Baicalein Relieves Ferroptosis-Mediated Phagocytosis Inhibition of Macrophages in Ovarian Endometriosis
and Zhi-Ling Zhu 1,4,*Round 1
Reviewer 1 Report
Before the manuscript is published, authors should complete it/answer the following questions:
- No Ethics Committee approval number in section 2.1. Patients and samples collection - please add
-both the study group and the control group are very small? authors should explain why (prepare a diagram of the criteria for inclusion in these groups) and add the period in which the samples were collected?
- Could not the reasons/diseases for which dilation and curettage were performed in the control group also affect the change in the expression of the tested genes; biological properties of macrophages or the biochemical composition of the tested and control PF .... and thus the impact on the results of all analyzes?
- Was the PF somehow protected after collection before it was used for research?
-whether this cell line is well suited to reflect macrophage behavior in endometriosis?
- Specification of the title 2.3. Extraction of macrophages from patients' PF - what next with the results of expression studies, e.g. genes from these macrophages?
- Description of RNA isolation, reverse transcription and qPCR - were there negative and positive controls in the reaction, what were they or were there any reference genes? Did the isolation and further research concern only cells from the lines or also those from patients? How were gene expressions calculated?
- specify the description of the treatment of cells with different concentrations of the tested compounds in the description of the methodology?
- did the authors carry out analyzes with a control PF? if so, when describing the results, it should be clarified whether it is PF from patients with endometriosis or a control PF?
- the description of the results regarding gene expression should be better described so that there is no doubt whether the authors tested in a cell line or in material from patients?
- was the level of Fe in the PF fluid from patients with endometriosis and the control PF fluid measured?
Author Response
To Reviewer #1:
Comments to the Author
- No Ethics Committee approval number in section 2.1. Patients and samples collection - please add
Response: Thank you for your suggestions. We added the Ethics Committee approval number in section 2.1. Patients and samples collection (2019-103).
- Both the study group and the control group are very small. authors should explain why (prepare a diagram of the criteria for inclusion in these groups) and add the period in which the samples were collected.
Response: Thank you for your suggestions. We used 8 paraffin sections of ovarian ectopic lesions and 8 paraffin sections of normal endometrium to prove that the ectopic lesions were affected by cystic fluid with obvious iron accumulation in the tissues by Prussian blue staining. The difference was very obvious among 8 pairs of samples. In addition, there are also supportive conclusions in the literature (PMID:33378529) that iron accumulation in ectopic lesions has been proved by Prussian blue staining. Therefore, we think that 8 pairs of samples are enough to explain this question. We added a diagram of the criteria for inclusion in these groups (table 2, page 14, lines 331-333).
- Could not the reasons/diseases for which dilation and curettage were performed in the control group also affect the change in the expression of the tested genes; biological properties of macrophages or the biochemical composition of the tested and control PF .... and thus the impact on the results of all analyzes?
Response: Thank you. We screened patients undergoing dilation and curettage with no endometrial organic lesions. Additionally, we use endometrium only for Prussian blue staining, a kind of iron staining. Theoretically, dilation and curettage had no excessive impact on their iron accumulation. Normal endometrial samples were not involved in gene expression detection in this article. In addition, the patients with endometriosis had no other complications and no hormones or other drugs before surgery. The patients with control peritoneal fluid had no other diseases except hysteromyoma or teratoma.
- Was the PF somehow protected after collection before it was used for research?
Response: Thank you for your suggestions. Peritoneal fluid was collected and immediately separated from macrophages with CD14 magnetic beads to ensure the vitality of macrophages. It was ensured that the CD14+ cell concentration was greater than 90% using Flow CytoMetry and then used to extract RNA. Therefore, we did not store the peritoneal fluid further. Chocolate-like cyst fluid, as an experimental reagent used to induce ferroptosis of macrophages in this study, was sucked from the patients' ovarian endometriosis vesicles under aseptic conditions and freeze it into -80℃ immediately for preservation. We have added the corresponding sampling details in the article (page 2, line 85).
- Whether this cell line is well suited to reflect macrophage behavior in endometriosis?
Response: Thank you. Our group has previously reported that the phagocytosis of peritoneal macrophages in patients with endometriosis is significantly reduced compared with the control (PMID: 31299634), and it has been confirmed that THP-1 cell line, a monocyte line derived from human leukemia, is the most widely used cell line in vitro studies to study the function of human primary macrophage cells. Therefore, we use the THP-1 cell lines to simulate the functional impact of macrophage in the high iron environment of cystic fluid.
- Specification of the title 2.3. Extraction of macrophages from patients' PF - what next with the results of expression studies, e.g. genes from these macrophages?
Response: Thank you for your comments. We found that the ferroptosis-related genes FTH1, HMOX1, FTL, and FPN of macrophages in ovarian endometriosis cyst wall were significantly up-regulated compared with normal endometrial tissue from single-cell sequencing. Therefore, we extracted macrophages from the peritoneal fluid of endometriosis patients to verify this phenomenon. Figure 2A is our verification result.
Figure 2 (A) Ctrl-pMϕ (n = 8) and EMs-pMϕ (n = 8) were extracted from the peritoneal fluid. The mRNA levels of HMOX1, FTH1, FTL, and FPN were measured with RT-qPCR.
- Description of RNA isolation, reverse transcription, and qPCR - were there negative and positive controls in the reaction, what were they or were there any reference genes? Did the isolation and further research concern only cells from the lines or also those from patients? How were gene expressions calculated?
Response: Thank you. We take ACTB as the internal reference gene. The main purpose of macrophage isolation is to detect whether ferroptosis-related genes of peritoneal macrophages in endometriosis are up-regulated compared with normal peritoneal macrophages. After that, we used the THP1 cell line in our experiments on macrophage-related functions in vitro. The main reason for replacing with the THP1 cell line is that the macrophage extracted from the abdominal cavity can not survive for more than three days in vitro, so it is difficult to use to conduct further experiments. Gene expression calculation is supplemented in the text (page 3, lines 104-105).
Specify the description of the treatment of cells with different concentrations of the tested compounds in the description of the methodology.
Response: Thank you for your suggestions. We added it in the Method (page 4, lines 153-157). The following is the revised version.All drugs and corresponding treatment concentration: Erastin (HY-15763), 10 μM; RSL3 (HY-100218A), 5μM; Ferrostatin-1 (HY-100579), 10 μM; Deferoxamine (HY-B0988), 200 μM; Necrostatin (HY-15760) 2 μM; ZVAD-FMK(HY-16658B), 5 μM were purchased from MedChemExpress (MCE). FeSO4 · 7H2O (F7002), 200μg/ml; Baicalein (465119), 20μM were purchased from Sigma.
- Did the authors carry out analyzes with a control PF? if so, when describing the results, it should be clarified whether it is PF from patients with endometriosis or a control PF.
Response: Thank you for your comments. We extracted PF only for gene expression verification of macrophages in PF (Please refer to the answer in comment 6). We observed the effects of a high iron environment in chocolate-like cystic fluid on the ferroptosis of macrophages in patients with ovarian endometriosis. Because chocolate-like cystic fluid only exists in patients with ovarian endometriosis, so we didn't set up a control group.
- The description of results regarding gene expression should be better described so that there is no doubt whether the authors tested in a cell line or material from patients.
Response: Thank you for your comments. We have indicated the cell type used in the text and figure legend.
- Was the level of Fe in the PF fluid from patients with endometriosis and the control PF fluid measured?
Response: Thank you for your comments. Our main research object is the high iron environment in the chocolate-like cystic fluid instead of peritoneal fluid. High iron concentration in the chocolate-like cystic fluid is greatly increased in a large number of literatures (e.g., PMID: 12372445, PMID: 18172249, PMID: 33780964, PMID: 18396284, PMID: 9692345). In Figure2C, after we treated macrophages with the cystic fluid, the iron probe showed obvious intracellular iron accumulation, which can indirectly explain the existence of high iron in the cystic fluid.
Figure2C
Author Response File: 
Author Response.docx
Reviewer 2 Report
The work presented to me for review concerns the influence of baicalein on macrophages in endometriosis, which is a chronic and incurable disease. To date, there is no ideal treatment for endometriosis. Endometriosis is a serious social disease in women.
The work is unique which significantly increases the value of the work. Introduction, materials and methodology written briefly and to the point, legibly. The results are clearly presented. A very exhaustive discussion, analyzing the latest literature.
In my opinion, the work presents an analysis of the influence of baicalein on macrophages in endometriosis, especially in patients with ferroptosis, in a very clear way, i.e. descriptive and graphic. The authors prove that the use of baiclein in patients with endometriosis who have significant ferroptosis may significantly contribute to the treatment of this chronic disease, and thus to the improvement of the quality of life in these patients.
Author Response
Thank you for your kind comments.
Reviewer 3 Report
In this article, the authors investigated the effects of iron-induced ferroptosis on the function of macrophage in endometriotic milium and explored the potential therapeutic effect of baicalein against iron-induced ferroptosis of macrophage in EMs in vitro. The manuscript is straightforward, well written, and concise and has a clear result. Definitely deserves to be published and is a valuable contribution to the “Current Issues in Molecular Biology” journal. Some comments need to be addressed before publication.
[1] “1. Introduction”, Page 2, Lines 48-49:
“Macrophages, as the main immune cells in the peritoneal cavity, play an important role in inflammatory exacerbation and development of EMs.”.
At that point, it should also be mentioned that the therapeutic target of different immune subsets, such as tumours-associated macrophages, may be relevant to making progress in ovarian cancer immunotherapy as well. PD-L1 is expressed in tumour-associated macrophages, and tumour cells.
Recommended reference: Revythis A, et al. Recent Insights into PARP and Immuno-Checkpoint Inhibitors in Epithelial Ovarian Cancer. Int J Environ Res Public Health. 2022;19(14):8577.
[2] “4. Discussion”, Page 13, Lines 336-338:
“Iron can shape M1-like macrophage polarization through mitogen-activated protein kinase (MAPK), NF-κB, and other cellular signaling pathways including ATF4, ROS, and NLRP3 inflammasome [31-35].”.
Here, it should be clarified that PI3K/Akt NF-kB pathway promotes cell survival, MAPK pathway promotes cell proliferation and the ERK pathway promotes cell proliferation, survival, differentiation, migration and angiogenesis. Through these signaling pathways, each of the VEGF family provides different actions.
Recommended reference: Ioannidou E, et al. Angiogenesis and Anti-Angiogenic Treatment in Prostate Cancer: Mechanisms of Action and Molecular Targets. Int J Mol Sci. 2021;22(18):9926.
Author Response
To Reviewer #3:
Comments to the Author
- [1] “1. Introduction”, Page 2, Lines 48-49:
“Macrophages, as the main immune cells in the peritoneal cavity, play an important role in inflammatory exacerbation and development of EMs.”.
At that point, it should also be mentioned that the therapeutic target of different immune subsets, such as tumors-associated macrophages, may be relevant to making progress in ovarian cancer immunotherapy as well. PD-L1 is expressed in tumor-associated macrophages and tumour cells.
Recommended reference: Revythis A, et al. Recent Insights into PARP and Immuno-Checkpoint Inhibitors in Epithelial Ovarian Cancer. Int J Environ Res Public Health. 2022;19(14):8577.
[2] “4. Discussion”, Page 13, Lines 336-338:
“Iron can shape M1-like macrophage polarization through mitogen-activated protein kinase (MAPK), NF-κB, and other cellular signaling pathways including ATF4, ROS, and NLRP3 inflammasome [31-35].”.
Here, it should be clarified that PI3K/Akt NF-kB pathway promotes cell survival, MAPK pathway promotes cell proliferation and the ERK pathway promotes cell proliferation, survival, differentiation, migration and angiogenesis. Through these signaling pathways, each of the VEGF family provides different actions.
Recommended reference: Ioannidou E, et al. Angiogenesis and Anti-Angiogenic Treatment in Prostate Cancer: Mechanisms of Action and Molecular Targets. Int J Mol Sci. 2021;22(18):9926.
Response: Thank you for your valuable suggestions. Thanks for the references, which are now included in the revised manuscript. We have made corresponding modifications to the references in the text, as follows
- Ioannidou, E.; Moschetta, M.; Shah, S.; Parker, J.S.; Ozturk, M.A.; Pappas-Gogos, G.; Sheriff, M.; Rassy, E.; Boussios, S. Angiogenesis and Anti-Angiogenic Treatment in Prostate Cancer: Mechanisms of Action and Molecular Targets. International journal of molecular sciences 2021, 22, doi:10.3390/ijms22189926.
- Revythis, A.; Limbu, A.; Mikropoulos, C.; Ghose, A.; Sanchez, E.; Sheriff, M.; Boussios, S. Recent Insights into PARP and Immuno-Checkpoint Inhibitors in Epithelial Ovarian Cancer. International journal of environmental research and public health 2022, 19, doi:10.3390/ijerph19148577.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Authors of the manuscript did not answer for the question about control group and number of samples of patients included to every investigated/control group (for example what was the reasen/medical condition for control group to be at this medical procedure like dilation and curettage and may by madical condition of this "control" patients may have imcpat for exaple on gene expression). They did not put the information about time period when these samples were tested. In my opinion these groups are two small to get any statsitical significantly informations and resultes, it was not only for Prissian blue staining becouse you have the results from qPCR and information that there are significant what is immposible for 8 samples.
In addition, the description of the methodology and the obtained results is still disordered, making it possible to get lost in which materials and why the subsequent analysis was performed.
The added Table 2 is important, but in my opinion it will not cover the inclusion/exclusion criteria for the study, but it contain the characteristics of the groups.
