Hydroxycitric Acid Alleviated Lung Ischemia-Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis through the Hif-1α Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article by Lu et al., is showing the effect of HCA on LIRI. However, following the review, I have a few concerns.

1. As the author mentioned in this manuscript, LIRI is one of the major causes of death in clinical settings.  So, what was the mortality in their case after inducing LIRI and during surgery?

2. If he found mortality in his mice, where is the effect of HCA on the survival rate of LIRI? He should show that.

3. Figure 1A, H&E, 2F, 4E, 5E & 5K, 7D, F & I images don't have a scale bar. It should be in images. It also needs to be clarified in which magnification the images were taken.

4. Surprisingly, the animal study data showed a minimal error bar, which shows the perfectness of the experiment. Thus, I am curious to see some of the raw values with analysis in the table form from Fig.1 D & 1E.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Lu et al, try to discern the mechanism by which hydroxycitric acid alleviates lung IRI. Hydroxycitric acid (HCA) is found in the fruit Garcinia cambogia and it is commonly used as a weight loss supplement. A role in having a therapeutic effect in macular degeneration by inhibiting HIF1a has also been reported.

The authors use a well-established model of mouse lung IRI where the mice were pre-treated with HCA one week before the surgery. The authors found that pre-treatment with HCA ameliorated both LIRI markers such us injury score, cytokine expression and wet/dry ratio, as well as ferroptosis and oxidative stress. The authors found that IRI increased the expression of HIF1a, which was reduced by pre-treatment with HCA. Experiments in vitro were performed using HUVEC endothelial cells. The authors found that HCA pre-treatment improved cell migration and expression of proteins involved with permeability and oxidative stress. Finally, a role for HIF1a was determined by using specific shRNA and over-expression.  

Comments

·         How many doses of HCA were administered, just one time? Please correct the route of administration as in methods it says gavage and in results injection.

·         The HCA control is missing in all experiments except when performing scratch assays. Actually, in the scratch assay the authors found that HCA could improve migration just by itself. This is suggestive of a mechanism independent of HIF1a.   Please, add the corresponding controls.

·         Title of Results section 3.1 should be re-written as it is pre-treatment with HCA and there is no reversal  but amelioration.

·         In Supplemental figure 2 the toxicity was found at 250 uM, thus when they write (line 333) “ did not induce significant cell death at concentrations up to 300 uM” should be changed to 200 uM.

·         line 336 HUVEC.

·         Line 338 100 or 200 uM.

·         Is endothelial permeability markers also changed with HCA alone?. A permeability assay using transwells should be performed.

·         432. The results do not suggest, the results show

·         There is no experimental data to suggest a role for HO-1 in the pathway. HO-1 is one of the many downstream targets of HIF1a and all should be decreased when HIF1a is decreased. Please eliminate HO-1 from the conclusion and title. The authors just proved a role for HIF1a

 

Comments on the Quality of English Language

I found the English appropriate. Minor review is needed.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has responded satisfactorily to my concerns.

Author Response

Thanks very much for your valuable suggestions

Thanks for your time! I really appreciated your help!

Reviewer 2 Report

Comments and Suggestions for Authors

The authors answered my questions except the one related to endothelial permeability.

Figure 4:

·         In Figure 4 Panels C,D and E are switched with F and G. please, fix the figure or arrange the text describing it accordingly.

·         The transwell migration experiments are not described in the methods section.

·         When I asked the authors to perform transwell assay, I referred to permeability assays not migration assay. The migration assay adds to the scratch assay, but the permeability one will add to the conclusion the author’s have with the decreased expression of VE-Cadherin and ZO-1. A protocol to perform these experiments can be found in PMCID: PMC6722679, among others.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have answered all my questions and fixed the manuscript accordingly