The Effect of Barley Bran Polyphenol-Rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague–Dawley Rats Fed a High-Fat and High-Cholesterol Diet

Round 1

Reviewer 1 Report

The manuscript needs supportive evidence that barley bran extract has protective ability against NASH. 

The manuscript by Omagari et al., entitled “Barley Bran ------------- Cholesterol Diet” deals with the use of Barley Bran extract for curing NASH in rat model.

Comments to the Author

Major Queries:

1.      Figure 2 is not complete as some figures are missing and are not readable.

2.      Figure 3 the histology images are not clear and better-quality images with good resolution and labelling of lesion will help the reader to understand the histology.

3.      The results section needs complete rewriting as the information is repeated much and needs edits. The significance can be written with p values such as (p<0.001, p<0.01 or p<0.05)

 instead of giving numbers.

4.      Each figures legend is describing the animal treatment as its already mentioned in materials and methods so repetition can be avoided.

5.      Figure 4 is not complete as some figures are missing such as mRNA expression of MTP, FAS, CPT-1, SREBP-1, LXR-alpha and FXR.

6.      As the BP prepared is crude its difficult to understand which chemical compound in that is have a beneficial effect against NASH.

7.      There is no significant difference between MO, ML, MH and PO, PL and PH between themselves so its not proving that BP has beneficial effects.

8.      The discussion section needs a figure explaining the mechanistic explanation of barley bran and its protection against NASH.

The manuscript has major flaws in the design as the results are not supportive that barley bran with different dose has protection against NASH. 

Author Response

RE: Manuscript ID livers-2914955

“The Effect of Barley Bran Polyphenol-rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague-Dawley Rats Fed a High-fat and High-cholesterol Diet” (revised from “Barley bran polyphenol-rich extracts prevent nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet”) by Katsuhisa Omagari et al.

 Thank you very much for your valuable comments. your comments have been very helpful in allowing us to revise our manuscript. In our revised manuscript, all revised portions are in red font.

Major queries:

1. Figure 2

      We are so sorry that some figures in the Figure 2 are missing. This is a technical error. Now all complete graphs are included in the revised Figure 2. 

2. Figure 3

      Thank you for your comment. Now the histology images are amended to be clearer with higher resolution, and inflammatory foci and hepatocyte ballooning are indicated by arrows and arrow heads, respectively, to make readers understand the histology.

3. The results section

       According to the Reviewer’s comment, the results section was re-edited to avoid repetition, and significance was written with p values when there was a statistically significant difference (lines 242-243, 252-255, 262-265, 273-274, and 285-286, in our revised manuscript). Because our results revealed no definite evidence of BP protective effects against NASH, the title is now amended to “The Effect of Barley Bran Polyphenol-rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague- Dawley Rats Fed a High-fat and High-cholesterol Diet” (lines 2-4 in our revised manuscript).

4. Figure legends

       According to the Reviewer’s comment, figures legends and table footnotes are amended to be more concisely to avoid repetition with the contents in the materials and methods section (figure legends in Fig 1, Fig 2, Fig 3, and Fig 4 and footnotes of Table 1, and Table 3 in our revised manuscript).

5. Figure 4

　　 We are so sorry that some figures in the Figure 4 are missing. This is a technical error. Now the mRNA expression of MTP, FAS, CPT-1, SREBP-1c, LXR-alpha and FXR graphs are included in the revised figure 4.

6. Beneficial ingredients for NASH treatment

        The Reviewer’s comments are quite right that the exact amounts of the effective ingredients were not confirmed in the present study. We speculate that procyanidins and/or proanthocyanidins are strong candidates, but we have no data to prove it right now. So, further studies are necessary to clarify the contents of proanthocyanidins and procyanidins in BP, their bioavailability, and their contributions to the physiological functions of BP. This is described in our manuscript (lines 456-466 in our revised manuscript).

7. No significant differences among MO, ML, and MH groups and among PO, PL, and PH groups

      As the Reviewer pointed out, there is no significant difference among MO, ML, and MH groups and among PO, PL, and PH groups in most of tested parameters. However, in the rat model of NASH with advanced hepatic fibrosis, high-dose BP prevented NASH development by ameliorating the histopathological findings of lobular inflammation. Also, BP tended to attenuate serum aspartate aminotransferase level in this model. In the rat model of NASH with mild-to-moderate hepatic fibrosis, BP tended to attenuate the serum levels of transaminases and ameliorated hepatic fibrosis, although there were no statistical differences. Also, BP-dose-dependent effects were revealed for several parameters, including MCP-1, TGF-β, and Mn-SOD gene expressions in the liver, although there were no statistical differences. Therefore, we suppose that BP may prevent NASH development or progression, presumably due to its anti-inflammatory and anti-oxidative properties. This is described in our manuscript (lines 472-488 in our revised manuscript).

8. Figure explaining the mechanism of BP protecting against NASH

       Thank you for your recommendation. Now we added a new Figure 5 showing the presumed mechanistic explanation of BP against NASH development in our rat model.

Reviewer 2 Report

The manuscript submitted for review examines the problem of non-alcoholic liver damage and steatohepatitis and the possibilities of influencing this pathology with natural products. The abstract is clear and informative. The introduction includes all the new evidence concerning the development of NASH, the mechanisms by which this pathology develops, and the protection of the liver by plant extracts.

The materials and methods used in this study are innovative, cutting-edge, and appropriate for the purpose the authors have set themselves. The results are presented with tables and graphs that are very informative and there is no need to read the text describing the results. The discussion is in agreement, both with the obtained results and with the studies of recent years studying the problem of NASH. I have some small remarks that the authors can keep in mind in the next experiments.

1. It would be good to measure some indicators related to oxidative stress, which is one of the leading causes in the pathogenesis of NASH, such as MDA, and GSH, and antioxidant enzymes such as GPx, CAT, SOD, etc.

2. It is not clear from the methodology in what doses isoflurane was used.

3. There is no positive control in the experiment with which to compare the effect of the extract used (e.g. silymarin or another phytoproduct with proven hepatoprotective and antioxidant properties).

4. How did you choose the number of animals you worked with?

5. What exactly is the low and high dose of the extract that you treat the experimental animals with (presented as mg/kg body weight of the rats)? What dose of the extract was each animal treated with and what was the route of administration of the extract?

The English language is at a very good scientific level, but there are some technical errors, such as on line 433 anti-viral should be anti-viral.

In lines 457, 462, and 465 the sentence "These results suggest that BP may have a...." is repeated 3 times

Author Response

RE: Manuscript ID livers-2914955

“The Effect of Barley Bran Polyphenol-rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague-Dawley Rats Fed a High-fat and High-cholesterol Diet” (revised from “Barley bran polyphenol-rich extracts prevent nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet”) by Katsuhisa Omagari et al.

Thank you very much for your positive evaluation. We are glad to revise our manuscript according to your recommendations. In addition, we checked and fixed some technical typographical errors and repetition (line 122, 154, 184, 395, lines 492-493, and line 507 in our revised manuscript).

1. Oxidative stress markers measurement

         Thank you for your comments. Actually, we measured hepatic mRNA expressions of CYP2E1, GPX-1, Mn-SOD to evaluate oxidative stress situation. In addition, HO-1 was also measured but there were no statistical differences in the rat model of NASH both with advanced hepatic fibrosis and mild-to-moderate hepatic fibrosis (data were not shown in our manuscript). Unfortunately, the remaining rat liver tissues run out. We are planning to measure other oxidative stress indicators such as MDA, GSH, and CAT in the next study, as the Reviewer’s recommendation.

2. Isoflurane dose

          Isoflurane was inhaled to rat until the rat does not move. Approximately 0.6 mL of liquid isoflurane per liter of chamber volume, but there were considerable individual differences. This is described in our manuscript (lines 119-121 in our revised manuscript).

3. Positive control

          As the Reviewer’s comment, there is no positive control group with known hepatoprotective or antioxidant agents in our study. As you know, there are currently no approved drugs for NASH treatment except for vitamin E. We assumed that it would be difficult to make strict comparison between BP and other known hepatoprotective or antioxidant agents. Therefore, positive control group was not set in our study. This is described in our manuscript (lines 452-456 in our revised manuscript).

4. Number of animals

          The number of rats per group was set as follows. In our last study (Omagari K, et al. J Nutr Sci Vitaminol 69: 357-369, 2023), hepatic triglyceride concentration, which is frequently accumulated in NAFLD/NASH liver, was 68.7 ± 22.6 mg/g liver in the rat model of NASH with mild-to-moderate hepatic fibrosis and 12.0 ± 7.0 mg/g liver in the Control group. when an α-error of 0.05 and a power of 80% were set, minimum required rat number is estimated to be three. In our present study, the number of rats per group was set to be 5 or 6 in order to deal with unexpected death during the rearing period. This is described in our manuscript (lines 99-105 in our revised manuscript).

5. Low and high dose of BP

         The daily doses of BP in this study were determined based on the human dose provided from the manufacturer of the BP, that is 3.5 g of BP per day for 60 kg of human body weight (approximately 58.3 mg/kg). Based on the dose conversion rate between rats and human (6.2; Nair AB, et al. J Basic Clin Pharma 7: 27-31, 2016), low dose of BP was set as 362 mg/kg body weight of the rats. The body weight of the rats between 9 to 18 weeks of age was expected to be 300-500 g, so 109-181 mg/day of BP should be fed to the rats. The daily food intake was expected to be 20-25 g/day. Therefore, the supplementation of approximately 0.7% (w/w) BP in the HFC diets would be good enough. In addition to this BP dose, we also used a 2.0% (w/w) BP dose as a dose that is roughly three-fold that of the human equivalent dose. The BP was fed by oral administration. This is described in our manuscript (lines 106-115 in our revised manuscript).

Reviewer 3 Report

1.   The manuscript is poorly written. There is no conclusion after each result section. A majority of the discussion is simply a repeat of the Result. Please make the discussion part more logistic.

2.   In Fig 1, the cumulative energy intake during the rearing period was significantly higher in the MO and MH groups than in the Control group, the food efficacy has no difference, but the body weight also has no difference. It does not make sense. For the cumulative energy intake, control group does not have error bar.

3.   In Fig 3, it is hard to see the staining clearly.

 Language needs to be improved throughout the manuscript. Some grammatical errors and typos are noted.

Author Response

RE: Manuscript ID livers-2914955

“The Effect of Barley Bran Polyphenol-rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague-Dawley Rats Fed a High-fat and High-cholesterol Diet” (revised from “Barley bran polyphenol-rich extracts prevent nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet”) by Katsuhisa Omagari et al.

Thank you very much for your valuable comments. Your comments have been very helpful in allowing us to revise our manuscript. In our revised manuscript, all revised portions are in red font.

1. The discussion part

        According to the Reviewer’s comment, the Results section was re-edited, and the Discussion section was also re-edited to avoid repetition and to make more logistic. Based on the statistical analyses, we concluded that in the rat model of NASH with advanced hepatic fibrosis, high-dose BP prevented NASH development by ameliorating the histopathological findings of lobular inflammation. Also, BP tended to attenuate serum aspartate aminotransferase level in this model. In the rat model of NASH with mild-to-moderate hepatic fibrosis, BP tended to attenuate the serum levels of transaminases and ameliorated hepatic fibrosis, although there were no statistical differences. Also, BP-dose-dependent effects were revealed for several parameters, including MCP-1, TGF-β, and Mn-SOD gene expressions in the liver, although there were no statistical differences. Therefore, we suppose that BP may prevent NASH development or progression, presumably due to its anti- inflammatory and anti-oxidative properties (lines 402-406, 408-419, 424-429, 431-451, and 472- 488 in our revised manuscript). Because our results revealed no definite evidence of BP protective effects against NASH, the title is now amended to “The Effect of Barley Bran Polyphenol-rich Extracts on the Development of Nonalcoholic Steatohepatitis in Sprague-Dawley Rats Fed a High-fat and High-cholesterol Diet” (lines 2-4 in our revised manuscript).

2. Cumulative energy intake, food efficacy, and body weight gain in the MO and MH groups

          As the Figure 1 indicates, the cumulative energy intake was significantly higher in the MO group than in the Control group (p = 0.003) and this was also higher in the MH group than in the Control group (p = 0.010). Because the food efficacy was not different among the Control, MO, and MH groups, body weight gain during the rearing period was higher in the MO group than in the Control group and this was also higher in the MH group than in the Control group, but there were no statistical differences (p = 0.235 and p = 0.853, respectively). The reason for these discrepant results are unclear but it may be related to the data variation (SD or SE). Indeed, standard error of the cumulative energy intake in the Control group was small (4917.5 ± 29.3 kcal) and therefore, error bar was hard to see in the original manuscript. Now we re-set this error bar more indicative.

3. Figure 3

        Now the histology images are amended to be clearer with higher resolution.

Round 2

Reviewer 1 Report

The manuscript can be accepted in its current form