Protective Effects of Resveratrol on Cytotoxicity of Mouse Hepatic Stellate Cells Induced by PM_2.5

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors evaluated the role of PM2.5 and the effects of RES in mHSC cells for 24 hours. They ran a series of experiments to evaluate their proposal. They reported that PM2.5 treatment increased the expression of reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH). NF-κB, NLRP3, Caspase1, and IL-1β were found to be enhanced, while SIRT1 was down-regulated. The RES therapy reduced oxidative stress and inflammation by inhibiting the SIRT1/NF-κB pathway. These is quite interesting data, and this manuscript deserves publication with revision.

Major Comments –

1)     The introduction should be strengthened. Please add studies on PM2.5 and NAFLD.

2)     Please mention the Procedure for IL-1β in Methods section.

3)     Please provide transcription (mRNA) data for NF-κB, NLRP3, Cleaved-Caspase1, IL-1β genes.

4)     Can you evaluate IL-18 levels in mHSC culture after treating with PM2.5 and resveratrol.  

5)     In statistical analysis section describe how many times each experiment was performed?

6)     Explain how Resveratrol was dissolved and used to treat cells.

7)     Can authors provide a graphical abstract?

8)      Did authors measure Caspase-3 activity in cells?

Minor comments –

1)     Please check the labelling of figures – 2A, 2C & 3A.

2)     In Material and Methods, please include a flow chart for PM2.5 preparation and collection.

3)     Please provide the composition of the protein lysis buffer.

Author Response

Responses to comments of the Editor and Reviewer (Ms. No.: atmosphere-2983501)

Dear Editors and reviewers,

Thank you very much for the revision request and the reviewers’ comments on our manuscript (Ms. ID.: atmosphere-2983501).

We are pleased to answer the comments that were pointed out by the reviewers as follows:

 

Reviewer 1

• The introduction should be strengthened. Please add studies on PM₅ and NAFLD.

Reply:

Thanks for the suggestion! We have added studies to the manuscript.

The added content is as follows: A prospective cohort study has shown that long-term exposure to PM_2.5 positively correlates with the risk of NAFLD. An increase of 1 μg/m³ in PM_2.5 concentration above 23.5 μg/m³ was associated with a hazard ratio (HR) of 1.06 (95% CI: 1.04-1.09) for NAFLD identified by the Fatty Liver Index (FLI), and an HR of 1.05 (95% CI: 1.03-1.07) for NAFLD identified by the Hepatic Steatosis Index (HSI) [9]. Prolonged exposure to PM_2.5 induces inflammation and oxidative stress, promoting lipid accumulation in the liver and ultimately increasing the risk of NAFLD [10].

[9] Sun, S.; Yang, Q.; Zhou, Q.; Cao, W.; Yu, S.; Zhan, S.; Sun, F. Long-term exposure to air pollution, habitual physical activity and risk of non-alcoholic fatty liver disease: A prospective cohort study. Ecotoxicol Environ Saf. 2022, 15(235), 113440.

[10] Xu, M.X.; Ge, C.X.; Qin, Y.T.; Gu, T.T.; Lou, D.S.; Li, Q.; Hu, L.F.; Feng, J.; Huang, P.; Tan, J. Prolonged PM_2.5 exposure elevates risk of oxidative stress-driven nonalcoholic fatty liver disease by triggering increase of dyslipidemia. Free Radic Biol Med. 2019, 130, 542-556.

• Please mention the Procedure for IL-1β in Methods section.

Reply:

We have already added the measurement steps for IL-1β in the methods section (2.5. Measurement of ROS, SOD, MDA, LDH and IL-1β levels in the cells).

• Please provide transcription (mRNA) data for NF-κB, NLRP3, Cleaved-Caspase1, IL-1β genes.

Reply:

Thank you for your comment. Gene expression typically undergoes transcription and translation processes to produce biologically active proteins. Differences between protein and mRNA levels are common in biological research. This study focuses on the protein expression of these genes, observing their regulatory roles at the protein level. We do not measure the expression levels of mRNA.

• Can you evaluate IL-18 levels in mHSC culture after treating with PM₅ and resveratrol.

Reply:

IL-18 and IL-1β are pro-inflammatory cytokines and crucial downstream effectors of the NLRP3 pathway. As shown in Fig. 4C, upon treatment with PM_2.5, the expression of IL-1β significantly increases compared to the control group, which is associated with elevated inflammation levels. However, when different concentrations of resveratrol combined with PM_2.5 were exposed to the cells, IL-1β levels notably decreased compared to those of the PM_2.5-treated group. This indicates that resveratrol exerts a positive anti-inflammatory effect by modulating the NLRP3/Caspase1/IL-1β pathway. We did not assess changes in IL-18. We appreciate the reviewer's comment and have removed the contents of IL-18 from the discussion part, which does not affect the results and discussion of this study.

• In statistical analysis section describe how many times each experiment was performed?

Reply:

In the “Materials and methods 2.7”, we have described that “The results were expressed as mean ± SD (n=4)”, so each experiment was performed four times.

• Explain how resveratrol was dissolved and used to treat cells.

Reply:

Thanks for your comment! Resveratrol was weighed and dissolved in DMSO to form two concentration solutions. Different concentrations of resveratrol solution were added to the cell culture medium in the dish to keep a final concentration of DMSO less than 0.1% in the culture medium. Such DMSO levels in the cells are non-toxic.

• Can authors provide a graphical abstract?

Reply:

Yes. We have added a graphical abstract at the end of the manuscript, as shown in the following figure.

8)Did authors measure Caspase-3 activity in cells?

Reply:

We have not measured the activity of caspase-3. The NLRP3 inflammasome is an upstream signaling factor that activates Caspase-1 and controls IL-1β release. Various external stimuli can activate the NLRP3 inflammasome, promoting the activation of pro-Caspase1. The key gene in the NLRP3 pathway is caspase1, which regulates inflammation and fibrosis independently of caspase3. Hence, we did not investigate the activity of caspase-3.

 

Minor comments –

• Please check the labelling of figures – 2A, 2C & 3A.

Reply:

We have checked the labelling of figures – 2A, 2C & 3A. Fig. 2C is correct. We have modified the legend of 2A to LDH activity, consistent with Fig. 2A. We also have revised Figs. 3A,4A and 4B, in which the labelling of ordinate should be “Protein expression (Fold change/control)”. We have updated the legends of Figs. 3A, 4A and 4B.

• In Material and Methods, please include a flow chart for PM₅ preparation and collection.

Reply:

We agree. As shown in the following figure, we have added a flow chart for PM_2.5 preparation in Fig. S1.

• Please provide the composition of the protein lysis buffer.

Reply:

The primary components of the protein lysis buffer include 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, as well as various inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, and leupeptin. We prepared the protein lysis buffer with PMSF at a ratio of 100:1.

 

The corresponding contents suggested by the reviewers have been revised and marked with red. Please check them. Thank you very much!

Very sincerely yours,

Ruijin Li, PhD

Institute of Environmental Science, Shanxi University, China

Email: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Protective Effects of resveratrol on Cytotoxicity of Mouse Hepatic Stellate Cells Induced by PM2.5” aimed to investigate ameliorative effect of resveratrol on mouse hepatic stellate cells (mHSCs) injury induced by PM2.5. The obtained results showed the cytotoxic effect of PM2.5 that was inhibited by resveratrol. The authors concluded that resveratrol is potentially protective against liver injury induced by air pollutants, and its regulatory mechanism is associated with the SIRT1/NF-κB/NLRP3 pathway.

 

Please give a reference for the cytotoxicity assay protocol.

 

Please briefly explain and give protocols for the oxidative stress biomarkers.

 

Authors could critically evaluate the downsides of resveratrol due to its bioavailability and actual impact on human health since the molecular targets of resveratrol are numerous and a dominant mechanism is elusive or non-existent.

 

Park EJ, Pezzuto JM. The pharmacology of resveratrol in animals and humans. Biochim Biophys Acta. 2015; 1852(6):1 071-113. doi: 10.1016/j.bbadis.2015.01.014.

 

Pezzuto JM. Resveratrol: Twenty Years of Growth, Development and Controversy. Biomol Ther (Seoul). 2019; 27(1): 1-14. doi: 10.4062/biomolther.2018.176.

 

Please rephrase the following sentence “MDA and SOD, as important lipid peroxidation products and antioxidant enzymes, play vital roles in oxidative stress processes” – MDA is a product of LPO while SOD is one of the antioxidant enzymes, the word “respectively” could do the trick.

 

Subchapter 6. Patents should go to 5. Conclusion section while 6. Patents could be erased.

 

Minor remarks:

 

Be consistent with brand names for equipment/chemicals used and give city and state information.

Line 357 – change to “However, the specific mechanism… “.

You could include Data Availability Statement: & Acknowledgments or erase them.

Comments on the Quality of English Language

Paper would benefit from the minor editing of English language.

Author Response

Responses to comments of the Editor and Reviewer (Ms. No.: atmosphere-2983501)

Dear Editors and reviewers,

Thank you very much for the revision request and the reviewers’ comments on our manuscript (Ms. ID.: atmosphere-2983501).

We are pleased to answer the comments that were pointed out by the reviewers as follows:

 

Reviewer 2

• Please give a reference for the cytotoxicity assay protocol.

Reply:

Thanks for the suggestion! We have added a reference for the cytotoxicity assay protocol. Please review the changes. The Reference is:

Li, Y.; Xu, X.; Wang, L.; Li, X.; Liu, R.; Zhang, L.; Xu, Y. REDD1 (regulated in development and DNA damage-1)/autophagy inhibition ameliorates fine particulate matter (PM_2.5) -induced inflammation and apoptosis in BEAS-2B cells. Bioengineered. 2021, 12(1), 1403-1414.

• Please briefly explain and give protocols for the oxidative stress biomarkers.

Reply:

The oxidative stress biomarkers include reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) in this manuscript. The ROS assay kit utilizes the fluorescent probe DCFH-DA to detect ROS. DCFH-DA is non-fluorescent by itself and, upon entering the cell, is hydrolyzed by intracellular esterases to generate DCFH. ROS within the cell can then oxidize the non-fluorescent DCFH to produce the fluorescent compound DCF. By measuring the fluorescence of DCF, the level of intracellular ROS can be determined. The SOD activity is determined using the xanthine oxidase method (hydroxylamine method) with a specific SOD assay kit. The MDA assay kit employs a colorimetric approach based on the reaction between MDA and thiobarbituric acid (TBA), which yields a red product. This is followed by spectrophotometric quantification of MDA in cell lysates. In brief, we first prepared the samples to be tested according to the instructions provided with the corresponding assay kit. Then, we strictly followed the procedures outlined in the kit manual.

• Authors could critically evaluate the downsides of resveratrol due to its bioavailability and actual impact on human health since the molecular targets of resveratrol are numerous and a dominant mechanism is elusive or non-existent.

Reply:

We agree. We notice that resveratrol's application has some limitations due to its low bioavailability (Shu et al., 2018). Scientists are also actively exploring strategies to enhance resveratrol's bioavailability, focusing on synergetic interactions, pro-drug designs, and formulation improvements to increase its bioaccessibility (Shu et al., 2018). This will facilitate the better application of resveratrol in treating various diseases. Furthermore, reinforcing fundamental research on resveratrol, such as investigating the optimal dosage and elucidating the mechanisms behind its antioxidative, anti-inflammatory, and antifibrotic effects on the liver, is essential and will actively promote its utilization. Researchers should drive the translation of resveratrol from basic research to clinical application, furnishing ample and compelling clinical evidence for its efficacy in treating liver diseases.

SIRT1 plays a crucial antioxidative and anti-inflammatory role in the liver. This study investigated the regulatory effect of PM_2.5 on SIRT1. PM_2.5 significantly suppressed the expression of SIRT1 in hepatic stellate cells.

Research indicates that resveratrol targets a protein family of Sirtuins (SIRTs) (Meng et al., 2020). As an agonist of SIRT1, it activates SIRT1, which in turn modulates the activity of antioxidant enzymes by negatively regulating the expression of nuclear factor-κB (NF-κB), thereby alleviating oxidative stress and inflammation in the liver (Rawat et al., 2021). We also found that resveratrol can activate SIRT1, alleviating oxidative damage and inflammatory responses in cells, and through the NF-κB/NLRP3 pathway, it reduces the expression of fibrotic genes. This may represent a key mechanism by which resveratrol mitigates liver injury induced by PM_2.5.

Thanks for your suggestion! We have added the literature discussion on the target (SIRT1) of resveratrol to our manuscript and supplemented information regarding the limitations of resveratrol in its applications. Please check the changes.

Shu, X.H. Resveratrol and its bioavailability. J. Dalian Med. Univ. 2018, 40(3), 193-197.

Meng, X.; Zhou, J.; Zhao, C.N.; Gan, R.Y.; Li, H.B. Health benefits and molecular mechanisms of resveratrol: a narrative review. Foods. 2020, 9(3), 340.

Rawat, D.; Chhonker, S.K.; Naik, R.A.; Koiri, R.K. Modulation of antioxidant enzymes, SIRT1 and NF-κB by resveratrol and nicotinamide in alcohol-aflatoxin B1-induced hepatocellular carcinoma. J Biochem Mol Toxicol. 2021, 35(1), e22625.

• Please rephrase the following sentence “MDA and SOD, as important lipid peroxidation products and antioxidant enzymes, play vital roles in oxidative stress processes” – MDA is a product of LPO while SOD is one of the antioxidant enzymes, the word “respectively” could do the trick.

Reply:

Thank you! We have revised the original text to read as follows: MDA is a product of LPO, while SOD is one of the antioxidant enzymes. They are important biomarkers of oxidative stress. Please check the changes.

• Subchapter 6. Patents should go to 5. Conclusion section while 6. Patents could be erased.

Reply:

This study does not involve patents; hence, the manuscript excludes patent content. The fifth subheading in this study is "Conclusions." There are no sixth or subsequent subheadings.

Minor remarks:

• Be consistent with brand names for equipment/chemicals used and give city and state information.

Reply:

Many thanks! We have proofread the manuscript to ensure consistency in using equipment and chemical brand names and have provided the city information.

• Line 404 – change to “However, the specific mechanism…”.

Reply:

We appreciate that you are helping us improve the manuscript! We have revised the related content to “However, the specific mechanism of SIRT1 on NLRP3 and how NLRP3 activates downstream factors to regulate HSC cell fibrosis require further in-depth investigation”.

• You could include Data Availability Statement: & Acknowledgments or erase them.

Reply:

We have added the contents of the Data Availability Statement & Acknowledgments.

 

The corresponding contents suggested by the reviewers have been revised and marked with red. Please check them. Thank you very much!

Very sincerely yours,

Ruijin Li, PhD

Institute of Environmental Science, Shanxi University, China; Email: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed the comments. Please check out the X-axis labeling in Figures 2C and 4A.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors responded to the raised question and changed their paper accordingly.

Comments on the Quality of English Language

Minor editing of English language required.