrected as your suggestion (page 4, line 185-187).

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have assessed a comprehensive range of parameters in a 4-armed study including finasteride treatment as positive control. Unfortunately, there are many concerns about the study, particularly about data presentation and interpretation.

Main comments:

1.      The testosterone model of BPH is commonly used and potentially useful. However, a major limitation of the model is that anything anti-androgenic intrinsically is effective in this model but not necessarily in at least some patients; on the other hand, something not targeting the androgen pathway may be ineffective in this model but nonetheless effective in some patients. This limitation of the model must be discussed.

2.      While rodent models are often used in BPH research and have some value, their general limitation is a difference in anatomy of the prostate between rodents and humans. The implications of such differences must explicitly be discussed as a study limitation.

3.      The Abstract claims that the study aim was to explore effects “of PD on BPH progression” (c.f., l. 12) whereas the main Introduction defines the study aim as investigation of “therapeutic efficacy” (c.f. l. 88); by the way, this turns into “protective effect” in l. 194 or “therapeutic potential” in l. 286. It may be a wording issue, but the study design intrinsically does neither allow determining effects on progression, which requires at least two time points being tested, nor of treatment efficacy. If I understand the design correctly, PD and testosterone treatment started concomitantly. This implies that the study has looked at the prevention of BPH in the testosterone model but neither at progression nor at treatment (the latter implying that PD administration would have started after BPH had developed). Therefore, the entire manuscript including the Abstract needs rewording to focus on prevention and remove references to progression and treatment.

4.      The current study design with concomitant start of testosterone and PD administration looks at BPH prevention. However, the present Introduction is based on treatment. Thus, the Introduction does not match what is being studied. Either the study must be repeated in a true treatment design (i.e., initiation of PD administration after BPH has developed) or the Introduction must be changed to providing a rationale why there is an unmet medical need for the prevention of BPH.

5.      The word “significant” is ambiguous because it means relevant, important, or similar in plain English but a calculated p-value smaller than the prespecified statistical alpha in a statistical context. The two are not necessarily linked. Accordingly, it remains unclear what the authors mean. Therefore, many statisticians recommend avoiding the word “significant” and replacing it with whatever is meant. Moreover, indications of statistical significance should never be mentioned without accompanying effect size indicators. Something may have a low p-value but nonetheless the effect size may be just 5% (probably irrelevant biologically) or 500% or anything in between and above. Please implement throughout the manuscript including the Abstract.

6.      Guidelines on data robustness recommend that scientific manuscripts should differentiate between studies designed to test a pre-specified statistical null hypothesis and all others, the latter by default being exploratory (Vollert et al., 2022; Vollert et al., 2020). To qualify as null hypothesis testing, a study must have a prespecified primary endpoint and a sample size based on pre-study power calculations. As this condition apparently is not met in the present study, it is an exploratory study by default. Of note, there is nothing wrong with well designed exploratory studies. However, the exploratory character must be stated explicitly, e.g., at the end of the main Introduction and/or in Methods.

7.      The manuscript draws cause-effect conclusions based on associations. This is scientifically not warranted. Different types of experiments would be needed to justify cause-effect conclusions.

8.      If flavonoids are key components of PD extracts, it is important to realize that flavonoids belong to the group of pan-assay interfering substance (PAINS) (Baell and Walters, 2014), which leads to many artefacts. This needs to be discussed as a study limitation.

9.      Please provide a dose justification primarily for the PD dose but also for finasteride.

10.  Sections 2.3 and 2.4 lack information on sample sizes, i.e., number of rats in each group. Please add.

11.  Histological examination is notoriously vulnerable for investigator bias. Please add an explicit statement whether the person selecting regions for viewing was blinded for group allocation.

12.  While GAPDH is frequently used to normalize RT-PCR findings, there are multiple examples where the expression of GAPDH itself is changed by an intervention. Therefore, please provide quantitative data on GAPDH expression (Ct values) in each group.

13.  Section 2.10: A) It follows from the exploratory character of the study that calculated p-values cannot be interpreted as hypothesis testing but only as descriptive. This must be stated explicitly. B) It did not become clear to me which inter-group comparisons were made based on the Dunnett post-tests. This should be stated explicitly.

14.  Based on seminal work by Weissgerber (Weissgerber et al., 2015) many leading journals including all journals of the Nature family mandate that continuous data are not presented as bar graphs but as scatter plots or bar graphs overlaid with scatter plots. Please implement.

15.  Figure 4B in comparison to figure 5 and table 2 creates a logical dilemma. How can prostate weight still be higher in the Fina and PD groups as compared to control if proliferation if anything is even less than in the control group? Only two logical explanations come to my mind. Either PCNA staining is a poor marker of cell proliferation, or finasteride and PD inhibit apoptosis relative to control (Figure 5 does not support this). I do not have a good explanation for this, but this dilemma must be discussed transparently as it has major implications for the overall interpretation of the results.

16.  It is interesting to note that both finasteride and PD have strong inhibitory effects on some parameters (bringing them down to control levels or even lower) but weaker effects on others. The implications of this observation need to be discussed.

17.  Pathologists define hyperplasia as an increase in cell number (as opposed to hypertrophy being an increase in cell size). Apparently, the authors feel strongly about hyperplasia (a histological diagnosis) but I did not find any assessment of cell number in the manuscript. Perhaps the only exception is PCNA staining, but this apparently has major problems (see above). Thus, what the authors have study is prostate enlargement (the human equivalent of which would be benign prostatic enlargement, BPE). Therefore, I feel strongly that conclusions on BPH are not warranted based on the currently presented data. Either robust data on changes in cell number must be presented, or conclusions on BPH must be changed to those on BPE. This is important because the degree of LUTS and prostate size are only poorly related, if at all.

Other comments

18.  The overall manuscript is understandable but nonetheless may benefit from proofreading by a native speaker or professional translator. For instance, articles are often missing before nouns. If a proofreader is engaged, the name of this person should be mentioned in the acknowledgement section.

19.  L. 10: This sentence is factually wrong because hyperplasia is often but not necessarily accompanied by prostatic enlargement. Please reword.

20.  L. 32: References 1 and 2 are used to support claims on the pathophysiology of BPH. A) Reference 1 is a review on efficacy and tolerability and does not have a focus on pathophysiology. B) Reference 2 is more than 25 years old. Do you expect this to reflect the state of the art of our understanding of the pathophysiology of BPH? I do not. I suggest replacing both references with more appropriate review articles.

21.  L. 56-57: I disagree with this statement for two reasons: Firstly, ARI have only shown efficacy in men with prostates greater than 40 ml. Thus, they are not pillars of BPH treatment in general. Second, ARI have a specific side effect profile. Other than adverse effects on sexual function, they may cause adverse effects on the brain (possibly as part of the post-ARI syndrome) and have been associated with an increased risk of developing type 2 diabetes in three studies. Please reword this statement.

22.  L. 57-58: What do you mean by “many 5-ARIs, including dutasteride and finasteride”? As far as I know, these are the only ones.

23.  L. 69: Do you really mean “feed” (not a noun) or rather “food”?

24.  L. 127-130: The preceding lines define the four study groups but do not clearly link them to the definitions of “negative” control and “positive” control. Please clarify. Moreover, this seems to me like a rather complex analysis of BPH. Why not simply report on prostate weight in each group?

25.  Wherever possible, please provide RRID of the animal strain, of kits, antibodies etc. Where unavailable, please add catalog numbers for unequivocal identification.

26.  L. 138 states that isolated prostate was placed in formalin, but l. 130 implies that only ventral lobe was used. Please clarify.

27.  Table 2: To better understand the data, an additional column should be added showing body weight in each group. Particularly, I am interested to see whether PD affects body weight.

28.  Figure 2C: There may be a mistake in the y-axis label, which currently reads “fold change”. A one-fold change is a doubling whereas no change (by definition true for the NC group) is a 0-fold change. Please either adapt the graph or change the y-axis label to e.g., “fold of control”. This comment also applies to other figures currently showing “fold changes” such as 4C.

29.  Figure 3A: Please add a scale bar to each panel.

References

Baell J and Walters MA (2014) Chemical con artists foil drug discovery. Nature 513:481-483.

 Vollert J, Macleod M, Dirnagl U, Kas MJ, Michel MC, Potschka H, Riedel G, Wever KE, Würbel H, Steckler T and Rice ASC (2022) The EQIPD framework for rigor in the design, conduct, analysis and documentation of animal experiments. Nat Methods 19:1334-1337.

 Vollert J, Schenker E, Macleod M, Bespalov A, Wuerbel H, Michel M, Dirnagl U, Potschka H, Waldron A-M, Wever K, Steckler T, van de Casteele T, Altevogt B, Sil A and Rice ASC (2020) Systematic review of guidelines for internal validity in the design, conduct and analysis of preclinical biomedical experiments involving laboratory animals. BMJ Open Science 4:e100046.

 Weissgerber TL, Milic NM, Winham SJ and Garovic VD (2015) Beyond bar and line graphs: time for a new data presentation paradigm. PLoS Biol 13:e1002128.

There are some issues but the text is clearly understandable.

Author Response

Editor Scientia pharmaceutica

January 13, 2024

Dear editor:

We have revised the manuscript entitled “Protective effect of Panicum dichotomiflorum in a rodent model of testosterone-induced benign prostatic hyperplasia” according to the suggestions made by reviewers. We have revised the text as indicated below, where we seek to address all of the reviewers’ comments in detail. I would like give sincere thanks to all reviewers for their time and effort reviewing this manuscript. All of their comments and suggestions were very constructive and critical which resulted in marked improvement of the manuscript. Responses to their inquiries are attached as below.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Hyo-Jung Kwun, D.V.M., Ph.D.

Associate Professor

Department of Veterinary Pathology, College of Veterinary Medicine

Chungnam National University

99 Daehak-ro, Yuseong-gu, Daejeon 305-764

South Korea

Tel: 82-42-821-6755

Fax: 82-42-821-8903

Email: [email protected]

Reviewer Comments:

Reviewer #3

The authors have assessed a comprehensive range of parameters in a 4-armed study including finasteride treatment as positive control. Unfortunately, there are many concerns about the study, particularly about data presentation and interpretation.

Main comments:

1. The testosterone model of BPH is commonly used and potentially useful. However, a major limitation of the model is that anything anti-androgenic intrinsically is effective in this model but not necessarily in at least some patients; on the other hand, something not targeting the androgen pathway may be ineffective in this model but nonetheless effective in some patients. This limitation of the model must be discussed.

Answer: We appreciate your comments. We have added the study limitations in discussion section (page 12, line 401-405).

2. While rodent models are often used in BPH research and have some value, their general limitation is a difference in anatomy of the prostate between rodents and humans. The implications of such differences must explicitly be discussed as a study limitation.

Answer: We appreciate your comments. We added the study limitations in discussion section (page 12, line 401-405).

3. The Abstract claims that the study aim was to explore effects “of PD on BPH progression” (c.f., l. 12) whereas the main Introduction defines the study aim as investigation of “therapeutic efficacy” (c.f. l. 88); by the way, this turns into “protective effect” in l. 194 or “therapeutic potential” in l. 286. It may be a wording issue, but the study design intrinsically does neither allow determining effects on progression, which requires at least two time points being tested, nor of treatment efficacy. If I understand the design correctly, PD and testosterone treatment started concomitantly. This implies that the study has looked at the prevention of BPH in the testosterone model but neither at progression nor at treatment (the latter implying that PD administration would have started after BPH had developed). Therefore, the entire manuscript including the Abstract needs rewording to focus on prevention and remove references to progression and treatment.

Answer: This is very critical point. We appreciate your comments. As your suggestion, we have revised the manuscript.

4. The current study design with concomitant start of testosterone and PD administration looks at BPH prevention. However, the present Introduction is based on treatment. Thus, the Introduction does not match what is being studied. Either the study must be repeated in a true treatment design (i.e., initiation of PD administration after BPH has developed) or the Introduction must be changed to providing a rationale why there is an unmet medical need for the prevention of BPH.

Answer: As your suggestion, we have changed the introduction (page 1, line 33-34, page 2, line 52-53, and page 2, line 89-91).

5. The word “significant” is ambiguous because it means relevant, important, or similar in plain English but a calculated p-value smaller than the prespecified statistical alpha in a statistical context. The two are not necessarily linked. Accordingly, it remains unclear what the authors mean. Therefore, many statisticians recommend avoiding the word “significant” and replacing it with whatever is meant. Moreover, indications of statistical significance should never be mentioned without accompanying effect size indicators. Something may have a low p-value but nonetheless the effect size may be just 5% (probably irrelevant biologically) or 500% or anything in between and above. Please implement throughout the manuscript including the Abstract.

Answer: This is critical point. As your comment, we have revised the description in manuscript.

6. Guidelines on data robustness recommend that scientific manuscripts should differentiate between studies designed to test a pre-specified statistical null hypothesis and all others, the latter by default being exploratory (Vollert et al., 2022; Vollert et al., 2020). To qualify as null hypothesis testing, a study must have a prespecified primary endpoint and a sample size based on pre-study power calculations. As this condition apparently is not met in the present study, it is an exploratory study by default. Of note, there is nothing wrong with well-designed exploratory studies. However, the exploratory character must be stated explicitly, e.g., at the end of the main Introduction and/or in Methods.

Answer: As your suggestion, we added the description in introduction (page 2, line 89-91).

7. The manuscript draws cause-effect conclusions based on associations. This is scientifically not warranted. Different types of experiments would be needed to justify cause-effect conclusions.

Answer: In present study, we examined the protective effect of PD extract on testosterone-induced BPH rat model. PD was administrated with testosterone simultaneously. As your comments, further experiment will be needed to confirm cause-effect conclusions. We appreciate your critical comments.

8. If flavonoids are key components of PD extracts, it is important to realize that flavonoids belong to the group of pan-assay interfering substance (PAINS) (Baell and Walters, 2014), which leads to many artefacts. This needs to be discussed as a study limitation.

Answer: As your suggestion, the study limitation was added in discussion (page 12, line 395-405).

9. Please provide a dose justification primarily for the PD dose but also for finasteride.

Answer: The dosage of PD extract and finasteride was determined based on the previous reports (Nutr Res Pract. 2018;12(5):378–386, Nutrients. 2017;9(10):1070. Lab Anim Res. 2020;36:26. Res Rep Urol. 2022;14:225-239).

10. Sections 2.3 and 2.4 lack information on sample sizes, i.e., number of rats in each group. Please add.

Answer: We have added the animal number per group in materials and methods (page 3, line 120).

11. Histological examination is notoriously vulnerable for investigator bias. Please add an explicit statement whether the person selecting regions for viewing was blinded for group allocation.

Answer: As your suggestion, we have added this statement in materials and methods (page 3, line 145).

12. While GAPDH is frequently used to normalize RT-PCR findings, there are multiple examples where the expression of GAPDH itself is changed by an intervention. Therefore, please provide quantitative data on GAPDH expression (Ct values) in each group.

Answer: Thank you for your important comment. The individual Ct value for Gapdh is presented as below table. There was no significant difference between all groups.

13. Section 2.10: A) It follows from the exploratory character of the study that calculated p-values cannot be interpreted as hypothesis testing but only as descriptive. This must be stated explicitly. B) It did not become clear to me which inter-group comparisons were made based on the Dunnett post-tests. This should be stated explicitly.

Answer: A) As your suggestion, we have added the description for exploratory study in introduction section (page 2, line 89-91). B). In addition, we have clarified the inter-group comparison in materials and methods (page 4, line 183-185).

14. Based on seminal work by Weissgerber (Weissgerber et al., 2015) many leading journals including all journals of the Nature family mandate that continuous data are not presented as bar graphs but as scatter plots or bar graphs overlaid with scatter plots. Please implement.

Answer: As your suggestion, we have revised all graphs to bar graphs overlaid with scatter plots.

15. Figure 4B in comparison to figure 5 and table 2 creates a logical dilemma. How can prostate weight still be higher in the Fina and PD groups as compared to control if proliferation if anything is even less than in the control group? Only two logical explanations come to my mind. Either PCNA staining is a poor marker of cell proliferation, or finasteride and PD inhibit apoptosis relative to control (Figure 5 does not support this). I do not have a good explanation for this, but this dilemma must be discussed transparently as it has major implications for the overall interpretation of the results.

Answer: This is critical point. We appreciate your comments.

BPH is defined histologically as “a disease process characterized by stromal and glandular epithelial cell hyperplasia of the prostate (Curr. Opin. Urol. 2011;21:5–12. Exp Ther Med. 2018;15(3):2703-2710). As your comment, finasteride and PD inhibited glandular epithelial cell proliferation (Figure 4B; the number of PCNA-positive cells) and increased apoptosis (Figure 5; the expression levels of Bcl-2 and cleaved caspase-3). However, prostate weight is still higher in the finasteride- and PD-treated groups as compared to control. This discrepancy may be due to the prostatic stromal cell hyperplasia. In the present study, we evaluated the cell proliferation and apoptosis only focused on glandular epithelial cell. This is similar with previous studies showing that although PCNA and Bcl-2 expressions are lower in finasteride-treated group than normal group, the prostate weight is still high in finasteride-treated group (Molecules. 2023;28(2):803. Mol Med Rep. 2015;12(2):1699-708). However, further study is needed to clarify whether PD treatment could decrease the prostatic stromal cell hyperplasia. We have now clarified the effects of PD on glandular epithelial cell proliferation (page 6, line 227-233) and added this dilemma of this study in discussion (page 11, line 345-349).

16. It is interesting to note that both finasteride and PD have strong inhibitory effects on some parameters (bringing them down to control levels or even lower) but weaker effects on others. The implications of this observation need to be discussed.

Answer: As your suggestion, we added the implication in discussion (page 12).

17. Pathologists define hyperplasia as an increase in cell number (as opposed to hypertrophy being an increase in cell size). Apparently, the authors feel strongly about hyperplasia (a histological diagnosis) but I did not find any assessment of cell number in the manuscript. Perhaps the only exception is PCNA staining, but this apparently has major problems (see above). Thus, what the authors have study is prostate enlargement (the human equivalent of which would be benign prostatic enlargement, BPE). Therefore, I feel strongly that conclusions on BPH are not warranted based on the currently presented data. Either robust data on changes in cell number must be presented, or conclusions on BPH must be changed to those on BPE. This is important because the degree of LUTS and prostate size are only poorly related, if at all.

Answer: We appreciate your critical comment. PCNA is a nuclear antigen that is a representative marker of cell proliferation, synthesized at the G1/S phase of the cell cycle (Clin Invest Med. 2008;31(1):E8-E15). Since abnormal proliferation in epithelial cells is a key feature of BPH development, we analyzed PCNA expression in the prostate of PD-treated group. Figure 4A and B shows that PD administration inhibited cell proliferation by reducing PCNA expression in the prostatic glandular epithelial cell. As your previous comment (No. 15), we have to clarify whether the decreased prostatic weight in PD group is associated with the proliferation of prostatic stromal cell, but this result could support the protective effect of PD on prostate hyperplasia.

I would like give sincere thanks to you. This comment is very constructive and critical which results in marked improvement of the manuscript. We have added the limitation of this study in discussion (page 11, line 345-349).

18. The overall manuscript is understandable but nonetheless may benefit from proofreading by a native speaker or professional translator. For instance, articles are often missing before nouns. If a proofreader is engaged, the name of this person should be mentioned in the acknowledgement section.

Answer: We appreciate your comment. Resubmitted manuscript was edited from English Manager Science Editing (https://sciencemanager.com).

19. L. 10: This sentence is factually wrong because hyperplasia is often but not necessarily accompanied by prostatic enlargement. Please reword.

Answer: We have now changed the sentence as your comment (page 1, line 10).

20. L. 32: References 1 and 2 are used to support claims on the pathophysiology of BPH. A) Reference 1 is a review on efficacy and tolerability and does not have a focus on pathophysiology. B) Reference 2 is more than 25 years old. Do you expect this to reflect the state of the art of our understanding of the pathophysiology of BPH? I do not. I suggest replacing both references with more appropriate review articles.

Answer: As your suggestion, we have changed the references 1 and 2.

21. L. 56-57: I disagree with this statement for two reasons: Firstly, ARI have only shown efficacy in men with prostates greater than 40 ml. Thus, they are not pillars of BPH treatment in general. Second, ARI have a specific side effect profile. Other than adverse effects on sexual function, they may cause adverse effects on the brain (possibly as part of the post-ARI syndrome) and have been associated with an increased risk of developing type 2 diabetes in three studies. Please reword this statement.

Answer: As your suggestion, we have changed the sentence (page 2, line 59-60, line 65-66).

22. L. 57-58: What do you mean by “many 5-ARIs, including dutasteride and finasteride”? As far as I know, these are the only ones.

Answer: As your suggestion, we have revised the description in introduction (page 2, line 59-60).

23. L. 69: Do you really mean “feed” (not a noun) or rather “food”?

Answer: We apologize for the confusion. We have corrected it as animal feed (page 2, line 70).

24. L. 127-130: The preceding lines define the four study groups but do not clearly link them to the definitions of “negative” control and “positive” control. Please clarify. Moreover, this seems to me like a rather complex analysis of BPH. Why not simply report on prostate weight in each group?

Answer: We apologize for the confusion. We have clarified normal and positive control group in the present study (page 3, line 125, line 133-134). In addition, we have added the prostatic weight in Table 2 (page 6).

25. Wherever possible, please provide RRID of the animal strain, of kits, antibodies etc. Where unavailable, please add catalog numbers for unequivocal identification.

Answer: As your suggestion, we have added all information in materials and methods section (page 3-4).

26. L. 138 states that isolated prostate was placed in formalin, but l. 130 implies that only ventral lobe was used. Please clarify.

Answer: As your comment, we have clarified the description (page 3, line 135-136)

27. Table 2: To better understand the data, an additional column should be added showing body weight in each group. Particularly, I am interested to see whether PD affects body weight.

Answer: We have now added body weight in Table 2 (page 6). There was no difference among all groups.

28. Figure 2C: There may be a mistake in the y-axis label, which currently reads “fold change”. A one-fold change is a doubling whereas no change (by definition true for the NC group) is a 0-fold change. Please either adapt the graph or change the y-axis label to e.g., “fold of control”. This comment also applies to other figures currently showing “fold changes” such as 4C.

Answer: As your suggestion, we have corrected the labels as “fold of control” in all figures.

29. Figure 3A: Please add a scale bar to each panel.

Answer: As your comments, we have added scale bar.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors generally addressed my points, but some minor concerns should be still addressed.


Please add information on randomization, castration and vehicle to the Materials and methods section.

Please check added manuscript pars for language as small errors are present, e.g., “Five resigns of ventral lobe” (line 147)

Author Response

Authors generally addressed my points, but some minor concerns should be still addressed.

Please add information on randomization, castration and vehicle to the Materials and methods section.

Answer: We appreciate your comments. As your suggestion, we added the information in materials and methods section (page 3, line 120, line 129-131)

Comments on the Quality of English Language

Please check added manuscript pars for language as small errors are present, e.g., “Five resigns of ventral lobe” (line 147).

Answer: We apologize for the confusion. We have carefully re-checked the entire manuscript and corrected the typographical errors.

Author Response File: Author Response.pdf

Reviewer 3 Report

All of my comments have been addressed adequately. Thank you.

Author Response

All of my comments have been addressed adequately. Thank you.

Answer: We appreciate your comment.