Sodium Butyrate Attenuates AGEs-Induced Oxidative Stress and Inflammation by Inhibiting Autophagy and Affecting Cellular Metabolism in THP-1 Cells

Round 1

Reviewer 1 Report

Overall comments:

The study focused on whether sodium butyrate could alleviate oxidative stress, inflammation, and metabolic dysfunction in THP-1 cells. Inflammation and oxidative stress caused by AGEs binding to receptors on the surface of macrophages have long-term consequences of possible kidney failure in people with diabetes. Their assertion therefore that the prevention and management of AGE-induced macrophage inflammation and oxidative stress are key to preventing diabetic kidney damage is sound. This study shows that sodium butyrate inhibits autophagy and NLRP3 inflammasome activation by blocking PI3K/Akt/NF-κB pathway. Overall, the study contributes important information to aid our understanding of the anti-inflammatory mechanisms of butyrate, and its potential application in managing diabetic nephropathy.

Introduction:

The introduction is well presented. The background provides relevant information needed to understand the context and rationale for the study.  

Materials and Methods:

1.     The methods and statistical tools used are appropriate/ satisfactory for the study.

2.     Some portions of the methods are however unclear. There is mention of four groups (line 97), but only three groups are described. Also, there is the presentation of results on different AGE concentrations used (lines 186 to 196; Figures 1A and 1B) but no method is described. It appears the treatment with varying concentrations of AGEs was a preliminary experiment prior to treatment with 400μg/L. If this is the case it would be helpful if it is stated as such.

Results:

1.     Figure 1A and 1B: it is unclear which AGE concentration was used for each of the outputs. The graph needs to show which bar refers to which AGE concentration.

2.     Figure 2A and 2B: Additional information should be provided under the figures to allow the reader to understand the graph without recourse to the text. The asterisk and hash symbols are not fully defined. The definitions should state clearly whether there’s a significant difference or not between the parameters compared.

3.     Table 1: Please provide information under the table to explain the content of the table.

Discussion:

1.     The discussion could be improved by limiting the background information, most of which have been adequately covered in the introduction. The first half of the discussion contains several examples of sentences of little relevance to the discussion.

 

2.     Lines 304-306: the sentence as phrased here is inaccurate, and not supported by the references cited. Intestinal bacterial endotoxin is not the main cause of insulin resistance and type two diabetes. 

Author Response

Reviewer 1#

The study focused on whether sodium butyrate could alleviate oxidative stress, inflammation, and metabolic dysfunction in THP-1 cells. Inflammation and oxidative stress caused by AGEs binding to receptors on the surface of macrophages have long-term consequences of possible kidney failure in people with diabetes. Their assertion therefore that the prevention and management of AGE-induced macrophage inflammation and oxidative stress are key to preventing diabetic kidney damage is sound. This study shows that sodium butyrate inhibits autophagy and NLRP3 inflammasome activation by blocking PI3K/Akt/NF-κB pathway. Overall, the study contributes important information to aid our understanding of the anti-inflammatory mechanisms of butyrate, and its potential application in managing diabetic nephropathy.

Dear Reviewer,

Thank you for your rapid response on our manuscript. The constructive criticism of you was much appreciated and we revised our manuscript accordingly. The suggestions were accepted, more information and details were included in the text, and the manuscript was revised thoroughly. All the modifications performed in the revised manuscript are highlighted in red. The enclosed document at the bottom of this letter contains a point-to-point reply to you comments.

 

Introduction:

The introduction is well presented. The background provides relevant information needed to understand the context and rationale for the study.

Response: Thanks for your comments.

 

Materials and Methods:

1. The methods and statistical tools used are appropriate/ satisfactory for the study.

Response: Thanks for your comments.

 

2. Some portions of the methods are however unclear. There is mention of four groups (line 97), but only three groups are described. Also, there is the presentation of results on different AGE concentrations used (lines 186 to 196; Figures 1A and 1B) but no method is described. It appears the treatment with varying concentrations of AGEs was a preliminary experiment prior to treatment with 400μg/L. If this is the case it would be helpful if it is stated as such.

Response: Thanks for your comments. There were indeed four experimental groups in our study: NC, NaB, AGE, and AGE+NaB group. For the purpose of our experiment, we explored the mechanism of inhibiting AGEs-induced inflammation in the NaB group. Therefore, our metabolomic analysis only detected the NC group, the AGEs group and the AGEs+NaB group. According to our previous studies, 400 mol/L NaB at the final concentration for 24 h had no inhibitory effect on THP-1 cells. The experimental design we used was consistent with your comments. In the Cell Culture and Treatment section, the verbal expression of the design was modified.

 

Results:

1. Figure 1A and 1B: it is unclear which AGE concentration was used for each of the outputs. The graph needs to show which bar refers to which AGE concentration.

Response: We are very sorry that we made a mistake in the logo of the picture when editing the Figure 1. We have edited the figure as you suggested.

 

2. Figure 2A and 2B: Additional information should be provided under the figures to allow the reader to understand the graph without recourse to the text. The asterisk and hash symbols are not fully defined. The definitions should state clearly whether there’s a significant difference or not between the parameters compared.

Response: Thanks for your comments. The detection of the oxidative stress factors MDA and SOD was omitted from the manuscript. We have now included it.

 

3. Table 1: Please provide information under the table to explain the content of the table.

Response: Revised.

 

Discussion:

1. The discussion could be improved by limiting the background information, most of which have been adequately covered in the introduction. The first half of the discussion contains several examples of sentences of little relevance to the discussion.

Response: Thanks for your comments. Revised.

 

2. Lines 344-346: the sentence as phrased here is inaccurate, and not supported by the references cited. Intestinal bacterial endotoxin is not the main cause of insulin resistance and type two diabetes.

Response: This is an inaccuracy of our expression. Chronic inflammation induced by endotoxins from gram-negative bacteria in the gut entering the blood system can only worsen insulin resistance and type 2 diabetes.

Author Response File: Author Response.doc

Reviewer 2 Report

AGEs produced in diabetic patients induce inflammation and oxidative stress in macrophages and direct these macrophages to the kidney to cause kidney damage. The dietary fiber metabolite butyrate is effective in the prevention and treatment of diabetic kidney damage. In this study, Yan et al. reveal that sodium butyrate (NaB) reversed AGEs-induced inflammation, oxidative stress and autophagy in human monocyte model THP-1 cells. They identify several metabolites altered after AGEs and NaB treatment by using metabolomics analysis, which could explain the reason underlying above physiological changes. This study provides new evidences for the mechanism of action of butyrate for treatment of type 2 diabetes. Although in the manuscript the cell model is a little inappropriate and the writing and data presentation need improvement, this manuscript could be considered to be accepted after adequate revision.

 

Major concerns:

1. Readers may not quickly grasp the idea of this paper before the long list of molecule markers in the abstract. IL-1β, NLRP3, Caspase-1 and NF-κB are associated with inflammation. LC3B and Beclin-1 are autophagy indicators. Malondialdehyde and SOD are also oxidative stress markers. What do “phosphatidylcholine, L-glutamic acid, UDP-N-acetylmuraminate, biotinyl-5'-AMP” represent? Try to make a concise and coherent summary of key findings in the introduction and attract more attention of potential readers.

2. Likewise, Authors depict the result in a matter-of-fact way. Especially in section 3.4, authors retell the names of altered metabolites following “The variation trends of the above differential metabolites were shown in Table 1”. Please emphasize key metabolites that support major conclusion. Tell readers who they are and what do they indicate. Current format let me confusing.

3. Given that AGEs results in kidney damage mainly through macrophage why authors directly use THP-1 cells but not differentiated macrophages from THP-1 cells using PMA and macrophage medium? Data from macrophage is true reflection of pathophysiological condition involving AGEs.

4. How many groups of samples are applied in UPLC-MS? All four groups or only NC, AGEs and AGEs+NaB? Please label sample names in Fig 4B-D and Fig 5A precisely so as to match corresponding description in figure legends.

5. In table 1, is it possible to list absolute content of each metabolite in each group or at least list fold change of metabolites in AGEs and AGEs+NaB groups compared with NC group, which is reflected by Fig 5A? Do not do pairwise comparison. Given NaB pre-treatment exerts protective effect in THP-1 cells treated with AGEs afterwards, authors should focus on metabolites whose contents are upregulated by AGEs and reversed by additional NaB. Not something up or down between two groups. In Fig 5A, UDP-N-acetylmuraminate, TXB2, SM_d18_116_0_, N-methylnicotinium meet such demand. What are their relationship with inflammation, autophagy, ROS and diabetes? Use reported studies (like Diabetes Care. 1998 Sep;21(9):1511-6) to correlate these finding with your major conclusion. Accordingly revise the last two paragraph in the Discussion section. In addition, please modify analysis method and redraw Fig 5B and C.

6. Under oxidative stress ratio of GSH:GSSH is reduced. Could authors explain why in Table 1 and Fig 5A, oxidized glutathione (GSSH) reduced after in AGEs-treated cells compared with NC cells?

7. I reckon that two first authors are familiar with related literatures. I just think the discussion section can be more refined. Every paragraph begins with a “mini-review” and ends with dispersed finding. Literature part contains less useful information and is separated with finding. Please provide a big picture perspective and reminder of the importance of this study at the beginning. Then please provide critical analysis of major findings combining with existing literatures as well as limitations and prospects.

 

Minor concerns:

1. Bar names on the upper right corner of Fig 1 are wrong. They should be AGEs concentration.

2. Sample number (n) should be provided in figure legend when corresponding panel contains error bar and p value. N is missing in Fig 1, 2 and 3.

3. There are some typographical errors. For example, row 75, remove “of”; row 209, “.” (full stop) after “THP-1 cells; row 395, remove “5” after inflammation; row 396, use abbreviation of uridine diphosphate-N- acetylglucosamine written in row 389; row 400-401, use full abbreviation of UDP-N-acetylglucosamine written in row 393.

4. Row 208, why authors choose cellular ROS, MDA and SOD as but not others as ROS indicators? Please briefly explain in the text.

5. Row 358-359, “Our hypothesis was that AGEs induce autophagy as a result of increased intracellular oxidative stress.” This can be quickly tested by treating AGEs-treated THP-1 cells with ROS scavengers like NAC and GSH and see if protein level of LC3B-II goes down.

Author Response

Reviewer 2#

AGEs produced in diabetic patients induce inflammation and oxidative stress in macrophages and direct these macrophages to the kidney to cause kidney damage. The dietary fiber metabolite butyrate is effective in the prevention and treatment of diabetic kidney damage. In this study, Yan et al. reveal that sodium butyrate (NaB) reversed AGEs-induced inflammation, oxidative stress and autophagy in human monocyte model THP-1 cells. They identify several metabolites altered after AGEs and NaB treatment by using metabolomics analysis, which could explain the reason underlying above physiological changes. This study provides new evidences for the mechanism of action of butyrate for treatment of type 2 diabetes. Although in the manuscript the cell model is a little inappropriate and the writing and data presentation need improvement, this manuscript could be considered to be accepted after adequate revision.

Dear Reviewer,

Thank you for your rapid response on our manuscript. The constructive criticism of you was much appreciated and we revised our manuscript accordingly. The suggestions were accepted, more information and details were included in the text, and the manuscript was revised thoroughly. All the modifications performed in the revised manuscript are highlighted in red. The enclosed document at the bottom of this letter contains a point-to-point reply to you comments.

 

Major concerns:

1. Readers may not quickly grasp the idea of this paper before the long list of molecule markers in the abstract. IL-1β, NLRP3, Caspase-1 and NF-κB are associated with inflammation. LC3B and Beclin-1 are autophagy indicators. Malondialdehyde and SOD are also oxidative stress markers. What do “phosphatidylcholine, L-glutamic acid, UDP-N-acetylmuraminate, biotinyl-5'-AMP” represent? Try to make a concise and coherent summary of key findings in the introduction and attract more attention of potential readers.

Response: Thanks for your comments. Revised.

 

2. Likewise, Authors depict the result in a matter-of-fact way. Especially in section 3.4, authors retell the names of altered metabolites following “The variation trends of the above differential metabolites were shown in Table 1”. Please emphasize key metabolites that support major conclusion. Tell readers who they are and what do they indicate. Current format let me confusing.

Response: Metabolomic analysis is currently limited to analyzing differential metabolites and metabolic pathways against a database. We have added the metabolic pathways involved in this differential metabolite in Table 1. The important differential metabolites involved in the function are also discussed in Discussion section.

 

3. Given that AGEs results in kidney damage mainly through macrophage why authors directly use THP-1 cells but not differentiated macrophages from THP-1 cells using PMA and macrophage medium? Data from macrophage is true reflection of pathophysiological condition involving AGEs.

Response: Please accept my deepest apologies for the oversight when we wrote the manuscript. In this study, we first added 100 ng/ml PMA into THP-1 cell culture medium to induce macrophage formation. After 24 h, the NC group, NaB group and AGEs group underwent follow-up treatment respectively, while the AGEs+NaB group was added with PMA and NaB at the beginning of the experiment, and then added with AGEs 24 h later. Relevant expressions have been added to the manuscript.

 

4. How many groups of samples are applied in UPLC-MS? All four groups or only NC, AGEs and AGEs+NaB? Please label sample names in Fig 4B-D and Fig 5A precisely so as to match corresponding description in figure legends.

Response: In the UPLC-MS experiment, there were 6 samples in each group. Only the NC, AGEs and AGEs+NaB groups were used for metabolomic analysis. We have modified the label to match the name in the manuscript.

 

5. In table 1, is it possible to list absolute content of each metabolite in each group or at least list fold change of metabolites in AGEs and AGEs+NaB groups compared with NC group, which is reflected by Fig 5A? Do not do pairwise comparison. Given NaB pre-treatment exerts protective effect in THP-1 cells treated with AGEs afterwards, authors should focus on metabolites whose contents are upregulated by AGEs and reversed by additional NaB. Not something up or down between two groups. In Fig 5A, UDP-N-acetylmuraminate, TXB2, SM_d18_116_0_, N-methylnicotinium meet such demand. What are their relationship with inflammation, autophagy, ROS and diabetes? Use reported studies (like Diabetes Care. 1998 Sep;21(9):1511-6) to correlate these finding with your major conclusion. Accordingly revise the last two paragraph in the Discussion section. In addition, please modify analysis method and redraw Fig 5B and C.

Response: The UPLC-MS devices used in untargeted metabolomics are excellent at identifying compounds, but not at quantifying them. Therefore, untargeted metabolomics cannot accurately detect the absolute concentration of this metabolic compound, but can only determine its relative concentration. UPLC-MS equipment has a detection limit for various compounds, and compounds below the detection limit cannot be detected. This may cause that the differential metabolites identified in the NC group/AGEs group and the AGEs group /AGEs+NaB group cannot be completely matched. In addition, the identification of compounds by UPLC-MS equipment is almost entirely dependent on molecular weight. The existing mass spectrometry technology still has errors in the detection of molecular weight of compounds, which also causes the differential metabolites between the groups in the experiment can not be completely matched. We revised the discussion following your suggestion.

 

6. Under oxidative stress ratio of GSH:GSSH is reduced. Could authors explain why in Table 1 and Fig 5A, oxidized glutathione (GSSH) reduced after in AGEs-treated cells compared with NC cells?

Response: As the exact mechanism of GSSH up-regulation remains unclear, it is thought to be related to cells' self-protection in the early stages, but a detailed understanding of the mechanism is necessary.

 

7. I reckon that two first authors are familiar with related literatures. I just think the discussion section can be more refined. Every paragraph begins with a “mini-review” and ends with dispersed finding. Literature part contains less useful information and is separated with finding. Please provide a big picture perspective and reminder of the importance of this study at the beginning. Then please provide critical analysis of major findings combining with existing literatures as well as limitations and prospects.

Response: Thanks for your comments. Revised.

 

Minor concerns:

1. Bar names on the upper right corner of Fig 1 are wrong. They should be AGEs concentration.

Response: Sorry for this mistake. We have modified it.

 

2. Sample number (n) should be provided in figure legend when corresponding panel contains error bar and p value. N is missing in Fig 1, 2 and 3.

Response: Thank you for your guidance, we have revised.

 

3. There are some typographical errors. For example, row 75, remove “of”; row 209, “.” (full stop) after “THP-1 cells; row 395, remove “5” after inflammation; row 396, use abbreviation of uridine diphosphate-N- acetylglucosamine written in row 389; row 400-401, use full abbreviation of UDP-N-acetylglucosamine written in row 393.

Response: We apologize for this mistake, revised. Thanks.

 

4. Row 208, why authors choose cellular ROS, MDA and SOD as but not others as ROS indicators? Please briefly explain in the text.

Response: An oxidative stress study usually utilizes SOD, MDA, and ROS as markers. ROS is a superoxide anion free radical produced by cell mitochondria, which is closely related to the inflammatory response level of macrophages and can induce cell damage. SOD is an important antioxidant enzyme that cleans superoxide anion free radicals in organism and can protect cells from the damage of oxygen free radicals. MDA is one of the products formed by the reaction between lipids and oxygen free radicals, and its content represents the degree of lipid peroxidation. Research has indicated that MDA levels have increased in patients with diabetes and SOD activity has decreased. ROS is related to the level of inflammatory response in patients and the occurrence of various diabetes complications. We revised the discussion following your suggestion.

 

5. Row 358-359, “Our hypothesis was that AGEs induce autophagy as a result of increased intracellular oxidative stress.” This can be quickly tested by treating AGEs-treated THP-1 cells with ROS scavengers like NAC and GSH and see if protein level of LC3B-II goes down.

Response: We have arranged to request additional experiments (Figure 2).

Author Response File: Author Response.doc

Reviewer 3 Report

1)    One of the major conclusions of the article is that sodium butyrate inhibits autophagy. However, only the formation of phagophore and autophagosome was analyzed. Therefore, it would be beneficial for the manuscript to check autophagy flux using BafA1. 

2)    Line 43, write diabetic patients instead of diabetics. 

3)    Reference(s) required in lines 67-68. 

4)    Line 96, is it 1 x 106 or 1 x 10⁶?

5)    Please rewrite line 98, only 3 groups are listed.

6)    Line 104 and 189, change qPCR to qRT-PCR. 

7)    Remove references from lines 105,116, and 135 unless using the same references somewhere else in the manuscript. qRT-PCR, WB, and ROS detection are common techniques. 

8)    Please provide catalogue # for primary and secondary antibodies in western blot section. 

9)    Line 143, please clarify that cell collection and preparation were done for which experiment?

10) Correct Figure 1A labels. You haven’t used any NaB in Figure 1A, therefore, correct it and provide concentration of AGEs. 

11) In Figure 1B, is IL-1β still significantly higher in AGEs+NaB group as compared to control group? Please provide a brief discussion. 

12) In Figure 2A, is MDA significantly higher in AGEs+NaB group as compared to control group? Please provide a brief discussion. You can include statistics comparing NC to AGEs+NaB in all the figures. 

13) Please provide high resolution image in 2B. Also, may be provide a better representative image as decrease in DCFH-DA is very little? Is it biological significant? 

14) In lines 222 and 225, rewrite treated to treatment. 

15) Provide molecular weight of all western blots in Figure 3.

16) In Figure 3B, provide a better representative image for caspase 1. All bands look similar. 

17) In figure legend for Figure 3, include inflammation. Write- The expression of autophagy related proteins and inflammation of THP-1 cells. 

18) Why No AGE+NAB group was included in Figures 4 and 5. 

19) Increase font size in Figure 4 because it is too difficult to read. 

20) Remove line 288. 

21) The use of a rodent (diabetes) model would have benefited the article. 

Author Response

Reviewer 3#

Dear Reviewer,

Thank you for your rapid response on our manuscript. The constructive criticism of you was much appreciated and we revised our manuscript accordingly. The suggestions were accepted, more information and details were included in the text, and the manuscript was revised thoroughly. All the modifications performed in the revised manuscript are highlighted in red. The enclosed document at the bottom of this letter contains a point-to-point reply to you comments.

1) One of the major conclusions of the article is that sodium butyrate inhibits autophagy. However, only the formation of phagophore and autophagosome was analyzed. Therefore, it would be beneficial for the manuscript to check autophagy flux using BafA1.

Response: Since the revision deadline is limited to 10 days, there are indeed some absence of experimental reagents due to this limitation. Therefore, we have not conducted BafA1 inhibitor studies, which is stated as a study limitation.

 

2) Line 43, write diabetic patients instead of diabetics.

Response: We apologize for this mistake, revised. Thanks.

 

3) Reference(s) required in lines 67-68.

Response: The Reference has been added.

 

4) Line 96, is it 1 x 106 or 1 x 106?

Response: We apologize for this mistake, revised. Thanks.

 

5) Please rewrite line 98, only 3 groups are listed.

Response: We apologize for this mistake, revised. Thanks.

 

6) Line 104 and 189, change qPCR to qRT-PCR.

Response: Thanks for your comments. Revised.

 

7) Remove references from lines 105,116, and 135 unless using the same references somewhere else in the manuscript. qRT-PCR, WB, and ROS detection are common techniques.

Response: These references have been deleted.

 

8) Please provide catalogue # for primary and secondary antibodies in western blot section.

Response: We had supplied.

 

9) Line 143, please clarify that cell collection and preparation were done for which experiment?

Response: Thanks for your comments. Revised.

 

10) Correct Figure 1A labels. You haven’t used any NaB in Figure 1A, therefore, correct it and provide concentration of AGEs.

Response: Thanks for your comments. Revised.

 

11) In Figure 1B, is IL-1β still significantly higher in AGEs+NaB group as compared to control group? Please provide a brief discussion.

Response: The exact reason for this is unknown. Our hypothesis is that IL-1β is not only secreted by macrophages via the NLRP3/Caspase-1/IL-1β pathway. There are also other pathways that activate cells.

 

12) In Figure 2A, is MDA significantly higher in AGEs+NaB group as compared to control group? Please provide a brief discussion. You can include statistics comparing NC to AGEs+NaB in all the figures.

Response: MDA is one of the products formed by the reaction between lipids and oxygen free radicals, and its content represents the degree of lipid peroxidation. Hypothetically, NaB would have little impact on lipid oxidation.

 

13) Please provide high resolution image in 2B. Also, may be provide a better representative image as decrease in DCFH-DA is very little? Is it biological significant?

Response: Thanks for your comments. Revised.

 

14) In lines 222 and 225, rewrite treated to treatment.

Response: Thanks for your comments. Revised.

 

15) Provide molecular weight of all western blots in Figure 3.

Response: Thanks for your comments. Revised.

 

16) In Figure 3B, provide a better representative image for caspase 1. All bands look similar.

Response: Thanks for your comments. When we were analyzing metabonomics data, the software could not enter "+". We have modified it manually.

 

17) In figure legend for Figure 3, include inflammation. Write- The expression of autophagy related proteins and inflammation of THP-1 cells.

Response: Thanks for your comments. Revised.

 

18) Why no AGE+NAB group was included in Figures 4 and 5.

Response: Thanks for your comments. Revised.

 

19) Increase font size in Figure 4 because it is too difficult to read.

Response: Thanks for your comments. Revised.

 

20) Remove line 288.

Response: Thanks for your comments. Removed.

 

The use of a rodent (diabetes) model would have benefited the article.

Response: Unfortunately, we have not conducted rodent studies, accordingly, we have added a paragraph to indicate the study limitations. In that paragraph, the beneficial effect of sodium butyrate on AGEs-induced diabetes rodent models are proposed for future studies.

Author Response File: Author Response.doc

Round 2

Reviewer 3 Report

Authors have addressed my concerns.