PONE-D-24-15138High-dose atorvastatin is significantly in mitigating LPS-induced vascular inflammation by inducing HO-1 expressionPLOS ONE

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Reviewers' comments:

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Rephrase title

Page 8, results section, it ill be good to add a list of the indices investigated. ...'we determined several biochemical indices' such as ......

Page 10, discussion, just wondering the unique and the new thing the current research brought on board. This is because much work on atorvastatin in mitigating LPS-induce vascular inflammation had been extensively explored. Kindly address this...

Reviewer #2: First Round Report

• The authors discuss an important disease vascular inflammation that is diagnosed in many different diseases and suggest different dosages for atorvastatin to relieve the inflammation effect.

• The authors presented a clear introduction sequentially to illustrate the relationship between vascular inflammatory injury and how it is related to lipopolysaccharide (LPS) and dose-dependent anti-inflammatory effects of atorvastatin on LPS-induced vascular inflammation and its mechanisms.

“Vascular inflammatory injury and how it is related to lipopolysaccharide (LPS), found in the cell wall of Gram-negative bacteria, triggers a vascular inflammatory response, causing endothelial dysfunction characterized by increased secretion of endothelin-1 (ET-1) and cytokines. Chronic inflammation inhibits endothelial nitric oxide synthase (NOS), reducing nitric oxide (NO) synthesis and increasing reactive oxygen species (ROS) production, leading to oxidative stress and endothelial dysfunction. Heme oxygenase 1 (HO-1) responds to ROS by exerting antioxidant effects. Atorvastatin, known for its cholesterol-lowering effects, also exhibits anti-inflammatory properties by modulating various pathways, including HO-1 expression”.

• In the Materials and methods, I found that the abbreviations mentioned as follows: “MDA, EPCR, NO, ET-1, vWF, and sTM concentrations” are unclear to me. So, I suggest adding the abbreviation after the abstract to easily guide the readers through the whole manuscript.

• In the results, the authors mentioned in the study design that the used rats were half male and half female, which was not shown in the description of the results, and as a result, the legends for the figures confused the readers about which one is referred to the male or the female.

• In the discussion, the authors address the outcomes clearly, but it is important to clarify the differences in the outcomes for males and females to avoid bias in showing and discussing the results.

Reviewer #3: 1. Is the manuscript technically sound, and do the data support the conclusion?

Yes

- Strength

- Technically it sounds good, the outcome is tested twice with CEC – circulating endothelial cells count and for tissue from direct descending aorta and both results are in coherence.

- For quantitative analysis of protein expression in the tissue the Coomassie Brilliant Blue method provides visual confirmation but may lack quantitative accuracy, but using high-power fields and high expertise could minimize the error. The ten 400x high power field where ten separate fields of view observed at a magnification of 400 times can ensure representative sampling of the tissue to increase the statistical robustness of the findings and it is enough as their tissue homogenate is homogenous.

- Typical sample size for experimental groups of Sprague-Dawley rats ranges from 8-12, of course it depends on statistical power, scientific validity and ethical consideration. The 10 sample size for each of four groups is enough sample size for this study. Controls are also used reverse control also being done by blocking HO-1 expression.

- The result, discussion and conclusion are inline and appropriate

- The biomarkers for endothelial injury and damage selected here (endothelial cell protein c receptors, endothelin-1, von Willbrand factor, and soluble thrombomodulin are appropriate, and others to measure the effect of atorvastatin such as HMG-CoA reductase for cholesterol TC, HO-1 expression and the LPS inflicted damage to the endothelial is also appropriate. The mechanism underlying the effect or atorvastatin through HMG-G inhibition and increasing HO-1 expression is also well explained.

- To be clarified or corrected

- The dosage of atorvastatin for low and high is 2 and 10 mg/kg/d. In researches involving Sprague-Dawley rats the dosage of atorvastatin; typically, or approximately range for low dose 1-5, medium 5-20 and high is above 20 mg/kg/day, here as the dose can vary based on specific research objective (here is to test high dose), duration of the study and weight of rats; it would be better if you mention the reason why you select the 2 and 10 mg dosage per kg/d, what is your background or could you reason out? Have you considered previous literature or do you have any dose-response studies to determine the most appropriate doses?

2. Has the statistical analysis been performed appropriately and rigorously?

Yes,

Strength

- one-way ANOVA and students t-test is used as their data was parametric, that is correct to compare the 3 or 2 groups, the right statistical methods used.

To be clarified or corrected

- Better to put the actual p value along with the cutting value 0.05 to get a highlight of how much it is significant in the result section instead of putting only like <0.05 or >0.05

- instead of saying the normally or approximately normally you should put the nature of distribution of your data aptly as normal or not or with the other words like parametric or non-parametric – this makes your analysis ambiguous so avoid the saying approximately here.

- In addition to this if multiple doses of atorvastatin are tested here, is it not possible to analyze dose-response relationships using regression analysis or non-linear modeling techniques– this is optional if you add this it will strengthen your study

- at what CI was the P value calculated it should have to be mentioned in the statistical methodology part at least once

3. Have the authors made all data underlying the findings in their manuscript fully available?

- No

- The average measurements were presented in the figures at the apex. The descriptive statistics part should be presented as additional data; the means, medians and variance measures should be available, according to PLOS data policy. It is better to be included here.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Yes

Strength

- The manuscript is presented with clear and standard English.

To be corrected or clarified

- The title “ ..is significantly in mitigating …“ is grammatically incorrect, the adjective significantly is used as a noun here. Use it as significant or if you prefer to use significantly you need to put it with its subordinate word such as high.– the third option is to restructure or change the word.

- In the introduction section “increased secretion of endothelin-1 (ET-1) from cells and a noticeable increase in cytokine levels, including malondialhdehyde (MDA), endothelial cell protein C receptor (EPCR), and ” in this sentence you should avoid including as it misleads that MDA is also a cytokine which it is not.

- In the abstract –“ based on heme oxygenase “ should be “ based on heme oxygenase expression”

- 1.3 statistical analysis

o “The normally or approximately normally distributed data” – this phrase has grammatical error instead of saying the normally you need to say the normal or approximately normal, this comment is from the language perspective but statistically you need to be apt, if it is parametric just say it is parametric and if not just put non-parametric .

o “meanSEM.One-way” – no space after full stop

o The confidence level should also be mentioned along with the p value results at least once

- 1.2.4

o “..CD31 positive cells.” Put the full stop.

- 2.2

o “following trend(Figure 2)” need to be following trend (Figure 2) - put the space

5. Review comments to the Author

- Generally, it is interesting to review a technically rich and thoroughly made study like this. The Authors has put great effort to make a significant contribution to the scientific community and to the existing body of knowledge. Technically the study is rich and the presentation also so much interesting. I am happy to read their manuscript to review it. It is well done and keep it up guys.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

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Submitted filename: Reviewer Attachment.docx

Atorvastatin Inhibits Lipopolysaccharide (LPS)-induced Vascular Inflammation to Protect Endothelium by Inducing Heme Oxygenase-1 (HO-1) Expression

PONE-D-24-15138R1

Dear Dr. Gong Chen,

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Kind regards,

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Academic Editor

PLOS ONE