Protective Effects of Theaflavins and Epigallocatechin Gallate against ZnO-NP-Induced Cell Apoptosis In Vitro
and Yan Li 1,2,*
Round 1
Reviewer 1 Report
This is an interesting study implying that TFs or EGCG possibly protect RTE cells from oxidative damage induced by ZnO-NPs through antiapoptotic and antioxidant effects. However, several issues must be figured out before its official publication.
1. Lines 43-45, dietary polyphenolics such as EGCG are sometimes not recommended to be used along with small-molecule anti-tumour drugs because of their interactive effects when sharing a similar metabolism pathway. So, it may be essential to specify which type of anti-tumour drug used to be delivered via nanomaterials should be the target of this study.
2. Line 64, the reference [24] is not related to the content of ‘antioxidant effect of mitochondria’; please confirm or correct it.
3. Line 75, a correction: ‘in previous studies’
4. Line 76, a correction: ‘could’
Please check the grammar and tense carefully for the rest of the article.
5. Lines 134-135, please give a brief description of the treatment of cells during the operation steps given in the kits used here.
6. Lines 121 and 132, what is the concentration of TFs and EGCG used? Is it also 10, 100 and 1000 μg/L? please specify the information.
7. Line 147, although the calculation method is referred to another study, it is still recommended to have a brief description of it here for a more direct reading experience.
8. Lines 193 and 194, why the sizes of ZnO-NPs’ at 30 and 90 nm were chosen for this study? Please consider adding 1-2 sentence(s) about this information in the paragraph.
9. Lines 335-349, please confirm or correct the tense used in this section.
10. Lines 347-348, which administration way (oral, intravenous, dermal, or inhaled) would you recommend using the TFs and EGCG to mitigate the toxicity of ZnO-NPs in lungs or other organs? Please consider briefing it with 1-3 sentence(s) about this question in the paragraph.
11. Figures 2-4, the experimental design for this study should consist of the blank group (the group with neither sample nor NPs treatment) and experimental groups (1. group with only NPs treatment; 2. groups with both NPs and sample treatment; 3. group with only sample treatment).
However, in this contribution, there is no information for the blank group and No. 3 of the experimental groups. Do you have related results to be supplemented or briefly described in this manuscript? Otherwise, how do you identify the relative gene expression levels in these ignored groups? which is necessary to confirm if ZnO-NPs are toxic and if EGCG and TFs have no cellular toxicity in current trials.
11. Please unify the hanging format of all the references.
11. Please unify the hanging format of all the references.
Author Response
Point 1: Lines 43-45, dietary polyphenolics such as EGCG are sometimes not recommended to be used along with small-molecule anti-tumour drugs because of their interactive effects when sharing a similar metabolism pathway. So, it may be essential to specify which type of anti-tumour drug used to be delivered via nanomaterials should be the target of this study.
Response 1: Thank you for this comment. For this section, we have reviewed the relevant reference, and it has now been changed as” However, in the medical field, where nanomaterials can be used for macromolecular drug delivery, targeted therapy of tumors. For example, nanoparticle CaH2 delivers cytotoxic T cells to induce tumor cell apoptosis and tumor microenvironment regulation, activate the immune system, promote the infiltration of immune cells into the tumor, and act together with immune checkpoint blockade to trigger a strong immune response, thereby inhibiting distal tumors [11]. So, it is particularly important to reduce its negative effects. Therefore, it is essential to find the necessary mitigation measures.” (Lines 46-53).
Point 2: Line 64, the reference [24] is not related to the content of ‘antioxidant effect of mitochondria’; please confirm or correct it.
Response 2: Thank you for this comment. The original reference [24] may focus on the anticancer effects of EGCG. Now we correct it as the final revised version “reference [25] Yi J, Chen C, Liu X, Kang Q, Hao L, Huang J, Lu J. Radioprotection of EGCG based on immunoregulatory effect and antioxidant activity against (60)Cogamma radiation-induced injury in mice. Food Chem Toxicol. 2020, 135, 111051. [PubMed]”.
Point 3: Line 75, a correction: ‘in previous studies’.
Response 3: Thank you for this comment. “In the previous study,” we correct it as”In previous studies,”(Lines 83-84).
Point 4: Line 76, a correction: ‘could’. Please check the grammar and tense carefully for the rest of the article.
Response 4: Thank you for this comment. We correct “can” as”could” (Line 84). The grammar and tense have been revised throughout in manuscript. We are sure there are no grammar and tense issues in the revised manuscript.
Point 5: Lines 134-135, please give a brief description of the treatment of cells during the operation steps given in the kits used here.
Response 5: Thank you for this comment. We add the MDA assay kit's brief description for treating cells:“Then scrape down the treated cells with a cell scraper, add the extraction reagent from the kit. Cells were sonicated, water bath at 95 °C for 40 min, running water cooled and 4000 rpm/min, centrifuged for 10 min, taken supernatant to be measured. The measurement of MDA content was at an absorbance of 532 nm according to the instructions of the MDA assay kit (Jiancheng Biotechnology, China).”(Lines 143-148).
Point 6: Lines 121 and 132, what is the concentration of TFs and EGCG used? Is it also 10, 100 and 1000 μg/L? please specify the information.
Response 6: Thank you for this comment. All the treatment of TFs and EGCG with the concentration of 10, 100 and 1000 μg/L. So we make some revise as : for line 131, “Then cells were treated with TFs or EGCG, respectively, and incubated for another 12 hours” was corrected as ” Then cells were treated with TFs or EGCG (10, 100 and 1000 μg/L), respectively, and incubated for another 12 hours”. For line 142, “The cells were treated with different concentrations of TFs or EGCG after exposure and incubated for 12 hours” was corrected as” The cells were treated with different concentrations of TFs or EGCG (10, 100 and 1000 μg/L) after exposure and incubated for 12 hours”.
Point 7: Line 147, although the calculation method is referred to another study, it is still recommended to have a brief description of it here for a more direct reading experience.
Response 7: Thank you for this comment. For this part, we add a brief description of the relative expression calculation of genes at the end of the paragraph after origiral line 147: ”The relative mRNA expression levels were following the method of Sedanza et al. [32] The expression of each gene was measured with Ct (threshold cycle) values. The relative expression levels of each gene were statistically analyzed in 2-(Ct tested gene - Ct reference gene). We use Rat GAPDH as the reference gene. ” (Lines 160-164)
At the same time, we add the quantitative primer information of gene Rat GAPDH in Table 1. (Lines 187-189)
Point 8: Lines 193 and 194, why the sizes of ZnO-NPs’ at 30 and 90 nm were chosen for this study? Please consider adding 1-2 sentence(s) about this information in the paragraph.
Response 8: Thank you for this comment. Through preliminary references research, we found that the size of the nanomaterials had profound effects on their toxicity. Based on the existing nanomaterials in our laboratory, we selected small size 30 nm and big size 90 nm ZnO-NPs as our experimental materials. For page5, “3.1. Uptake of zinc by RTE cells and the proliferation inhibition efficiency of RTE cells” section, “All exposed groups showed a significant increase in Zn content compared to the con-trol group (0 μg/L concentration group, P < 0.05, Figure 1.A), implying that ZnO-NPs could enter the cell membrane and accumulate within cells, 30 nm ZnO-NPs could en-ter RTE cells more easily than 90 nm ZnO-NPs” is revised as ” All exposed groups showed a significant increase in Zn content compared to the control group (0 μg/L concentration group, P < 0.05, Figure 1.A), indicated that the amount of ZnO-NPs entering the cell increased with an increase in exposure concentration. It implying that ZnO-NPs could enter the cell membrane and accumulate within cells, 30 nm ZnO-NPs could enter RTE cells more easily than 90 nm ZnO-NPs, and it also had more opportunities to interact with intracellular substances then induced cytotoxicity. Wang et al. [34] and Shalini et al. [35]’s studies reaffirmed that smaller size of NPs had more biotoxic effects” (Lines 199-206).
Point 9: Lines 335-349, please confirm or correct the tense used in this section.
Response 9: Thank you for this comment. In the conclusion section, we have revised the tense as follows:” To conclude, when ZnO-NPs entered RTE cells, cell proliferation was inhibited and oxidative stress was induced, resulting in large amounts of ROS and MDA. With the disruption of the redox balance in RTE cells, cells underwent an inflammatory response, resulting in the ex-pression of inflammation-related cytokines and chemokines. Subsequently, the in-flammatory response was triggered in RTE cells, and genes associated with the mito-chondrial apoptotic pathway (CytoC, Caspase 3, Caspase 8 and Caspase 9) were acti-vated. When TFs or EGCG were added, the ROS and MDA levels in RTE cells were controlled, and the intracellular redox balance was stabilized. After the intracellular redox system was stabilized, the inflammatory response of the RTE cells was also ef-fectively alleviated. Along with the elimination of intracellular inflammation, the gene expression related to the control of apoptosis in RTE cells were down-regulated, and approach to normal levels. TFs and EGCG could be novel approaches which could mitigate the emerging pollutants toxicity in such as nanoparticles in organisms, poten-tially slowing down the destruction of biodiversity.” (Lines 354-367)
Point 10: Lines 347-348, which administration way (oral, intravenous, dermal, or inhaled) would you recommend using the TFs and EGCG to mitigate the toxicity of ZnO-NPs in lungs or other organs? Please consider briefing it with 1-3 sentence(s) about this question in the paragraph.
Response 10: Thank you for this comment. At the end of the text, we add setence as:“ While in lungs, maybe inhalable drug of TFs and EGCG will mitigate the toxicity of ZnO-NPs; while in stomach, the oral drug maybe suitbile.” (Lines 367-369)
Point 11: Figures 2-4, the experimental design for this study should consist of the blank group (the group with neither sample nor NPs treatment) and experimental groups (1. group with only NPs treatment; 2. groups with both NPs and sample treatment; 3. group with only sample treatment).
However, in this contribution, there is no information for the blank group and No. 3 of the experimental groups. Do you have related results to be supplemented or briefly described in this manuscript? Otherwise, how do you identify the relative gene expression levels in these ignored groups? which is necessary to confirm if ZnO-NPs are toxic and if EGCG and TFs have no cellular toxicity in current trials.
Please unify the hanging format of all the references.
Response 11: Thank you for this comment. In Figures 2-4, we re-inserted the data of CK (the group with neither sample nor NPs treatment), and we found that after the treatment of NPs-treated cells, expression of cellular oxidative stress, inflammatory factors, and apoptotic genes all rose sharply, suggesting that NPs have strong biotoxic effects, in accord with Wang et al. [34] and Shalini et al. [35]’s studies. Through the treatment of EGCG or TF, the expression of related genes was reduced, which once again reflected the antioxidant, anti-inflammatory and anti-apoptotic effects of EGCG and TF. Thank you for your suggestion, in the future when we design experiments will pay attention to the No. 3 group, and take it into account.
We unify the hanging format of all the references. We are sure there are no abbreviation issues in the revised manuscript.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The present manuscript focus on Protective Effects of Theaflavins and Epigallocatechin gallate against ZnO-NP-induced Cell Apoptosis in vitro. The subject frame of the work is well constructed. So, in this respect and this article should be contributed to present research. I recommended this work for publication after the following minor revisions.
1. There are several typographical mistakes as well in whole manuscript. Therefore, the author’s thoroughly careful check the language and typo mistake to minimize the error.
2. The abstract should be beginning with a sentence about the background of concept and the aims as well as novelty of study should be mentions. What exactly is the novelty of this study? The abstract is poorly written and should be improved. Abbreviations must be avoided in abstract. Parenthesis should be avoided in abstract - this is poor writing. Please improve.
3. Introduction; Check and format the citations in the whole manuscript. Also, Appropriate references must be provided to explained the background, what is already done and why this study carried out. Other vise the novelty of this research is still poorly presented. This is important especially for the high IF journals. The scientific style should be used. What exactly is the aim of this work? Hypothesis statement is missing in the introduction section.
4. All figures are of poor technical quality and not suitable for publication, especially in a high reputed journal. Font size and kind is too small and must be unified in all figures. Small writings are unreadable. All figures must be self-explanatory.
5. I suggest first time write full name rather than abbreviation; revise throughout in manuscript
Author Response
Point 1: There are several typographical mistakes as well in whole manuscript. Therefore, the author’s thoroughly careful check the language and typo mistake to minimize the error.
Response 1: Thank you for this comment. The language and typo mistake have been carefully checked and revised.
Point 2: The abstract should be beginning with a sentence about the background of concept and the aims as well as novelty of study should be mentions. What exactly is the novelty of this study? The abstract is poorly written and should be improved. Abbreviations must be avoided in abstract. Parenthesis should be avoided in abstract - this is poor writing. Please improve.
Response 2: Thank you for this comment. Based on your thoughtful reminder, the abstract has been rewritten. First, the abstract begin with a sentence about the applications and toxicity of Zinc oxide nanoparticles (ZnO-NPs), but how to resist ZnO-NPs-induced toxicity is still unknown. This study aims to evaluate the protective effects of theaflavins (TFs) or epigallocatechin gallate (EGCG) against ZnO-NP-induced cytotoxicity. Second, the novelty is the comparative protective effects of TFs and EGCG against ZnO-NP-induced toxicity, which provided novel approaches which mitigate the emerging nanoparticle toxicity in organisms. Third, some unnecessary abbreviations and parenthesis have been removed. The abstract has been rewritten as follows:
“Zinc oxide nanoparticles (ZnO-NPs) are commonly used in various commercial applications, causing toxic effects on organisms and destroying biodiversity, but information about its protective approaches remains unknown. This study aims to evaluate the protective effects of theaflavins (TFs) or epigallocatechin gallate (EGCG) against ZnO-NP-induced cytotoxicity in rat tracheal epithelial (RTE) cells. Herein, RTE cells were exposed to 100 μg/L ZnO-NPs for 12 h, then treated with 0, 10, 100, and 1000 μg/L TFs or EGCG for another 12 h; subsequently, oxidative stress, inflammation, and apoptosis analyses were conducted. Relative to the control groups, TFs and EGCG treatment significantly inhibited the levels of reactive oxygen species and malondialdehyde content. Exposure to 1000 μg/L TFs or EGCG treatment downregulated cytochrome C gene expression level by 59.10% and 77.27%, Caspase 3 gene expression by 50.03% and 60.01%, Caspase 8 gene expression by 45.11% and 55.57%, and Caspase 9 gene expression by 51.33% and 66.67%, respectively. Meanwhile, interleukin 1β, interleukin 6, tumor necrosis factor-α, and the other inflammatory chemokines like C-C motif chemokine 2, C-X-C motif chemokine 8 expressions were gradually rescued after the addition of TFs or EGCG. These results implied that TFs or EGCG possibly ameliorated ZnO-NPs-induced toxicity through antiapoptotic, antioxidant, and anti-inflammatory effects. This study provided novel approaches which mitigate the emerging nanoparticle pollutants toxicity in organisms, potentially slowing down the destruction of biodiversity”. (Lines 13-29)
Point 3: Introduction; Check and format the citations in the whole manuscript. Also, Appropriate references must be provided to explained the background, what is already done and why this study carried out. Other vise the novelty of this research is still poorly presented. This is important especially for the high IF journals. The scientific style should be used. What exactly is the aim of this work? Hypothesis statement is missing in the introduction section.
Response 3: Thank you for this comment. The citations has been checked in the whole manuscript. The aims and novelty of this research has been supplemented in the “abstract” and “introduction” section. Furthermore, hypothesis statement has been supplemented in the revised manuscripts as follows:
“Herein we exposed RTE cells to 100 μg/L ZnO-NPs for 12 h, then treated with 0, 10, 100, and 1000 μg/L TFs or EGCG for another 12 h; furthermore, to explore whether TFs and EGCG ameliorate ZnO-NPs-induced toxicity and elucidate its potential mechanism, we performed oxidative stress oxidative stress, inflammation, and apoptosis analyses.” (Lines 88-91)
Point 4: All figures are of poor technical quality and not suitable for publication, especially in a high reputed journal. Font size and kind is too small and must be unified in all figures. Small writings are unreadable. All figures must be self-explanatory.
Response 4: Thank you for this comment. The font size of figure 1-4 have been enlarged from 16 to 18, and unified.
Point 5: I suggest first time write full name rather than abbreviation; revise throughout in manuscript.
Response 5: Thank you for this comment. The abbreviation has been revised throughout in manuscript. We are sure there are no abbreviation issues in the revised manuscript.
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
Lines 161-162, ‘Then scrape down the treated cells with a cell scraper, add the extraction reagent from the kit’ may be changed to ‘Then scarping down the treated cells with a cell scraper, and mixing them with extraction reagent of the kit’
Line 162-164, ‘Cells were sonicated, water bath at 95 °C for 40 min, running water cooled and 4000 rpm/min, centrifuged for 10 min, taken supernatant to be measured.’ may be changed to ‘cells were sonicated in the water bath at 95 °C for 40 min, then cooled by running water and centrifuged at 4000 rpm/min, for 10 min, the supernatant was collected for MDA measurement’
Lines 221-224, are there any method or name for significance analysis used in the study? Please consider specifying them.
Line 231, correction, ‘it implies that…’
Line 413, correction, ‘suitable’?
Author Response
Point 1: Lines 161-162, ‘Then scrape down the treated cells with a cell scraper, add the extraction reagent from the kit’ may be changed to ‘Then scarping down the treated cells with a cell scraper, and mixing them with extraction reagent of the kit’.
Response 1: Thank you for this comment. “Then scrape down the treated cells with a cell scraper, add the extraction reagent from the kit”, we correct it as “Then scarping down the treated cells with a cell scraper, and mixing them with extraction reagent of the kit”. (Lines 143-144)
Point 2: Line 162-164, ‘Cells were sonicated, water bath at 95 °C for 40 min, running water cooled and 4000 rpm/min, centrifuged for 10 min, taken supernatant to be measured.’ may be changed to ‘cells were sonicated in the water bath at 95 °C for 40 min, then cooled by running water and centrifuged at 4000 rpm/min, for 10 min, the supernatant was collected for MDA measurement’.
Response 2: Thank you for this comment. “Cells were sonicated, water bath at 95 °C for 40 min, running water cooled and 4000 rpm/min, centrifuged for 10 min, taken supernatant to be measured” , we correct it as “Cells were sonicated in the water bath at 95 °C for 40 min, then cooled by running water and centrifuged at 4000 rpm/min, for 10 min, the supernatant was collected for MDA measurement”. (Lines 144-146)
Point 3: Lines 221-224, are there any method or name for significance analysis used in the study? Please consider specifying them.
Response 3: Thank you for this comment. We explain for the content of significance analysis in lines 193-196 as ”Levene’s test and Kolmogorov–Smirnov one-sample test were used to evaluate the homogeneity of variance and normality of the data. One-way analysis of variance based on Tukey post hoc tests was applied to evaluate significant differences between groups”.
Point 4: Line 231, correction, ‘it implies that…’.
Response 4: Thank you for this comment. “It implying that”, we correct it as “it implies that”. (Line 206)
Point 5: Line 413, correction, ‘suitable’?
Response 5: Thank you for this comment. Line 373 “suitbile”, we correct it as”suitable”.
Author Response File: Author Response.docx
