Review Reports - European Bilberry Extract Ameliorates Dietary Advanced Glycation End Products-Induced Non-Alcoholic Steatohepatitis in Rats via Gut Microbiota and Its Metabolites

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of Manuscript ID: nutrients-4006346

Title: European bilberry extract ameliorates dietary advanced glycation end products-induced non-alcoholic steatohepatitis in rats via the gut microbiota and its metabolites

Authors: Lihui Shen, Ruijie Cheng, Wenwen Chen, Hongjie Liu, Xinyu Wang, Ruikun He, Xiaoxing Mo, Liegang Liu.

General comment: the study reports the beneficial effect of administration of a European bilberry extract (EBE) to rats submitted for 80 weeks to a diet enriched in advanced glycation end products (AGEs) that provokes a non-alcoholic steatohepatitis. The long-term high AGEs diet caused liver inflammation, steatosis, fibrosis, and dysfunction in rats, and EBE treatment attenuated or prevented all these processes. EBE regulated gut dysbiosis and promoted production of short chain fatty acids and regulated their receptor in high AGEs diet-fed rats. Finally, at molecular level, EBE inhibited the activation of HMGB1/RAGE/NF-κB signaling pathway in the liver of high AGEs diet-fed rats. All these effects significantly alleviated the non-alcoholic steatohepatitis induced by a high AGE diet in rats. Hypothesis and objectives are sound and clearly exposed, methods are adequate and results are clear, well organized and correctly discussed. Finally, bibliography is very suitable and updated. The main achievement of the study is the extraordinary long period of assay, 80 weeks, that facilitates the potential translation of results to humans; and the major concern is the lack of information of some parameters at the end of such long term. Some specific comments are detailed below:

Specific comments:

Lines 90-97; protocol number for animal experimentation should be included.
Material and methods; several epigraphs are out-of-place.
Line 110; the word/term SCFASCFA should be explained or removed.
Figure 1; the reason why body weight and food intake exposed in this figure were evaluated just for 21 weeks whereas the experiment lasted up to 80 weeks should be exposed. The long-term effect of AGE diet on both parameters and the potential preventive effect of EBE treatment could be very informative; indeed, the long-term effect is the main goal of the present study since the rest of determinations were performed at the end of the feeding/treatment period. Furthermore, when the authors expose the potential limitations of the study, they assume that heating of the diet might reduce the concentration of vitamins and evoke the production of acrylamide, two conditions that could easily affect food intake and body growth over time.
Figure 2, incomplete statistical analysis; all three data within any assay in panels B and C, control, AGEs and EBE, should be compared among them, not only comparing control and EBE to AGEs, but data from controls and EBE should also be compared to reveal if the recovery to untreated controls is complete or partial. The same comment applies to the rest of figures.
Figure 7; there seems to be a discrepancy between band density and densitometry data for RAGE; while densitometry in panel B indicates a 50 % increase in RAGE, band intensity in panel A clearly exceeds that percent. Although western blot exposure and photograph is very clean, it must be representative of the densitometry data obtained from several assays. The authors should find a picture more representative or re-evaluate the densitometry from the different assays.

Author Response

Specific comments:

Comments 1: Lines 90-97; protocol number for animal experimentation should be included.

Response 1: Thank for these precious comments and suggestions. We have added the protocol number for animal experimentation in lines 95-98 with red front in revised version.

Comments 2: Material and methods; several epigraphs are out-of-place.

Response 2: Thank for these precious comments and suggestions. We have checked all epigraphs and modified them in the revised version.

Comments 3: Line 110; the word/term SCFASCFA should be explained or removed.

Response 3: Thank for these precious comments and suggestions. We apologize for the oversight of erroneously including “SCFASCFA” in the text. It has now been removed in the revised version.

Comments 4: Figure 1; the reason why body weight and food intake exposed in this figure were evaluated just for 21 weeks whereas the experiment lasted up to 80 weeks should be exposed. The long-term effect of AGE diet on both parameters and the potential preventive effect of EBE treatment could be very informative; indeed, the long-term effect is the main goal of the present study since the rest of determinations were performed at the end of the feeding/treatment period. Furthermore, when the authors expose the potential limitations of the study, they assume that heating of the diet might reduce the concentration of vitamins and evoke the production of acrylamide, two conditions that could easily affect food intake and body growth over time.

Response 4: Thank you for your suggestion. We feel sorry for our carelessness. In this study, after a one-month acclimation period, the rats underwent a 20-month (80-week) dietary intervention, resulting in a total experimental duration of 21 months. As showed in Figure S1, we have changed “weeks” to “months”.

Comments 5: Figure 2, incomplete statistical analysis; all three data within any assay in panels B and C, control, AGEs and EBE, should be compared among them, not only comparing control and EBE to AGEs, but data from controls and EBE should also be compared to reveal if the recovery to untreated controls is complete or partial. The same comment applies to the rest of figures.

Response 5: Thank for these precious comments and suggestions. We aimed to investigate the protective effects of EBE under conditions of high dietary AGEs exposure in this study. The comparison between the Control group and the high-AGEs diet group exclusively involved high-AGEs diet as the single variable, with the objective of investigating its impact on non-alcoholic steatohepatitis in rats. And the sole variable between the high-AGEs diet group and the EBE group was the EBE intervention, which aimed to investigate the ameliorative effect of EBE on high-AGEs-induced non-alcoholic steatohepatitis. However, if comparing control and EBE group, two variables (high-AGEs diet and EBE intervention) are simultaneously introduced, thus precluding direct comparison between these groups.

Comments 6: Figure 7; there seems to be a discrepancy between band density and densitometry data for RAGE; while densitometry in panel B indicates a 50 % increase in RAGE, band intensity in panel A clearly exceeds that percent. Although western blot exposure and photograph is very clean, it must be representative of the densitometry data obtained from several assays. The authors should find a picture more representative or re-evaluate the densitometry from the different assays.

Response 6: Thank for these precious comments and suggestions. We sincerely apologize for the incorrect calculation of the densitometry of RAGE due to our carelessness. We have recalculated the densitometry for RAGE. Please see Figure 6.

Reviewer 2 Report

Comments and Suggestions for Authors

This study describes the pernicious effects of a long-term high AGEs (Advanced 2 Glycation End Products) diet-on the liver health and the ameliorating action of EBE (European Bilberry Extract) simultaneous ingestion.

The action seems to be mediated a change in the microbiota composition, although several complementary biochemical markers (cytokines, butyrate receptors and HMGB1/RAGE/NFκB signalling pathway) are also measured. Authors indicate that the effect of EBE on the dietary AGEs-induced liver damage is poorly known, so that performing this study is convenient and necessary.

The work is complete, methods are appropriate, and the paper is well referenced. Conclusion is consistent and congruent with the results. However, some points to be addressed before acceptance for clarifying some minor points.

In the 4.2.4section, 30 rats were involved (3 groups of 10 rats), but 14 of those rats were excluded due to tumors, intestinal obstruction or chronic infections. The number of casualties is too high. These high number of exclusions would be justified to discard possible side circumstances during the study.

Figure 1 indicates the body weight and food intake for 21 weeks, but the rats were followed for 80 weeks (according to 2.4). Please, clarify this discrepancy or partial information.

On the use of the expression “AGEs diet” and “High AGEs diet”.

At 3.2 it is stated that EBE reduces the AGEs accumulation in rats fed with "High AGEs diet" (line 219). The groups are control chow and “high AGEs diet”. The denomination “high AGEs diet” is maintained in 3.3, 3.4 and Figures 2, 3 and 4. The expression “high AGEs diet” could be assumable, but at 3.5 , lines 289-297, there are a description of bacterial genus reduced (beneficial group) and increased (deleterious group) after (1) “AGEs diet” versus controls; and (2) “High AGEs diet” versus EBE group. Both bacterial sets are very similar, but the denomination “AGE group” and “High AGEs group” puzzles to me and possible future readers. There are no two AGEs group described at the methods. According to section 2.3, there are two groups, normal and high AGEs diet, and according to 2.4, three groups, control, AGEs and EBE groups are presented. The use of the term “High AGEs” is maintained at the section 3.6 and 3.7. I suggest that the term “AGEs diet” and “High AGEs diet” would be uniformed to avoid confusion between two groups with an “AGEs” and “High AGEs” diets that were never created..

Minor points

The agent “nine fluorovaleric” acid is mentioned at lines 136, 142. Please, check this agent. I know 2-fluorovaleric acid, but I do not know 9-fluorovaleric, and valeric acid contains less than 9 carbons.

Line 175: PFBBr is an alkylation agent (pentafluorobenzyl bromide). Please, check this addition and indicate the concentration and other details. Alternatively, give some reference about the protocol or the role of that agent in the SCFA derivation for GC/MS analysis.

Author Response

Comments 1: In the 4.2.4section, 30 rats were involved (3 groups of 10 rats), but 14 of those rats were excluded due to tumors, intestinal obstruction or chronic infections. The number of casualties is too high. These high number of exclusions would be justified to discard possible side circumstances during the study.

Response 1: Thank for these precious comments and suggestions. Since this study investigates the impact of long-term high-AGEs diet on non-alcoholic steatohepatitis using a naturally aging rat model, it should be noted that during the aging process, various physiological functions decline while the risk of developing multiple diseases (including tumors) significantly increases. In the natural aging process, approximately 20% - 40% animals would be lost [1]. The mortality in rats from causes such as tumors, intestinal obstruction, or chronic infection were confirmed by veterinarians. Besides, in cases of tumor-related deaths, we collected tumor tissue for pathological analysis to further verify the cause of mortality.

Comments 2: Figure 1 indicates the body weight and food intake for 21 weeks, but the rats were followed for 80 weeks (according to 2.4). Please, clarify this discrepancy or partial information.

Response 2: Thank you for your suggestion. We feel sorry for our carelessness. In this study, after a one-month acclimation period, the rats underwent a 20-month (80-week) dietary intervention, resulting in a total experimental duration of 21 months. As showed in Figure S1, we have changed “weeks” to “months”.

Comments 3: On the use of the expression “AGEs diet” and “High AGEs diet”.

At 3.2 it is stated that EBE reduces the AGEs accumulation in rats fed with "High AGEs diet" (line 219). The groups are control chow and “high AGEs diet”. The denomination “high AGEs diet” is maintained in 3.3, 3.4 and Figures 2, 3 and 4. The expression “high AGEs diet” could be assumable, but at 3.5 lines 289-297, there are a description of bacterial genus reduced (beneficial group) and increased (deleterious group) after (1) “AGEs diet” versus controls; and (2) “High AGEs diet” versus EBE group. Both bacterial sets are very similar, but the denomination “AGE group” and “High AGEs group” puzzles to me and possible future readers. There are no two AGEs group described at the methods. According to section 2.3, there are two groups, normal and high AGEs diet, and according to 2.4, three groups, control, AGEs and EBE groups are presented. The use of the term “High AGEs” is maintained at the section 3.6 and 3.7. I suggest that the term “AGEs diet” and “High AGEs diet” would be uniformed to avoid confusion between two groups with an “AGEs” and “High AGEs” diets that were never created.

Response 3: Thank for these precious comments and suggestions. To avoid ambiguity, we have changed the “AGEs group” to “high-AGEs diet group” throughout the text and figures. All the revisions were labeled in red font in revised manuscript, and can be tracked in revision mode.

Minor points

Comments 4: The agent “nine fluorovaleric” acid is mentioned at lines 136, 142. Please, check this agent. I know 2-fluorovaleric acid, but I do not know 9-fluorovaleric, and valeric acid contains less than 9 carbons.

Response 4: Thank you for your suggestion. We feel sorry for our carelessness. We have corrected the reagent name to “Nonafluorovaleric Acid”. Nonafluorovaleric Acid were obtained from Tokyo Chemical Industry, CAS RN: 2706-90-3.

Comments 5: Line 175: PFBBr is an alkylation agent (pentafluorobenzyl bromide). Please, check this addition and indicate the concentration and other details. Alternatively, give some reference about the protocol or the role of that agent in the SCFA derivation for GC/MS analysis.

Response 5: Thank for these precious comments and suggestions. Our group has previously established a well-validated method for SCFA detection, which has been documented in some published articles [2-4]. He et al. also used 100mM PFBBr for SCFA derivation [5]. We have added the concentration and dosage of PFBBr in the revised version with red front in line 173.

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[3]Mo X., Sun Y., Liang X., Li L., Hu S., Xu Z., Liu S., Zhang Y., Li X. and Liu L. Insoluble yeast β-glucan attenuates high-fat diet-induced obesity by regulating gut microbiota and its metabolites. Carbohydr Polym. 2022; 281: 119046. doi: 10.1016/j.carbpol.2021.119046.

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Reviewer 3 Report

Comments and Suggestions for Authors

This study investigates the effects of EBE on NASH induced by long-term dietary AGEs and its underlying mechanisms. However, the following issues require revision:

Introduction:

Why was EBE selected for this study? What are the unique advantages of EBE?

Methods:

Why was 150 mg/kg EBE chosen as the treatment concentration? Is there any scientific basis for this specific dose, especially since EBE concentrations of 100–400 mg/kg are commonly used? Why were no dose-response groups included?

Which companies supplied the commercial assay kits and ELISA kits?

The manuscript formatting is problematic. The organization of Sections 2.12 and 2.13 is unclear.

Data on body weight changes and food intake are not critical and should be moved to the supplementary materials.

Line 203: Is there an extra space after the comma?

Please check punctuation usage, for example, in Line 207.

Section 3.4:

The authors should provide a detailed description of the images instead of merely stating that EBE ameliorated injury and steatosis.

A brief explanation of certain indicators, such as ALT and AST, and their relevance to liver pathology should be included.

Why were total SCFA levels in blood and feces not measured?

Discussion:

The authors did not measure AGEs levels in the intestines. Why was this not done? Without such data, what is the basis for emphasizing that most AGEs are transferred to the colon(doi.org/10.1016/j.phrs.2025.107819)? Is there any scientific evidence to support this claim?

Some bacterial names are not italicized.

Lines 375–380: Is it necessary to reiterate the results here? How does this differ from the Results section?

What is meant by "Elegant studies"?

Lines 380–383: These points are not the focus. The emphasis should be on why EBE modulates gut microbiota. For instance, which components of EBE promote gut microbiota growth, or do other extracts similar to EBE have the same effect(DOI: 10.26599/FSHW.2025.9250732)? By comparing relevant studies, which components in EBE are believed to play a key role in balancing gut microbiota? Additionally, while OGTT and other indicators were measured (doi.org/10.1002/fft2.70122), it is unclear why, as this study focuses on the liver rather than diabetes.

Figure 2:

Is "The" necessary in the axis labels?

Author Response

This study investigates the effects of EBE on NASH induced by long-term dietary AGEs and its underlying mechanisms. However, the following issues require revision:

Introduction:

Comments 1: Why was EBE selected for this study? What are the unique advantages of EBE?

Response 1: Thank for these precious comments and suggestions. The EBE was primarily composed of 15 anthocyanins and 5 anthocyanidin. Phytochemicals, particular anthocyanins, can be used to inhibit protein glycation and AGEs production [1]. Our preliminary research has found that EBE, which was composed of abundant anthocyanins, reduced the accumulation of AGEs in serum and different tissues. After a single oral administration of 100 mg/kg EBE, the anthocyanins present in EBE were absorbed into systemic circulation after 15-30 mins consumption; they were mainly distributed in the liver and kidney and then excreted into urine over the following 24 h [2]. Gui et al. also reported that the anthocyanins present in EBE could be detected in the systemic circulation, jejunum, liver, kidney, and brain after oral administration of EBE [3] . Moreover, anthocyanins inhibited AGEs accumulation in the serum and liver [4, 5] Numerous studies have shown that EBE, particularly anthocyanins, can distribute in the liver and reduce the accumulation of AGEs in the liver. Furthermore, our previous studies have demonstrated that EBE could ameliorate AD-like pathological changes induced by high AGEs diet[6] and decrease the AGEs accumulation in the liver[7]. We hypothesized that it could improve liver function by antagonizing AGEs. Therefore, under conditions of a high-AGEs diet, we further investigated the effects of EBE on non-alcoholic steatohepatitis. Corresponding revisions have been made to the introduction section in the revised version in red front.

Methods:

Comments 2: Why was 150 mg/kg EBE chosen as the treatment concentration? Is there any scientific basis for this specific dose, especially since EBE concentrations of 100–400 mg/kg are commonly used? Why were no dose-response groups included?

Response 2: Thank for these precious comments and suggestions. It is helpful to improve our ability of logic thinking. Firstly, we summarized the relevant articles of EBE on its related metabolic disorders in the following Table 1. The dose range of EBE was 25-4400 mg/kg in animals. Then, the LD (50) value of EBE was greater than 5000 mg/kg 16328973 in the acute toxicity test, and no observed adverse effect level (NOAEL) was determined, after subchronic oral administration of 1000 mg/kg EBE in rat 34812659. Our group’s previously published research has demonstrated that EBE can effectively reduce the internal exposure levels of AGEs [6]. Nevertheless, we fully agree with your suggestion and will design multiple dose groups in subsequent experiments to investigate the effects of different EBE doses on AGEs.

Table 1 Studies of EBE its-related metabolic disorders.

Species	Dose	Intervention	Time	Outcome	Reference
Rat	150 mg/kg	EBE	80 weeks	EBE can reduce the accumulation of AGEs in tissues.	[7]
Rat	150 mg/kg	Bilberry extract	15 months	BE alleviated typical AD-like pathological changes.	[6]
Rat	400 mg/kg	Bilberry extract	24 hours	The distribution of anthocyanins.	[1]
Rat	165 mg/kg	Purple potato extract	14 days	Suppressed malondialdehyde levels, and restored antioxidant enzyme activities in diabetic rats.	[5]
Rat	25, 50 mg/kg	Anthocyanins	8 weeks	50 mg/kg anthocyanins improved energy production, glucose homeostasis, and oxidative stress.	[8]

Comments 3: Which companies supplied the commercial assay kits and ELISA kits?

Response 3: Thank for these precious comments and suggestions. The commercial assay kits to detect total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT) and aspartate transaminase (AST) were obtain from Elabscience Biotechnology Co. Ltd. The ELISA kits to detect inflammatory cytokines were obtained from R&D Systems. This information was added in line 150-152 in red front.

Comments 4: The manuscript formatting is problematic. The organization of Sections 2.12 and 2.13 is unclear.

Response 4: Thank you for your suggestions. We are so sorry for the problematic formatting cause by our carelessness. In the revised version, we have carefully reviewed the formatting throughout the entire text and have adjusted all formatting errors accordingly.

Comments 5: Data on body weight changes and food intake are not critical and should be moved to the supplementary materials.

Response 5: Thank you for your suggestion. We have moved the body weight changes and food intake in Figure S1.

Comments 6: Line 203: Is there an extra space after the comma?

Response 6: Thank you for your suggestions. We are so sorry for our carefulness. We have deleted the extra spaces in line 203.

Comments 7: Please check punctuation usage, for example, in Line 207.

Response 7: Thank you for your suggestions. We apologize for these mistakes and have corrected all punctuation misusages in the whole text.

Section 3.4:

Comments 8: The authors should provide a detailed description of the images instead of merely stating that EBE ameliorated injury and steatosis.

Response 8: Thank for these precious comments and suggestions. We have added detailed descriptions of Figure 3 A-D. The specific content was shown as follows: To explore the effect of high AGEs diet on the liver, we conducted liver pathological examination. Compared with control group, evident lipid droplets and inflammatory cell infiltration were observed in livers in high AGEs diet-fed rats (Fig. 3A-B). However, EBE reduced liver inflammation and steatosis in high AGEs diet-fed rats (Fig. 3A-B). According to Sirius red staining and α-SMA staining, increased α-SMA expression and fibrogenesis were induced by high AGEs diet, whereas EBE inhibited liver fibrosis in high AGEs diet-fed rats (Fig. 3C-D). Please see the revised manuscript section 3.3.

Comments 9: A brief explanation of certain indicators, such as ALT and AST, and their relevance to liver pathology should be included.

Response 9: Thank for these precious comments and suggestions. The relevant content has been incorporated in the revised version with red front.

Comments 10: Why were total SCFA levels in blood and feces not measured?

Response 10: Thank for these precious comments and suggestions. The major SCFAs formed by the gut bacteria are acetate, propionate, and butyrate which account for approximately 80% of all SCFAs [9]. By measuring acetate, propionate, and butyrate levels separately, we can more accurately evaluate the targeted effect of EBE on each individual short-chain fatty acid. Furthermore, the measurement of acetate, propionate, and butyrate levels is commonly employed in the extensive existing literature on SCFA [10-12]. Nevertheless, your suggestion has been truly insightful. We will proceed with detect total SCFA by GC-MS/MS further.

Discussion:

Comments 11: The authors did not measure AGEs levels in the intestines. Why was this not done? Without such data, what is the basis for emphasizing that most AGEs are transferred to the colon (doi.org/10.1016/j.phrs.2025.107819)? Is there any scientific evidence to support this claim?

Response 11: Thank for these precious comments and suggestions. In our previous research, we had already measured the AGEs content in the intestines and found that EBE intervention significantly reduced AGEs levels in colon and duodenum [7]. Corresponding part has been added in line 362-363 in red front.

Comments 12: Some bacterial names are not italicized.

Response 12: Thank you for your suggestion. We are so sorry for our carelessness. We have checked the entire text carefully and italicized all the bacterial names.

Comments 13: Lines 375–380: Is it necessary to reiterate the results here? How does this differ from the Results section?

Response 13: Thank for these precious comments and suggestions. This section provides a concise summary of the research findings and builds upon them to develop the discussion.

Comments 14: What is meant by "Elegant studies"?

Response 14: Thank you for your suggestion. We are so sorry for our carelessness of mistakenly writing “Elegant” instead of “Emerging”. We have corrected it in the revised version.

Comments 15: Lines 380–383: These points are not the focus. The emphasis should be on why EBE modulates gut microbiota. For instance, which components of EBE promote gut microbiota growth, or do other extracts similar to EBE have the same effect (DOI: 10.26599/FSHW.2025.9250732)? By comparing relevant studies, which components in EBE are believed to play a key role in balancing gut microbiota? Additionally, while OGTT and other indicators were measured (doi.org/10.1002/fft2.70122), it is unclear why, as this study focuses on the liver rather than diabetes.

Response 15: Thank for these precious comments and suggestions. We have rewritten the relevant section of the discussion in the revised version with red front. The details are as follows: “We proposed that anthocyanins, the key active components in EBE, functioned as prebiotic-like substances by reaching the colon intact and selectively promoting the growth of beneficial bacteria. This notion was supported by our findings and consistent with previous studies showing that anthocyanins from other botanical sources (e.g., blueberry, purple red rice) served as favorable carbon sources for taxa like Bifidobacterium and Lactococcus [13-15]. Furthermore, a recent comparative study on polyphenol-rich extracts also confirmed that flavonoid components, particularly anthocyanins, played a pivotal role in balancing gut microbiota [16].”

Despite comparable fasting blood glucose levels, EBE could maintain glucose tolerance and insulin sensitivity. Numerous studies investigating hepatic steatosis also include measurements of glucose tolerance-related indicators [17-19].

Figure 2:

Comments 16: Is "The" necessary in the axis labels?

Response 16: Thank you for your suggestion. We have deleted “the” in the axis labels.

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have conveniently addressed all my comments and queries, thus, in my opinion the revised version should be accepted for publication at Nutrients.

Author Response

Comments 1: The authors have conveniently addressed all my comments and queries, thus, in my opinion the revised version should be accepted for publication at Nutrients.

Response 1: We appreciate your valuable suggestions. We have accordingly revised the figures（Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6) and addressed the details you mentioned, specifically by optimizing the font sizes and the spacing and alignment between subfigures, as suggested, to achieve a more polished presentation.