Effect of Porcine Placental Extract Mixture on Alcohol-Induced Hepatotoxicity in Rats

Round 1

Reviewer 1 Report

This study aimed to investigate the effect of porcine placenta extract mixture on alcoholic-induced hepatotoxicity in rats. Overall, this experimental study could be of potential scientific interest, however numerous corrections would be needed to make it acceptable. The main problem resides in the methodologies and results sections. In particular, the preparation and composition of the placenta materials used here should be properly detailed. The choice of experimental animal groups (section 2.2) should be rationalized as well as experimental procedures are not clear. Furthermore, histopathological evaluation of hepatic lesions should be performed on a representative number of tissue sections per animal and also histopathological evaluation takes into account a scoring system used for nonalcoholic fatty liver disease (Kleiner et al., 2005), and also its application is incorrect in this study. Although a statistical analysis is included in the materials and methods section, no significance value is reported in the main text, as well as the results are incomplete or unclear. For example, data on food intake, any adverse events, body weight, euthanasia, physiological ranges for haematological parameters, etc, are missing. Finally, discussion section include unnecessary data (lines 212-222), or results from other studies, but no results obtained in this study is discussed in the section. 

Author Response

(My response to reviewer’s opinion by point to points)

• Main problems are in methodology and results sections: Especilally, the preparation and configuration of the placenta material used here should be appropriately detailed in scetion 2.1. Materials.
• The choice of experimental animal groups (section 2.2) should be rationalized as well as experimental procedures are not clear: Experimental animal and groups selection was rewritten as a descrition instead of the table.
• Furthermore, histopathological evaluation of hepatic lesions should be performed on a representative number of tissue sections per animal and also histopathological evaluation takes into account a scoring system used for nonalcoholic fatty liver disease (Kleiner et al., 2005), and also its application is incorrect in this study.

Reseponse> Because the pathologic findings of alcoholic liver disease and nonalcoholic hepatitis are similar, pathological evaluation also applied the same pathological score system, and there was no problem, and I added a reference to it to similar tissue evaluation criteria. Additional references were added (26, Sakhuja P, Pathology of alcoholic liver disease, can it be differentiated from nnalcoholic steatohepaptis? World J Gastroenterol. 2014, 20, 16464-16479). Animal liver tissue was observed to be representative of each of the three fragments in three of each group.

4. The statistical part added significance in the main text, and the statistical method(ANOVA) is a powerful method for assaying the homogeneity of the set of a set of aggregates. We have applied mainly Duncan's Multiple-Range Test among several ways to assign the significance of the group consisting of homogeneous average, and Tukey HSD and Scheffe Test were conducted but it was not much different among them.
5. Food intake, side effects, and body weight were generally considered, and euthanasia was planned according to IACUC standards, but all animals completed the experiment.

Response> The following was added to the results section: During the experimental period, body weight was measured once a week, and the overall animal condition was examined in detail while measuring daily food intake. The pattern of weight change and food intake in each group was similar, and there was no case of death due to an abnormal condition in each group.

6. The unnecessary part (lines 212-222) was deleted and the additional discussed.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have reported that porcine placenta extract mixture (PPEM) with different concentrations can decrease the expression level of ALT and AST in rat blood. High PPEM group can increase the enzymatic activity of hepatic ALDH. The mild inflammation and fatty lesions were observed in control and attenuated by PPEM. In addition, the PPEM showed activities in attenuating the alcoholic hepatotoxicity on the serological markers, hepatic ADH and ALDH and pathologic findings. However, some issues and concerns about the generalizability and usefulness of the model should be clarified or need to have further revision.

1. The authors present that the increasing expression of AST and ALT in alcohol-induced rat, and PPEM can reduce the level of AST and ALT. However, the ratio of AST:ALT is also the important index in human liver disease. In the table 2, all of the AST:ALT ratio is >1.5~2.4, it is meant that all of these rat models are similar to alcoholic liver disease (the clinical index shows ~92% of alcoholic liver disease contain ratio >1). Could the authors clarify this issue?
2. For statistic analysis, why do the author choose the Duncan’s MRT? How about the result using other statistic strategies for this analysis? Please explain.
3. The resolution of Figure 3 is not good. Please upload the new figure with higher resolution. In addition, the figure caption is not clear.
4. All of the abbreviations should have the full name.

Author Response

1. The authors present that the increasing expression of AST and ALT in alcohol-induced rat, and PPEM can reduce the level of AST and ALT. However, the ratio of AST:ALT is also the important index in human liver disease. In the table 2, all of the AST:ALT ratio is >1.5~2.4, it is meant that all of these rat models are similar to alcoholic liver disease (the clinical index shows ~92% of alcoholic liver disease contain ratio >1). Could the authors clarify this issue?

Response> In clinical practice, AST/ALT ratio is well known that alcoholic liver disease, chronic liver disease such as cirrhosis, and hepatotoxicity are important indicators. However, the AST/ALT ratio was found to be 2.4 at the mean normal values (AST=81.37, ALT=33.66) of rats in this experiment, and each value was also different from the human values (AST=0~40, ALT=0~40). Therefore, rather than applying the ratio as it is, it was thought that the change of each value should be viewed as factors for hepatic damage. Therefore, in this study, the change in each value was evaluated rather than the ratio of AST and ALT.

2. For statistic analysis, why do the author choose the Duncan’s MRT? How about the result using other statistic strategies for this analysis? Please explain.

Response> The statistical part added significance in the main text, and the statistical method(ANOVA) is a powerful method for assaying the homogeneity of the set of a set of aggregates. We have applied mainly Duncan's Multiple-Range Test among several ways to assign the significance of the group consisting of homogeneous average, and Tukey HSD and Scheffe Test were conducted but it was not much different among them.

3. The resolution of Figure 3 is not good. Please upload the new figure with higher resolution. In addition, the figure caption is not clear.

Response> upload the more clear new figure and caption.

4. All of the abbreviations should have the full name.

Response> In the abbreviation, the whole word was written.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

This study aimed to investigate the effect of porcine placenta extract mixture on alcohol-induced hepatotoxicity in rats. Overall, the manuscript has been significantly improved, in particular, data relating to the preparation and composition of the placenta materials used here has been added, as well as detailed information on experimental animal groups and serum indicators of liver damage. Furthermore, the statistical analysis and the correct application of the histological scoring system were added (section 3.3., and Table 3). Only few minor concerns to make the manuscript suitable for publication. 

Table 1. Has the amino acid composition of the pPEM formulation been determined?

Furthermore, nutritional strategy are considered key factors in preventing liver diseases. Though the introduction section falls within the topic of the study, Authors should enhance this section adding more information concerning the field of nutritional strategy, and thus, emphasize the growing interest showed by scientific community on diet improvement to optimize animal health status and welfare. On this regards, in the introduction section Authors could write: "Nowadays, the diet composition improvement represents a key factor to enhance the health status and welfare of animals; indeed, within the scientific community, the diet supplementation has been widely accepted as useful strategy to modulate and/or optimize the biochemical and molecular pathways which orchestrate the metabolic responses of the animal to both physiological and pathological conditions (Giannetto et al., 2020; Abbate et al., 2020)."

Giannetto A, Esposito E, Lanza M, Oliva S, Riolo K, Di Pietro S, Abbate JM, Briguglio G, Cassata G, Cicero L, Macrì F. Protein Hydrolysates from Anchovy (Engraulis encrasicolus) waste: In Vitro and In Vivo Biological Activities. Marine Drugs 2020, 18, 86; doi:10.3390/md18020086. 

Abbate JM, Macrì F, Capparucci F, Iaria C, Briguglio G, Cicero L, Salvo A, Arfuso F, Ieni A, Piccione G, Lanteri G. Administration of Protein Hydrolysates from Anchovy (Engraulis encrasicolus) waste for twelve weeks decreases Metabolic Dysfunction-Associated Fatty Liver Disease Severity in ApoE-/- Mice. Animals 2020; 10, 2303; doi:10.3390/ani10122303. 

Author Response

We are very grateful for the valued comments of the reviewer, and we submit new revised paper(cimb-1679991_v2) in addition to the cover-letter(v2).  

Author Response File: Author Response.docx