BushenHuoxue formula promotes osteogenic differentiation via affecting Hedgehog signaling pathway in bone marrow stem cells to improve osteoporosis symptoms

Yuqi Chen; ZhiYong Wei; HongXia Shi; Xin Wen; YiRan Wang; Rong Wei

doi:10.1371/journal.pone.0289912

Peer Review History

Original SubmissionJanuary 24, 2023
7 Jun 2023 Decision Letter - Gary S. Stein, Editor PONE-D-23-01680BushenHuoxue Formula improves osteoporosis by promoting Osteogenic Differentiation of Bone Marrow Stem CellsPLOS ONE Dear Dr. Wei, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Comments to authors: This manuscript reported an interesting topic that the Chinese traditional medicine BSHX promotes osteoporosis. However, there are multiple concerns to be addressed. Specific comments: 1. The supplementary materials were missed. 2. The title may need to be formatted. 3. Grammar and some expressions need to be corrected and simplified. 4. Mammals have three Hedgehog homologues, you only mentioned IHH and SHH, how about DHH in Hedgehog signaling pathway? 5. The description of BSHX was missed. What components are in BSHX? Which are effective? 6. Line 132 and 133, the first “after” should be removed and what is “every t3 days”? 7. Line 141, correct the typo “10-8 mol/L”. 8. Reformat line 168-172. 9. Lack of evidence that supports the relationship between SHH, IHH, GLI2, RUNX2 and pathogenesis of OP. 10. With OD, qRT-PCR, ELISA analysis, you concluded that BSHX may affect the Hedgehog signaling pathway by altering protein levels of IHH and GLI2 in bone tissue, but how BSHX alter IHH and GLI2? And how about SHH and RUNX2? 11. Side effects and toxicity of BSHX are missed. This part is important. Reviewer #2: Introduction There was no aim of the stıdy and hypothesis. Please add the end of the introduction section. Surgery and sacrificing procedures should be explained in more detail. Material and methods “Briefly, 10 kg of antler, epimedium, psoralen, salvia, astragalus, radix rehmanniae, white peony root, and other Chinese medicines were mixed with 1,500 mL boiling (100°C) distilled water and incubated by shaking at room temperature for 1 h”. This subject is not clear. How many kilograms of plants were used? Was it a total of 10 kg or 10 kg of each used? Is this mixture designed for this study or is it a previously used mixture for treatment? Add references, please. İn 126. What does mean 10 female rats? Which group? Please add references in section 2.5 Results Figures 3 and 5: It is too hard to understand which result is significant or which is not. Figure 3 and 5 need to be redesigned. İn 348. Why did you compare others to the calcium group? İt should be explained Discussion Runx 2 is a very important marker in bone metabolism and osteoporosis. Runx 2 needs to be more discussed in the discussion section. The following article should be used in this section J Bone Miner Metab 2023 Apr 8. doi: 10.1007/s00774-023-01416-z. Resveratrol prevents ovariectomy-induced bone quality deterioration by improving the microarchitectural and biophysicochemical properties of bone İn 420 and 421. “We observed that BSHX effectively 421 improved trabecular bone and reticular structure without any observable side effects.” How did you decide that BSHX does not effect any other tissue? In addition to these, the following articles should be used in the discussion section. Role of Traditional Chinese Medicine in Bone Regeneration and Osteoporosis Zhicai Peng, Ronghua Xu, Qinjian You Front Bioeng Biotechnol. 2022; 10: 911326 Identification of gene biomarkers in patients with postmenopausal osteoporosis Chenggang Yang, Jing Ren, Bangling Li, Chuandi Jin, Cui Ma, Cheng Cheng, Yaolan Sun, Xiaofeng Shi Mol Med Rep. 2019 Feb; 19(2): 1065–1073 Therapeutic Anabolic and Anticatabolic Benefits of Natural Chinese Medicines for the Treatment of Osteoporosis Jianbo He, Xiaojuan Li, Ziyi Wang, Samuel Bennett, Kai Chen, Zhifeng Xiao, Jiheng Zhan, Shudong Chen, Yu Hou, Junhao Chen, Shaofang Wang, Jiake Xu, Dingkun Lin Front Pharmacol. 2019; 10: 1344 There are a large number of grammatical errors in the manuscript. These errors need to be fixed ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
18 Jul 2023 Author Response Dear Editors and Reviewers: Thank you for your letter and for the reviewers’ comments about our manuscript entitled “BushenHuoxue Formula improves osteoporosis by promoting Osteogenic Differentiation of Bone Marrow Stem Cells” (PONE-D-23-01680). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meeting with approval. Revised portions are highlighted in the paper. The main corrections in the paper and the responds to the reviewer's comments are as flowing: Responds to the reviewer's comments: Reviewer #1: 1. The supplementary materials were missed. Response: Thanks for your revisions. We have submitted all the supplementary materials involved in the article, and some new experimental results as you suggested were added as supplementary materials. 2. The title may need to be formatted. Response: Yes, thanks for your comments. And the title was revised as “BushenHuoxue formula promotes osteogenic differentiation via affecting Hedgehog signaling pathway in bone marrow stem cells to improve osteoporosis symptoms.” 3. Grammar and some expressions need to be corrected and simplified. Response: Thanks for your comments. We have corrected the related errors of grammar and expression. 4. Mammals have three Hedgehog homologues, you only mentioned IHH and SHH, how about DHH in Hedgehog signaling pathway? Response: Good suggestion. As you suggested, we performed related experiments to determine the expression of DHH in our experimental conditions, and the results are shown in the figure S1. Indeed, previous studies have suggested that the key factors of hedgehog signaling pathway involved in osteoporosis are SHH, GLI and IHH. And DHH only acts on gonads, including sperm and ovarian granulosa cells. Therefore, we did not detect DHH in the initial experiment. As you suggested, we found that DHH may also involve in mechanisms of BSHX and osteoporosis. 5. The description of BSHX was missed. What components are in BSHX? Which are effective? Response: Thanks. We have added on the description of BSHX in “Introduction” and the components of BSHX in tableS1. Moreover, we further performed network pharmacology analysis to identify the main bioactive metabolites of BSHX in improving osteoporosis. And the related results were added in Fig. 6, we found that quercetin might be the main bioactive compounds in BSHX. Numerous proteins that involved in osteoporosis and hedgehog signaling pathway were the candidate targets of quercetin, including SHH, IHH, Gli2, etc. 6. Line 132 and 133, the first “after” should be removed and what is “every t3 days”? Response: Thank you for your suggestion. We have modified the mistakes. 7. Line 141, correct the typo “10-8 mol/L”. Response: Thanks. We have corrected it. 8. Reformat line 168-172. Response: Thanks for your suggestion. We have redescribed the expression. 9. Lack of evidence that supports the relationship between SHH, IHH, GLI2, RUNX2 and pathogenesis of OP. Response: Thanks for your suggestion. We have added the evidence and references that support the relationship between OP and SHH, IHH, GLI2, RUNX2. The contents were shown in lines538-44 of the manuscript. 10. With OD, qRT-PCR, ELISA analysis, you concluded that BSHX may affect the Hedgehog signaling pathway by altering protein levels of IHH and GLI2 in bone tissue, but how BSHX alter IHH and GLI2? And how about SHH and RUNX2? Response: Thanks for your reminding. To clarify how BSHX alter IHH and GLI2, we constructed the metabolic fingerprint of BSHX using LC-MS/MS platform. We have found that BSHX mainly perform its function in improving OP through quercetin. Quercetin could target and bind with several functional proteins that involved in OP and Hedgehog signaling pathway. RT-qPCR assay detects that quercetin could affected the expression levels of hedgehog-related genes, including IHH, Gli2, SHH and Runx2, then leading changes in Hedgehog signaling pathway, and OP pathogenesis. Remarkably, IHH and GLI2 were also identified as the targets of quercetin. Therefore, we considered that quercetin is the bioactive compounds of BSHX in improving OP via affecting Hedgehog signaling pathway. 11. Side effects and toxicity of BSHX are missed. This part is important. Response: Yes, thanks for your reminding, we have carried out the MTT assay. MTT results showed that BSHX extracts did not affect the cell growth of mesenchymal stem cells (Figure S2). Thus, we proposed that BSHX might exert small side-effect in affecting human health or cell growth. Reviewer #2: 1. There was no aim of the study and hypothesis. Please add the end of the introduction section. The surgery and sacrificing procedures should be explained in more detail. Response: Thanks for your suggestion. We have added the aim of the study and hypothesis. Furthermore, the surgery and sacrificing procedures have been supplemented in the manuscript. 2. “Briefly, 10 kg of antler, epimedium, psoralen, salvia, astragalus, radix rehmanniae, white peony root, and other Chinese medicines were mixed with 1,500 mL boiling (100°C) distilled water and incubated by shaking at room temperature for 1 h”. This subject is not clear. How many kilograms of plants were used? Was it a total of 10 kg or 10 kg of each used? Is this mixture designed for this study or is it a previously used mixture for treatment? Add references, please. Response: Thanks for your reminding, we have added the dose for each plant. And we have added the reference to show that BSHX was a previously used mixture. 3. İn 126. What does mean 10 female rats? Which group? Response: Thank you for your suggestion. We have revised the expression. What 's more, this part of the rats was used to obtain BMSCs from SD rats at 6 weeks of age and were not grouped. 4. Please add references in section 2.5 Response: Thanks. We have added the reference in section 2.5. 5. Figures 3 and 5: It is too hard to understand which result is significant or which is not. Figures 3 and 5 need to be redesigned. Response: Thanks for your suggestion. The Figure 3 mainly verifies the expression of SHH, IHH, Gli2, and Runx2 in bone tissue. The Figure 5 mainly describes the expression of factors in BMSCs. Our aim was to verify in vitro and in vivo that BSHX can improve osteoporosis by influencing the expression of some cytokines in the Hedgehog signaling pathway. 6. İn 348. Why did you compare others to the calcium group? İt should be explained. Response: Thank you for your suggestion. Calcium supplement is the most basic treatment for osteoporosis. In this study, we compared other groups with calcium supplement group to investigate the effects of these treatment in improving OP symptoms. 7. Runx2 is a very important marker in bone metabolism and osteoporosis. Runx2 needs to be more discussed in the discussion section. Response: Thanks for your reminding, we have added more references to discuss the function of Runx2 in OP pathogenesis and BSHX-treatment in the discussion section, and the references that you suggested have been cited in the new manuscript. 8. İn 420 and 421. “We observed that BSHX effectively 421 improved trabecular bone and reticular structure without any observable side effects.” How did you decide that BSHX does not effect any other tissue? Response: Thanks for your suggestion. We have modified the inappropriate expression and made a more appropriate description. 9. In addition to these, the following articles should be used in the discussion section. Response: This is a good proposal. We've read the corresponding literatures and cited them. 10. There are a large number of grammatical errors in the manuscript. These errors need to be fixed. Response: Thanks for your comments. As your comments, I have recorrected some errors in grammar and expression. Attachments Attachment Submitted filename: Response to Reviewers-DuRev.docx https://doi.org/10.1371/journal.pone.0289912.r002
31 Jul 2023 Decision Letter - Gary S. Stein, Editor BushenHuoxue formula promotes osteogenic differentiation via affecting Hedgehog signaling pathway in bone marrow stem cells to improve osteoporosis symptoms PONE-D-23-01680R1 Dear Dr. Wei, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Gary S. Stein Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0289912.r003
Formally Accepted
3 Aug 2023 Acceptance Letter - Gary S. Stein, Editor PONE-D-23-01680R1 BushenHuoxue formula promotes osteogenic differentiation via affecting Hedgehog signaling pathway in bone marrow stem cells to improve osteoporosis symptoms. Dear Dr. Wei: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Gary S. Stein Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289912.r004

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