Lactic Acid Bacteria Exert a Hepatoprotective Effect against Ethanol-Induced Liver Injury in HepG2 Cells
Round 1
Reviewer 1 Report
This study tested the Lactic acid bacteria strains protective effect in ethanol-induced HepG2 cells including the ethanol-induced oxidative stress measured by the lipid peroxidation and glutathione content, hepatocyte injury level measured by ALT and AST levels. In addition, the research further investigated the mRNA expression level associated with antioxidant enzyme and lipid metabolism to demonstrate the underlying molecular pathway. The antibiotic susceptibility, hemolysis, survival rate in simulated gastrointestinal tract stability and adhesion ability were also tested for the evaluation of the safety for considering the strains as potential candidates for alcohol-induced liver disease.
As authors already clearly pointed out that the further investigation and in vivo study are needed; however, this study provides useful information on further investigation of the potential beneficial function of lactic acid bacteria in ALFD. Overall, the study is well-designed and the data are worthy to publish and will provide additional information and data to the community for further investigation and exploration.
Some concerns are required to be revised to improve the quality of the manuscript.
Majors:
In Table 2 Erythromycin for MG4237, the MIC value is not consistent with the (S) based on the cut-off value, should it be (R)? please double-check. Therefore, the description in lines 276-277 is not totally correct.
In Figure 6, what is the relationship of CYP2E1 with antioxidant enzyme and oxidative stress, inhibiting or promoting, illustrating it with an appropriate arrow.
In section 3.5, please indicate the gene expression of enzymes, since only mRNA expression was detected not the protein levels. In addition, the description in lines 260-261 is not totally correct, revise it.
In lines 358 and 290, making it consistent, 98% or 99%?
Minors:
Line 10: add punctuation mark before “Therefore”.
Line 26: suggest change” includes” to “includes alcohol-induced”, as liver fibrosis and cancer can be induced by other factors. Similarly, in the Discussion section.
Line 110: “100 Ø” change to 100 mm, put the unit.
Line 191: ***p < 0.001, three stars
Line 198: 10x > 10×.
Line 222: there is an additional space before the ALT.
Line 257: suggest change “all lipid oxidation factors” to all tested lipid oxidation factors”.
Line 259: suggest change “all factors” to “all tested factors”.
Table S2: n. r. please indicate the meaning ’not required”.
In addition, there are some tracked red markers in the manuscript, in lines 361 and 363, and Table 3.
Author Response
Response to Reviewer 1 Comments
Dear reviewer in Microorganisms
We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “Lactic acid bacteria exert hepatoprotective effect against ethanol-induced liver injury in HepG2 cells”, manuscript number microorganisms-1358365. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Microorganisms.
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<GENERAL COMMENTS>
This study tested the Lactic acid bacteria strains protective effect in ethanol-induced HepG2 cells including the ethanol-induced oxidative stress measured by the lipid peroxidation and glutathione content, hepatocyte injury level measured by ALT and AST levels. In addition, the research further investigated the mRNA expression level associated with antioxidant enzyme and lipid metabolism to demonstrate the underlying molecular pathway. The antibiotic susceptibility, hemolysis, survival rate in simulated gastrointestinal tract stability and adhesion ability were also tested for the evaluation of the safety for considering the strains as potential candidates for alcohol-induced liver disease.
As authors already clearly pointed out that the further investigation and in vivo study are needed; however, this study provides useful information on further investigation of the potential beneficial function of lactic acid bacteria in ALFD. Overall, the study is well-designed and the data are worthy to publish and will provide additional information and data to the community for further investigation and exploration.
Some concerns are required to be revised to improve the quality of the manuscript.
Majors:
Point 1: In Table 2 Erythromycin for MG4237, the MIC value is not consistent with the (S) based on the cut-off value, should it be (R)? please double-check. Therefore, the description in lines 276-277 is not totally correct.
Response 1: Thank you for your comment. As a result of checking the value again, it was confirmed that it was (R), so we changed “S” to “R” in Table 2. So, we add “~MG5311 and erythromycin from L. fermentum MG4237” in line 280.
Point 2: In Figure 6, what is the relationship of CYP2E1 with antioxidant enzyme and oxidative stress, inhibiting or promoting, illustrating it with an appropriate arrow.
Response 2: Thank you for your comment. CYP2E1 is known to inhibit the expression of antioxidant enzymes by expressing ROS. Thus, we illustrate an appropriate arrow between CYP2E1 and antioxidant enzyme.
Point 3: In section 3.5, please indicate the gene expression of enzymes, since only mRNA expression was detected not the protein levels. In addition, the description in lines 260-261 is not totally correct, revise it.
Response 3: Thank you for your comment. We change section 3.5 “~ Enhancing Antioxidant Enzyme ~” to “~ Enhancing mRNA Expression of Antioxidant Enzyme ~”. In addition, we change “~ ethanol-induced HepG2 cells have been shown to activate CYP2E1~ and adipose metabolism-related factors~” to “~ all LAB have been shown to inactivate CYP2E1 ~ and modulate adipose metabolism-related factors ~ in ethanol-induced HepG2 cells” in lines 263-265.
Point 4: In lines 358 and 290, making it consistent, 98% or 99%?
Response 4: Thank you for your comment. As a result of checking the value again, it was confirmed that it was 98%, so we changed to 98% in lines 358.
Minors:
Point 5: Line 10: add punctuation mark before “Therefore”.
Response 5: Thank you for your comment. We add punctuation mark before “Therefore” in Line 10.
Point 6: Line 26: suggest change” includes” to “includes alcohol-induced”, as liver fibrosis and cancer can be induced by other factors. Similarly, in the Discussion section.
Response 6: Thank you for your comment. We change” includes” to “includes alcohol-induced” in Line 26-27 and Discussion section.
Point 7: Line 110: “100 Ø” change to 100 mm, put the unit.
Response 7: Thank you for your comment. We change” 100 Ø” to “100 mm” in Line 111.
Point 8: Line 191: ***p < 0.001, three stars
Response 8: Thank you for your comment. We change to ***p < 0.001 in Line 193.
Point 9: Line 198: 10x > 10×.
Response 9 Thank you for your comment. We change “10x “ to “10×”in Line 200.
Point 10: Line 222: there is an additional space before the ALT.
Response 10: Thank you for your comment. We remove space before the ALT in Line 224.
Point 11: Line 257: suggest change “all lipid oxidation factors” to all tested lipid oxidation factors”.
Response 11: Thank you for your comment. We change “all lipid oxidation factors” to” all tested lipid oxidation factors” in Line 259.
Point 12: Line 259: suggest change “all factors” to “all tested factors”.
Response 12: Thank you for your comment. We change “all factors” to “all tested factors” in Line 260.
Point 13: Table S2: n. r. please indicate the meaning ’not required”.
Response 13: Thank you for your comment. We add ’not required” for indicate n.r. in Table S2.
Point 14: In addition, there are some tracked red markers in the manuscript, in lines 361 and 363, and Table 3
Response 14: Thank you for your comment. We remove tracked red markers in lines 361 and 363, and Table 3.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The protective properties of lactic acid bacteria (LAB) strains, including Lactobacillus brevis, Limosilactobacillus reuteri, and Limosilactobacillus fermentum, were evaluated in ethanol-induced HepG2 cells. Even though this is just Cell culture data, mice models would have been added to increase the impact of the studies. Considereing the cell culture data the manuscript comes with a great concept of protection by LAB strains on ALFD. T The results are presented well and the study design is good with minor corrections.
- Addition of Gram staining will give clarity to the images in Figure -2.
- Please provide any staining to show Adipose or lipid deposition. Oil-O-Red staining can be done to the HePG2 cells to confirm the fatty changes.
Author Response
Dear reviewer in Microorganisms
We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “Lactic acid bacteria exert hepatoprotective effect against ethanol-induced liver injury in HepG2 cells”, manuscript number microorganisms-1358365. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Microorganisms.
<GENERAL COMMENTS>
The protective properties of lactic acid bacteria (LAB) strains, including Lactobacillus brevis, Limosilactobacillus reuteri, and Limosilactobacillus fermentum, were evaluated in ethanol-induced HepG2 cells. Even though this is just Cell culture data, mice models would have been added to increase the impact of the studies. Considereing the cell culture data the manuscript comes with a great concept of protection by LAB strains on ALFD. T The results are presented well and the study design is good with minor corrections.
Point 1: Addition of Gram staining will give clarity to the images in Figure -2
Response 1: Gram staining, which is used to stain microbial cells, specifically stains peptidoglycan in the cell membrane so that microorganisms are clearly visible. However, since HepG2 is animal-derived cells and hepatocytes, it is not a suitable method to check the cell shape through gram staining. It is mainly confirmed by MTT staining, and the results are presented in Table 1.
Point 2: Please provide any staining to show Adipose or lipid deposition. Oil-O-Red staining can be done to the HePG2 cells to confirm the fatty changes
Response 2: Cell apoptosis was confirmed after ethanol induction, so it was not suitable to perform oil red o staining. However, lipogenesis-related factors were identified through mRNA expression.
Author Response File: Author Response.docx
