Attenuation of Pulmonary Damage Associated with COPD in a Cadmium-Exposed Model Due to the Administration of a siRNA Targeting PAD4

Round 1

Reviewer 1 Report

The provided scientific article investigates the effects of a PAD4-targeted siRNA on respiratory function in a rat model of COPD induced by cadmium exposure. The study outlines the prevalence and severity of COPD, emphasizing its association with environmental factors like cigarette smoke containing cadmium. The focus shifts to PAD4, an enzyme implicated in lung damage via neutrophil extracellular traps (NETs) formation, suggesting it as a potential target for intervention.

The study is well-written and well-designed, however, there are several concerns that should be addressed before accepting the manuscript:

1.  

2.  

3. - The statistical methods and rationale behind sample size determination are briefly mentioned but need more depth and justification. The methodology section requires more details on the experimental procedures, controls, randomization, blinding, and potential biases.

4.  

5. - Although the article mentions obtaining ethical approval, there's limited information on the ethical considerations regarding the use of animals, welfare, and adherence to guidelines for animal studies, including efforts to minimize suffering.

- I found several minor english errors throughout the manuscript. Please have a deep language revision.

1.  

2. -The methodology for siRNA design and administration needs further elaboration and validation. Details on the specificity, off-target effects, efficiency, and validation of the siRNA need to be included.

3.  

4. -The conclusion lacks a succinct summary of the findings and their implications. It should reiterate the main results and their significance in the context of the research question.

5.  

6. -The discussion section provides some interpretation but lacks depth in discussing the broader context of the findings, mechanisms involved, potential clinical relevance, and future directions for research.

revision needed. 

Author Response

Reviewer, we appreciate your comments and have made all the suggested changes.

Comments and Suggestions for Authors

The provided scientific article investigates the effects of a PAD4-targeted siRNA on respiratory function in a rat model of COPD induced by cadmium exposure. The study outlines the prevalence and severity of COPD, emphasizing its association with environmental factors like cigarette smoke containing cadmium. The focus shifts to PAD4, an enzyme implicated in lung damage via neutrophil extracellular traps (NETs) formation, suggesting it as a potential target for intervention.

The study is well-written and well-designed, however, there are several concerns that should be addressed before accepting the manuscript:

- The statistical methods and rationale behind sample size determination are briefly mentioned but need more depth and justification. The methodology section requires more details on the experimental procedures, controls, randomization, blinding, and potential biases.

We appreciate your comment, in this regard, we added some sentences to clarify. Line 97-10.

The number of animals allocated per group was based on the principle of 3Rs (re-duce, refine and replace) and statistical parameters. A significance level of α=0.05 was used as this value is common in biomedical studies to minimise the probability of type 1 errors [22]. Additionally, a statistical power (β) of 0.2 was used to detect significant differ-ences while limiting the number of animals used to avoid unnecessary excesses. A variance of 5.985 and an allowed difference of 3 were used. Sample Size Calculations in Clinical Research was used to calculate the sample size [23]. The sample number obtained was 6.

22. Berkson, J. Tests of Significance Considered as Evidence*. Int J Epidemiol 2003, 32, 687–691, doi:10.1093/ije/dyg255.
23. Chow, S.-C.; Shao, J.; Wang, H.; Lokhnygina, Y. Sample Size Calculations in Clinical Research: Third Edition; Chow, S.-C., Shao, J., Wang, H., Lokhnygina, Y., Eds.; Chapman and Hall/CRC: Third edition. | Boca Raton : Taylor & Francis, 2017. | Series: Chapman & Hall/CRC biostatistics series | “A CRC title, part of the Taylor & Francis im-print, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.,” 2017; ISBN 9781315183084.

- Although the article mentions obtaining ethical approval, there's limited information on the ethical considerations regarding the use of animals, welfare, and adherence to guidelines for animal studies, including efforts to minimize suffering.

We appreciate your observation; we added the Mexican NOM. We have added a sentence regarding this (line 90–91).

- I found several minor english errors throughout the manuscript. Please have a deep language revision.

We appreciate your comments about the need to check grammar and spelling in our manuscript. We sent the text to a professional language correction service to ensure that these aspects were carefully reviewed and corrected (Proof-Reading-Service.com. Attached English certificate).

-The methodology for siRNA design and administration needs further elaboration and validation. Details on the specificity, off-target effects, efficiency, and validation of the siRNA need to be included.

We appreciate your observation; we have extended the methodology description (see lines 168–176). We have also added a sentence and some tables (line 180-187 and table 6,7).

-The conclusion lacks a succinct summary of the findings and their implications. It should reiterate the main results and their significance in the context of the research question.

We appreciate your observation; we added a summary and extended the conclusion. See lines 361-369 and 383-387.

In summary, the effect of cadmium administration in our model caused an increase in the expression of PAD4, which is related to the formation of NETs and lung damage in COPD. However, our siRNA targeting PAD4 reduced the expression of PAD4 and partially improved the parameters measured in our assay. These findings suggest a possible relationship between cadmium and increased PAD4 expression in the pathogenesis of COPD and the use of siRNA as a potential therapeutic strategy to attenuate the harmful effects of cadmium in the respiratory system. More research is still required to fully under-stand the mechanisms involved as well as further studies that corroborate the clinical relevance of these findings.

5. Conclusions

In conclusion, our study provides significant evidence that siRNA targeting PAD4 may be an effective therapeutic approach to mitigate lung damage in the rat cadmium-induced COPD model. These results are an important step towards understanding COPD at the molecular level and they offer a promising direction for future research and therapeutic applications.

-The discussion section provides some interpretation but lacks depth in discussing the broader context of the findings, mechanisms involved, potential clinical relevance, and future directions for research.

We appreciate your observation; we have deepened the discussion in lines 294–298 and 370–380

Cadmium has been implicated as one of the components present in cigarettes which may responsible for causing lung damage in smokers [26] but there is little information regarding the mechanisms involved in this damage. Our results show that PAD4 may be part of one of the signalling pathways involved in this damage, likely due to an increase in the formation of NETs.

The clinical relevance of our study lies in its potential for development of more effective and targeted interventions for patients with COPD, especially those whose condition is exacerbated by environmental factors such as cadmium exposure from cigarette use. Given the emerging role of PAD4 in the pathogenesis of COPD, our findings suggest that siRNA therapy could be a promising strategy, although further studies are still needed to establish its safety and efficacy in clinical settings. 

Future work should therefore focus on longitudinal studies and more complex mod-els to validate these results and explore how PAD4 modulation may influence long-term COPD progression, as well as its role in other lung diseases such as COVID. It would also be interesting to test our siRNAs in other types of lung disease [44].

Reviewer 2 Report

In this interesting study authors demonstrate that administration of siRNA targeting PAD4 can improve respiratory function by decreasing lung and heart damage. I only have 2 concerns to be addressed prior to be considered for publication in this journal:

1) Authors should include the statistical analyses performed in each figure legend.

2) Authors should complete de section “Statistical analysis”  with more information about the statistical analyses performed.

Author Response

Reviewer, we appreciate your comments and have made all the suggested changes.

Comments and Suggestions for Authors

In this interesting study authors demonstrate that administration of siRNA targeting PAD4 can improve respiratory function by decreasing lung and heart damage. I only have 2 concerns to be addressed prior to be considered for publication in this journal:

1) Authors should include the statistical analyses performed in each figure legend.

We appreciate your observation; we added the statistical analyses in each figure legend. We added “One-way ANOVA was used for the comparisons between the different groups” in each figure.

2) Authors should complete de section “Statistical analysis” with more information about the statistical analyses performed.

We appreciate your observation; we have added a definition and explained the statistical methods in line 206. We added the sentence “Values are expressed as the mean ± SEM (Standard Error of the Mean)”

One-Way Analysis of Variance (ANOVA): We used one-way ANOVA to compare the means between the different experimental groups and the control group. This method evaluates whether there are statistically significant differences between groups. When the ANOVA results indicated significant differences, we proceeded with post-hoc analyses to specifically identify where these differences lay.

Tukey's Post-hoc Test: After finding significant differences with ANOVA, we applied Tukey's post-hoc test. This test determines which specific groups differ from one other, providing a detailed comparative analysis between multiple experimental groups and the control.

Reviewer 3 Report

1.       Authors should be consistent. Abstract uses cadmium, and in methods employ cadmium chloride.

2.       The authors used cadmium chloride as a means of inducing COPD. While the abstract indicated intraperitoneal injections were administered every 14 hours, the procedures really state every 24 hours. Kindly make the correction.

3.       If the authors can include PAD4 protein expression data (western blot, IHC) for the various treatment groups. It will give the manuscript more weight.

4.       Elaborate the COPD model/ animal model in the methods section with different groups, number of animals in each group. For better understanding of readers.

5.       Authors neglected to explain how the data were utilized for statistical analysis, such as the average, mean, or median, in the statistics sections.

6.       Authors should elaborate the conclusion part.

7.       In the manuscript, authors should carefully check for a few grammatical and spelling errors.

In the manuscript, authors should carefully check for a few grammatical and spelling errors.

Author Response

Reviewer, we appreciate your comments and have made all the suggested changes.

Comments and Suggestions for Authors

1. Authors should be consistent. Abstract uses cadmium, and in methods employ cadmium chloride.

We appreciate your observation about the need to maintain consistency in the use of terms in our manuscript, particularly regarding the use of “cadmium” in the abstract and “cadmium chloride” in the methods section. We have carefully reviewed the manuscript and made any necessary corrections to ensure consistency throughout the document.

2. The authors used cadmium chloride as a means of inducing COPD. While the abstract indicated intraperitoneal injections were administered every 14 hours, the procedures really state every 24 hours. Kindly make the correction.

We appreciate your observation about the inconsistency in the information regarding the administration of cadmium chloride in our manuscript. We have reviewed and corrected this discrepancy to ensure accuracy in the description of our experimental procedures clarifying that it was every 24 hours.

3. If the authors can include PAD4 protein expression data (western blot, IHC) for the various treatment groups. It will give the manuscript more weight.

We really appreciated your commentary. We used real-time PCR based on the study of Wu et al., who showed that decreased mRNA is associated with decreased protein (Wu et al., 2004) and the study of Fire and Melo who showed that siRNA decreases protein expression (Fire et al., 1998)

Weilin Wu 1, Emily Hodges, Jenny Redelius, Christer Höög A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells. Nucleic Acids Res . 2004 Jan 22;32(2):e17. doi: 10.1093/nar/gnh010.

A Fire 1, S Xu, M K Montgomery, S A Kostas, S E Driver, C C Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11. doi: 10.1038/35888.

4. Elaborate the COPD model/ animal model in the methods section with different groups, number of animals in each group. For better understanding of readers.

We appreciated your comments. We have explained the groups and number of animals in lines 124–135.

Control group: Rats not exposed to cadmium chloride. Administered only with 0.9% saline solution 1 ml every 24 hours for 5 days. N=6 rats.

Cadmium group: Rats exposed to cadmium chloride to induce COPD, with administrations of 5 mg/kg body weight by IP administration every 24 hours for 5 days. N=6 rats.

Cadmium + vehicle group: Rats exposed to cadmium chloride to induce COPD with administrations of 5 mg/kg body weight by IP administration every 24 hours for 5 days. On day 7, the administration of transfection vehicle was performed. N=6 rats.

Cadmium + siRNA group: Rats exposed to cadmium chloride to induce COPD, with administrations of 5 mg/kg body weight by IP administration every 24 hours for 5 days. On day 7, the vehicle administration was performed by administering transfection + 5 µL of the siRNA solution with the single dose turbofect. N=6 rats.

5. Authors neglected to explain how the data were utilized for statistical analysis, such as the average, mean, or median, in the statistics sections.

We appreciated your comments. We added the sentence “Values are expressed as the mean ± Standard Error of the Mean (SEM)” (line 204).

6. Authors should elaborate the conclusion part.

We appreciated your comments. We have elaborated in the conclusion. See lines 377-381.

In conclusion, our study provides significant evidence that siRNA targeting PAD4 may be an effective therapeutic approach to mitigate lung damage in the rat cadmi-um-induced COPD model. These results are an important step towards understanding COPD at the molecular level and they offer a promising direction for future research and therapeutic applications.

7. In the manuscript, authors should carefully check for a few grammatical and spelling errors.

We appreciate your comments about the need to check grammar and spelling in our manuscript. We have sent the text to a professional language correction service to ensure that these aspects are carefully reviewed and corrected (Proof-Reading-Service.com. Attached English certificate).

Comments on the Quality of English Language

In the manuscript, authors should carefully check for a few grammatical and spelling errors.

We appreciate your comments about the need to check grammar and spelling in our manuscript. We have sent the text to a professional language correction service to ensure that these aspects are carefully reviewed and corrected (Proof-Reading-Service.com. Attached English certificate).

Round 2

Reviewer 1 Report

authors replied to my comments in a satisfactorily way. Ok to accept for me

Reviewer 2 Report

Authors addressed all my comments.