Retinoic Acid Potentiates the Therapeutic Efficiency of Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) against Cisplatin-Induced Hepatotoxicity in Rats
, Abdelaziz M. Hussein 2,*, Basma H. Marghani 1,3, Nashwa M. Barakat 4, Mohsen M. M. Khedr 5 and Nabil Abu Heakel 1Round 1
Reviewer 1 Report
Authors here demonstrate the hepato-protective effects of BM-MSCs and RA against Cisplation toxicity through their antioxidant, anti-inflammatory, and anti-apoptotic properties. The manuscript as whole is well written and has some interesting results. I have few suggestions to increase the scientific rigor of the experimental design here and to strengthen the claims made in this manuscript. The manuscript may be accepted following major revision.
Comments,
- Please provide references for the dose selection of BM-MSCs and RA (1mg/kg).
- Please provide more details for the scores of hepatic lesions (L161) Please refer to Front. Pharmacol., 10 November 2020 | https://doi.org/10.3389/fphar.2020.563750; PLoS ONE 11(3): e0151649
- It would be interesting to see the expression of p53, levels of Bcl-2 to check the antiapoptotic effect of BM-MSCs and RA against Cis-induced hepatotoxicity.
- Another important assay would be to check the levels of proinflammatory cytokines IL-10, IL-6, IL-1β to assess the anti-inflammatory property of BM-MSCs and RA against Cis-induced hepatotoxicity.
Author Response
Reviewer’s # 1
Authors here demonstrate the hepato-protective effects of BM-MSCs and RA against Cisplatin toxicity through their antioxidant, anti-inflammatory, and anti-apoptotic properties. The manuscript as whole is well written and has some interesting results. I have few suggestions to increase the scientific rigor of the experimental design here and to strengthen the claims made in this manuscript. The manuscript may be accepted following major revision.
Comments,
Comment
- Please provide references for the dose selection of BM-MSCs and RA (1mg/kg).
Response
The reference for dosing of RA and BM-MSCs was added to the revised manuscript
Comment
- Please provide more details for the scores of hepatic lesions (L161) Please refer to Front. Pharmacol., 10 November 2020 | https://doi.org/10.3389/fphar.2020.563750; PLoS ONE 11(3): e0151649
Response
We would like to thank the reviewer for this comment. The above-mentioned reference used a scoring system for hepatic lesion in chronic active hepatitis in human not in animals and the scoring system. We added more details about the scoring system of histological changes from reference # 30 with maximum score of 12.
Comment
- It would be interesting to see the expression of p53, levels of Bcl-2 to check the antiapoptotic effect of BM-MSCs and RA against Cis-induced hepatotoxicity.
Response
Immunostaining for p53 and Bcl2 expression by immunostaining was added to revised version of the manuscript
Comment
- Another important assay would be to check the levels of proinflammatory cytokines IL-10, IL-6, IL-1β to assess the anti-inflammatory property of BM-MSCs and RA against Cis-induced hepatotoxicity.
Response
We the expression of IL-1beta by immunostaining in liver tissues was added in the revised manuscript
Author Response File: 
Author Response.doc
Reviewer 2 Report
The study by Azam MMME et al examines the potential for addition of retinoic acid to bone marrow mesenchymal stem cells in protecting against cisplatin-induced liver injury in rats. Though the authors have carried out several experiments, issues with clarity and consistency make interpretation of the data problematic. Additionally, there is a lack of clinical relevance for the study and several very important controls are missing as well.
Major points
1) Several important control groups seem to be missing from the study- namely rats given BM-MSC or retinoic acid alone, without cisplatin. Since the MSC were injected in culture media, an additional control (Cisplatin+culture media alone) is also missing. These are critical to interpret the data.
2) The introduction contains no proper justification for carrying out these studies. It seems to be merely a collection of paragraphs discussing cisplatin toxicity, bone marrow MSC and retinoic acid, without any connection between them, or a rationale for why it was tested. For example, what percentage of patients on cisplatin get liver injury? Which pathways of cisplatin toxicity in the liver have MSC shown to target? How do the authors align their study with the fact that retinoic acid has in fact been shown to potentiate the cytotoxic action of cisplatin (PMID: 23867314)? None of these pertinent issues are discussed.
3) This is all the more important since cisplatin induced hepatotoxicity is rare at standard doses and transient even at higher doses in humans. So why is it clinically relevant to study this? What effect does the BM-MSC+retinoic acid at this dose have on nephrotoxicity due to cisplatin, which seems to be more clinically relevant?
4) What was the justification for choice of cisplatin dose in the study? How does this compare to what is given to humans?
5) How were the bone-marrow MSC characterized? What assays were run to confirm that the cell population isolated was indeed MSC? No information on this very important point is provided.
6) The time course data for all parameters should be presented as either a line graph or bar graph rather than tables, which would significantly improve understanding of the data.
7) What are the units for measurement of MDA, GSH and Catalase activity? This needs to be included.
8) The gene expression data is difficult to interpret due to the significant variation. The authors should present the data as a scatter plot, which would help determine whether a few data points are skewing the results.
9) The images showing liver histology need to be repeated since the current images are of very poor quality and cannot be readily interpreted. ALT values of around 300 IU/L would indicate some liver necrosis, which seems to be not evident in the provided images. Lower magnification images should be provided to confirm that the damage was widespread and not localized to small regions.
10) The caspase 3 immunostaining also does not seem to be accurate since the signals seem to be non-specific staining independent of cellular location and do not always seem within hepatocytes. In addition, the images under higher magnification show no morphological signs of apoptosis in cells which seem to have high caspase 3 signal.
Minor points
1) Page 3, line 102- ….”soaked for two minutes in a 70% (vol/vol) solution”. A solution of what? The sentence implies that the whole rats were soaked. Is this correct, or only the limbs?
2) Page 4, line 175- Leica DFC280 seems to be the camera, and it would be useful to also provide detail of the type of microscope used.
Author Response
Reviewer’s # 2
The study by Azam MMME et al examines the potential for addition of retinoic acid to bone marrow mesenchymal stem cells in protecting against cisplatin-induced liver injury in rats. Though the authors have carried out several experiments, issues with clarity and consistency make interpretation of the data problematic. Additionally, there is a lack of clinical relevance for the study and several very important controls are missing as well.
Major points
Comment
1) Several important control groups seem to be missing from the study- namely rats given BM-MSC or retinoic acid alone, without cisplatin. Since the MSC were injected in culture media, an additional control (Cisplatin+culture media alone) is also missing. These are critical to interpret the data.
Response
A group of cisplatin and vehicle or culture media was added to the revised manuscript
Comment
2) The introduction contains no proper justification for carrying out these studies. It seems to be merely a collection of paragraphs discussing cisplatin toxicity, bone marrow MSC and retinoic acid, without any connection between them, or a rationale for why it was tested. For example, what percentage of patients on cisplatin get liver injury? Which pathways of cisplatin toxicity in the liver have MSC shown to target? How do the authors align their study with the fact that retinoic acid has in fact been shown to potentiate the cytotoxic action of cisplatin (PMID: 23867314)? None of these pertinent issues are discussed.
Response
Few sentences were added to the introduction of revised manuscript to clarify all requested points and the following references were added
- Abd Rashid, N., Abd Halim, S. A. S., Teoh, S. L., Budin, S. B., Hussan, F., Ridzuan, N. R. A., & Jalil, N. A. A. (2021). The role of natural antioxidants in cisplatin-induced hepatotoxicity. Biomedicine & Pharmacotherapy, 144, 112328.
- Sarhan, M., El Serougy, H., Hussein, A. M., El-Dosoky, M., Sobh, M. A., Fouad, S. A., ... & Elhusseini, F. (2014). Impact of bone-marrow-derived mesenchymal stem cells on adriamycin-induced chronic nephropathy. Canadian journal of physiology and pharmacology, 92(9), 733-743.
Comment
How do the authors align their study with the fact that retinoic acid has in fact been shown to potentiate the cytotoxic action of cisplatin (PMID: 23867314)?
Response
This study was invitro not in vivo study which concluded that ATRA could significantly improve the effect of cisplatin, which is at least partially attributed to ATRA-induced differentiation of HCC TICs, and the subsequent decrease in this chemo-resistant subpopulation, however in the current study we investigated the effect of BM-MSCs-which are pretreated or preconditioned with ATRA in culture before using it for treated. We think there is no controversy between this study and our work as we postulated that ATRA could enhance differentiation of BM-MSCs hence improve its therapeutic efficacy
Comment
3) This is all the more important since cisplatin induced hepatotoxicity is rare at standard doses and transient even at higher doses in humans. So why is it clinically relevant to study this? What effect does the BM-MSC+retinoic acid at this dose have on nephrotoxicity due to cisplatin, which seems to be more clinically relevant?
Response
The dose of BM-MSCs given in the current study was 1x106 i.e. one million cells which seems to be effective in a previous study by Khedr et al. 2022 against cisplatin induced nephrotoxicity. Also, previous clinical studies used the same dose of BM-MSCs i.e. 1x106 such as Kantarcioglu et al. 2012 (they administrated stem cells via peripheral veins in liver cirrhosis) and El-Ansary et al. 2012 (they administrated stem cells via intrasplenic vein and peripheral veins in patients with chronic liver disease)
- Khedr, M., Barakat, N., El-Deen, I. M., & Zahran, F. (2022). Impact of preconditioning stem cells with all-trans retinoic acid signaling pathway on cisplatin-induced nephrotoxicity by down-regulation of TGFβ1, IL-6, and caspase-3 and up-regulation of HIF1α and VEGF. Saudi Journal of Biological Sciences, 29(2), 831-839.
- Kantarcioglu M. Efficacy of Exvivo Expanded Autologous Mesenchymal Stem Cell Transplantation Via Peripheral Vein in Patients With Liver Cirrhosis. clinicaltrials.gov; 2012. Available from: https://clinicaltrials.gov/ct2/show/NCT01499459.
- El-Ansary M, Abdel-Aziz I, Mogawer S, et al. Phase II trial: undifferentiated versus differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis. Stem Cell Rev Rep 2012;8:972-81
Comment
4) What was the justification for choice of cisplatin dose in the study? How does this compare to what is given to humans?
Response
In previous studies we found 6 doses for cisplatin to induce hepatoxicity in rats , 6 mg/kg (Khedr et al., 2022), 7 mg/kg (Cüre et al., 2016) and 10 mg/Kg (Taghizadeh et al., 2021), so we did a pilot study in which we used 3 doses 6 mg/Kg, 7 mg/Kg and 10 mg /Kg and found 7 mg /kg produced significant impairment in liver tissues without great loss in rats as 10 mg/Kg produces 90% mortality in rats.
- Cüre MC, Cüre E, Kalkan Y, Kırbaş A, Tümkaya L, Yılmaz A, Türkyılmaz AK, Şehitoğlu İ, Yüce S. Infliximab Modulates Cisplatin-Induced Hepatotoxicity in Rats. Balkan Med J. 2016 Sep;33(5):504-511. doi: 10.5152/balkanmedj.2016.150576. Epub 2016 Sep 1. PMID: 27761277; PMCID: PMC5056652.
- Taghizadeh F, Hosseinimehr SJ, Zargari M, Karimpour Malekshah A, Mirzaei M, Talebpour Amiri F. Alleviation of cisplatin-induced hepatotoxicity by gliclazide: Involvement of oxidative stress and caspase-3 activity. Pharmacol Res Perspect. 2021 May;9(3):e00788. doi: 10.1002/prp2.788. PMID: 34003600; PMCID: PMC8130655.
Comment
5) How were the bone-marrow MSC characterized? What assays were run to confirm that the cell population isolated was indeed MSC? No information on this very important point is provided.
Response
Characterization of BM-MSCs was added to the revised manuscript
Comment
6) The time course data for all parameters should be presented as either a line graph or bar graph rather than tables, which would significantly improve understanding of the data.
Response
All data were represented in graph form
Comment
7) What are the units for measurement of MDA, GSH and Catalase activity? This needs to be included.
Response
The units for measurement of these markers were added to revised manuscript
Comment
8) The gene expression data is difficult to interpret due to the significant variation. The authors should present the data as a scatter plot, which would help determine whether a few data points are skewing the results.
Response
New clear graphs for the gene expression results were made in the revised manuscript after adding the new group.
Comment
9) The images showing liver histology need to be repeated since the current images are of very poor quality and cannot be readily interpreted. ALT values of around 300 IU/L would indicate some liver necrosis, which seems to be not evident in the provided images. Lower magnification images should be provided to confirm that the damage was widespread and not localized to small regions.
Response
New images for the histopathological changes including lower magnification images were added to the revised manuscript
Comment
10) The caspase 3 immunostaining also does not seem to be accurate since the signals seem to be non-specific staining independent of cellular location and do not always seem within hepatocytes. In addition, the images under higher magnification show no morphological signs of apoptosis in cells which seem to have high caspase 3 signal.
Response
New images for caspase-3 were added to the revised manuscript
Comment
Minor points
1) Page 3, line 102- ….”soaked for two minutes in a 70% (vol/vol) solution”. A solution of what? The sentence implies that the whole rats were soaked. Is this correct, or only the limbs?
Response
Great thanks for this comment. This typing error was corrected in revised manuscript
Comment
2) Page 4, line 175- Leica DFC280 seems to be the camera, and it would be useful to also provide detail of the type of microscope used.
Response
Great thanks the microscope and its version was added to the revised version.
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
Manuscript may be accepted
Author Response
Reviewer’s # 1
Comment
Manuscript may be accepted
Response
Great thanks you
Author Response File: Author Response.doc
