Chitosan from Crabs (Scylla serrata) Represses Hyperlipidemia-Induced Hepato-Renal Dysfunctions in Rats: Modulation of CD43 and p53 Expression
, Kunle Ogungbemi 1,3, Adewale Segun James 1, Ayodele Peter Folorunsho 1, Samuel Olanrewaju Abolade 1, Stella Onajite Ajamikoko 1, Eniola Olapeju Atayese 1 and Omowunmi Victoria Adedeji 1Round 1
Reviewer 1 Report
Review
This manuscript examines an interesting topic about the effect of chitosan in high-fat diets (HFD) on the liver (ALT, AST, ALP) and kidney (ALT, AST, urea, and creatinine) biomarkers, as well as the expression of CD43 and p53 proteins in these organs, in a murine model. The objective of the study was to evaluate the effect of chitosan in rats with a hyperlipidemic diet. The results show interesting data on the effect of chitosan in HFD; however, it is necessary to specify and expand information from the experiment, results, and discussion that allows a better understanding of the knowledge generated and which are detailed below.
Abstract
Line 23. Results of body weight and food intake must be reported for better understanding.
Line 28. A conclusion specifying the better concentration of chitosan in the high-fat diet (HFD) should be included in congruence with the best effects observed on liver and kidney biomarkers.
Materials and methods
Line 83. Drying, grinding, and drying conditions must be provided.
Line 89. A brief description of chitosan extraction should be provided.
Lines 92 -101. Conditions of the animals must be provided such as temperature, relative humidity, feeding, and water consumption during the acclimatization period and later during the experimental period. Also, indicate if the animals had individual boxes.
Lines 103-108. Detailed information in this section is necessary and very important such as: Indicate the reference model for the normal diet. Indicate the reference model for the HFD. Indicate the nutrient that provided each ingredient and the nature of these ingredients, if they were of commercial origin or of special design for animal diets, it is necessary to specify the brand and/or the supplier. A growth premix is ​​indicated, it is convenient to describe what it consists of this growth mix? Also, a salt is mentioned, what type of salt was used? The authors are encouraged to include a table describing the composition of the normal diet and HFD diets, including their energy provided by each diet and the proportion of their ingredients. In addition, the composition of the chitosan extract is important to include. Indicate if the diets were adjusted by adding chitosan and, what nutrients were adjusted?
Line 111. Detailed and very important information should be provided by the authors in this section. Indicate if the animals were divided randomly or some criteria were followed. Indicate, how the animals were managed in relation to their diets (daily amount provided)? How was food intake evaluated? Indicate, how was monitored the bodyweight of the animals? and, how often each animal was weighed?
Line 117. Indicate the method as were sacrificed animals. If you have obtained a blood sample, indicate the place and method of collection.
Lines 121-123. Specify whether these biomarkers (ALP, AST, ALT) were determined in serum, plasma, or in the tissue itself, briefly indicating, how the samples were prepared? Specify if the method was spectrophotometric and the equipment used.
Lines 126-136. Indicate, how the proportion of positive cells, location (nuclear, cytoplasmic, membranous), and intensity was determined? Was the comparison made with any specific control?
Line: 141. Indicate if multiple comparisons were made with ANOVA and the method used.
Results
Lines 147-151. This part is very confusing. On the one hand, the authors refer to the weight gained by the animals in the experimental period, but later they refer to the increase and decrease in body weight. It should be noted that there is no reduction in body weight, but there is a lesser increase in the weight of the animals. I strongly suggest to the authors that Table 1 report the initial, final, and weight gain for all experimental groups.
It is also necessary to include a table on the initial and final food intake of the animals and refer to these results. This topic is not reported in the actual manuscript.
Lines 176-183. In addition to the results cited by the authors, there is an ALT-lowering effect on normal diet + chitosan 5%, which is an event that the authors should point to as an effect antagonist of chitosan when HFD is not provided. A discussion about it should be included.
Line 189. It is very confusing when the authors refer to “the percentage positivity of hepatic CD43 is shown in Figure 3”. I suggest reviewing and clearly writing these results, exposing the positive cases observed, and relating these with the photomicrographs and the CD43 expression graph.
Figure 3. Indicate the scale on the photomicrographs. What does “**” mean?
Line 197. The authors should explain, why in Figures 3, 4, 5, and 6 the n = 3? What happened to the other animals?
Line 198. Include significance: p <0.05.
Line 201. The authors should also point out the effect of chitosan in the normal diet + 5% chitosan, its antagonistic effect in this diet compared to the HFD is interesting. A discussion should be included about it.
Figure 4. Indicate the scale on the photomicrographs.
Line 204. Include significance: p <0.05.
Line 209. It is very confusing when the authors refer to a “considerable decrease in the CD43 positivity”. I suggest reviewing and clearly writing these results, exposing the positive cases observed, and relating these with the photomicrographs and the CD43 expression graph.
Figure 5. Include significance: p <0.05.
Lines 215-218. Again, the authors should also point out the effect of chitosan in the normal diet + 5% chitosan, its antagonistic effect in this diet compared to an HFD is interesting. A discussion should be included about it.
Figure 6. Indicate the scale on the photomicrographs.
Line 221. Include significance: p <0.05.
Discussion
Lines 246-253. It is necessary that the authors include in the results the food intake in the different treatments and relate in the discussion the effect of this consumption and the effect of chitosan to clearly establish the relationship of each of these variables with respect to body weight gain. In addition, it is important to know the composition of the diets and the chitosan extract to relate it to this part of the discussion and this discussion should be expanded.
The discussion in relation to satiety cannot be linked in this study since the contribution of fiber from the diets and chitosan extract is not known. Then the authors should incorporate these results and the discussion should be expanded on this topic.
Line 264. Correct “enxymes” to “enzymes”
Line 285. Correct “P53” to “p53”
Line 317. Reference should be included in “deficient of CD43 (CD43 - / - BMT mice) did not present with atherosclerosis follow-ing sixteen weeks of high-fat diet consumption”.
Further discussion is needed on the effect of chitosan in a normal diet supplemented with 5% chitosan reducing ALT and p53 (Liver) and p53 (Kidney).
Lines 327-331. It is important that the authors specify the percentage of chitosan supplementation that shows the best results on CD43 and p53 on HFD.
Comments for author File: 
Comments.pdf
Author Response
REVIEWER 1
Line 23. Results of body weight and food intake must be reported for better understanding.
RESPONSE
The result of the bodyweight gain has been added to the abstract section, however, feed intake and performance data have been reported elsewhere (Ogungbemi et al., 2020).
Line 28 A conclusion specifying the better concentration of chitosan in the high-fat diet (HFD) should be included in congruence with the best effects observed on liver and kidney biomarkers.
RESPONSE
A wholesome look at the results shows that the inclusion of 1-3 % chitosan in the diet appeared to be effective; therefore, this observation has been included in the conclusion aspect of the abstract and the main manuscript.
Line 83. Drying, grinding, and drying conditions must be provided.
RESPONSE
The drying, grinding, and other necessary methods of chitosan preparation have been included under the methodology section.
Line 89. A brief description of chitosan extraction should be provided.
RESPONSE
A description of the extraction procedure has been included in the methodology section
Lines 92 -101. Conditions of the animals must be provided such as temperature, relative humidity, feeding, and water consumption during the acclimatization period and later during the experimental period. Also, indicate if the animals had individual boxes.
RESPONSE
The experimental condition has been included in the manuscript. See the experimental animals' sub-section in the methodology. The animals were housed in their respective cages per group.
Lines 103-108. Detailed information in this section is necessary and very important such as: Indicate the reference model for the normal diet. Indicate the reference model for the HFD. Indicate the nutrient that provided each ingredient and the nature
of these ingredients, if they were of commercial origin or of special design for animal diets, it is necessary to specify the brand and/or the supplier. A growth premix is indicated, it is convenient to describe what it consists of this growth mix? Also, a salt is mentioned, what type of salt was used? The authors are encouraged to include a table describing the composition of the normal diet and HFD diets, including their energy provided by each diet and the proportion of their ingredients. In addition, the composition of the chitosan extract is important to include. Indicate if the diets were adjusted by adding chitosan and, what nutrients were adjusted?
RESPONSE
The experimental diet used during the acclimatization period was purchased from an animal feed vendor, while the HFD was specially formulated for the experiment. Furthermore, the chitosan was added to the feed by reductive inclusion method at 1-, 3-, and 5% levels respectively. Table 1 depicts the diet composition as suggested by the reviewer.
Line 111. Detailed and very important information should be provided by the authors in this section. Indicate if the animals were divided randomly or some criteria were followed. Indicate, how the animals were managed in relation to their diets (daily amount provided)? How was food intake evaluated? Indicate, how was monitored the bodyweight of the animals? and, how often each animal was weighed?
RESPONSE
The animals were allotted into their respective groups using simple randomisation method. The amount of feed provided daily was 100 g. The HFD was offered to the animals in the HFD and HFD + Chitosan groups, whereas the control diet (normal diet was offered to the animals in the normal control and the normal diet + 5% chitosan groups). The animal bodyweight was taken weekly using a small animal weighing balance.
Line 117. Indicate the method as were sacrificed animals. If you have obtained a blood sample, indicate the place and method of collection.
RESPONSE
The animals were sacrificed using ketamine (35 mg/kg)/ xylazine (5 mg/kg) (Oguntoye and Oke, 2014). This has been included in the animal sacrifice subsection in the methodology section.
Lines 121-123. Specify whether these biomarkers (ALP, AST, ALT) were determined in serum, plasma, or in the tissue itself, briefly indicating, how the samples were prepared? Specify if the method was spectrophotometric and the equipment used.
RESPONSE
The biomarkers were measured spectrophometrically, using commercial lab kits. The title axis on figure 2 shows hepatic ALP, AST, and ALT. The post-nuclear fraction obtained from the homogenisation of 1 g of tissue in 9 mL of phosphate buffer, pH 7.4 was used following centrifugation at 3000 rpm.
Lines 126-136. Indicate, how the proportion of positive cells, location (nuclear, cytoplasmic, membranous), and intensity was determined? Was the comparison made with any specific control?
RESPONSE
The degree of positivity of the proteins (CD43 and p53) in the hepatic and renal cytoplasm was quantified using Image J software (Schneider et al., 2012). The photomicrograph is a representative slide from five animals, from which 3 slides were quantified. The comparison was made across the six groups.
Line: 141. Indicate if multiple comparisons were made with ANOVA and the method used.
RESPONSE
One-way analysis of variance was used to analyse the data, followed by Duncan Multiple range Test to ascertain the level of significance among the groups. See the statistical analysis section.
Lines 147-151. This part is very confusing. On the one hand, the authors refer to the weight gained by the animals in the experimental period, but later they refer to the increase and decrease in body weight. It should be noted that there is no reduction in body weight, but there is a lesser increase in the weight of the animals. I strongly
suggest to the authors that Table 1 report the initial, final, and weight gain for all experimental groups.
RESPONSE
Lines 176-183. In addition to the results cited by the authors, there is an ALT-lowering effect on normal diet + chitosan 5%, which is an event that the authors should point to as an effect antagonist of chitosan when HFD is not provided. A discussion about it should be included.
RESPONSE
The interpretation of the reduced ALT activity in the normal + 5% chitosan has been included. See the results section (3.3). A discussion has been included as well to depict the possible antagonistic effect of chitosan when HFD is not present.
Line 189. It is very confusing when the authors refer to “the percentage positivity of hepatic CD43 is shown in Figure 3”. I suggest reviewing and clearly writing these results, exposing the positive cases observed, and relating these with the photomicrographs and the CD43 expression graph.
RESPONSE
The data in figure 3 has been explained more clearly
Figure 3. Indicate the scale on the photomicrographs. What does “**” mean?
RESPONSE
The scales of all the photomicrographs have been included. ** was an oversight and has been corrected.
Line 197. The authors should explain, why in Figures 3, 4, 5, and 6 the n = 3? What happened to the other animals?
RESPONSE
The photomicrographs shown in figures 3, 4, 5, and 6 were representative of the five animals in the group. The quantification of the protein was analysed thrice using image J, this was why n=3 was inserted to indicate that the data used to plot the graphs below the slide were taken in triplicates
Line 198. Include significance: p <0.05.
(p < 0.05) has been inserted into line 198 and other necessary places
Line 201. The authors should also point out the effect of chitosan in the normal diet + 5% chitosan, its antagonistic effect in this diet compared to the HFD is interesting. A discussion should be included about it.
RESPONSE
Thank you for pointing this out. The antagonistic effect of chitosan when included at 5% in the normal feed has been indicated in the results and discussed accordingly.
Figure 4. Indicate the scale on the photomicrographs.
RESPONSE
The scale has been added
Line 204. Include significance: p <0.05.
RESPONSE
p <0.05 has been inserted.
Line 209. It is very confusing when the authors refer to a “considerable decrease in the CD43 positivity”. I suggest reviewing and clearly writing these results, exposing the positive cases observed and relating these with the photomicrographs and the CD43 expression graph.
RESPONSE
The word ‘’ considerable’’ has been replaced with ‘’ significantly’’
Figure 5. Include significance: p <0.05.
RESPONSE
p <0.05 has been inserted.
Lines 215-218. Again, the authors should also point out the effect of chitosan in the normal diet + 5% chitosan, its antagonistic effect in this diet compared to an HFD is interesting. A discussion should be included about it.
RESPONSE
The antagonistic effect has been shown and a discussion has been included
Figure 6. Indicate the scale on the photomicrographs.
RESPONSE
The scale has been inserted.
Line 221. Include significance: p <0.05.
RESPONSE
p <0.05 has been inserted.
Lines 246-253. It is necessary that the authors include in the results the food intake in the different treatments and relate in the discussion the effect of this consumption and the effect of chitosan to clearly establish the relationship of each of these variables with respect to body weight gain. In addition, it is important to know the composition of the diets and the chitosan extract to relate it to this part of the discussion and this discussion should be expanded.
RESPONSE
Thank you for this observation. Firstly, the result of the feed intake has been reported elsewhere (Ogungbemi et al., 2020). Admittedly, the data presented in table 1 is identical to the table 2 in the paper published by Ogungbemi et al. 2021 and has been inserted due to human error. The result that meant to be presented was of body animal body weight as against the cumulative weight gain which was presented. We understand the implication and severity of plagiarism. To this end, the table 1 has been re-presented as body weight and NOT cumulative weight gain.
Secondly, a comprehensive analysis of the diet and chitosan inclusion has been included and cited properly in the manuscript. A brief discussion on the effects of chitosan on body weight has been inserted into the discussion section. Furthermore, the diet composition has been presented on table 1 to show the components
The discussion in relation to satiety cannot be linked in this study since the contribution of fiber from the diets and chitosan extract is not known. Then the authors should incorporate these results and the discussion should be expanded on this topic.
RESPONSE
It has been reported that chitosan acts like prebiotics (fibres), and fibres are known to produce satiety sensation especially when used to treat metabolic diseases such as obesity and hyperlipidemia. This was the reason why it was suggested in the paper. However, the proximate analysis of the diet reported by Ogungbemi et al. (2020) shows that, diet with chitosan inclusion had higher fibre content than the normal diet.
Line 264. Correct “enxymes” to “enzymes”
RESPONSE
Enxymes changed to enzymes
Line 285. Correct “P53” to “p53”
RESPONSE
The P53 has been changed to p53
Line 317. Reference should be included in “deficient of CD43 (CD43 - / - BMT mice) did not present with atherosclerosis follow-ing sixteen weeks of high-fat diet consumption”.
RESPONSE
The references have been included
Further discussion is needed on the effect of chitosan in a normal diet supplemented with 5% chitosan reducing ALT and p53 (Liver) and p53 (Kidney).
RESPONSE
The discussion has been inserted
Lines: 327-331. It is important that the authors specify the percentage of chitosan supplementation that shows the best results on CD43 and p53 on HFD.
RESPONSE
Inclusion levels between 3-5% produced the best modulatory effects on p53 and CD43 expression. This has been included in the conclusion section of the paper.
Reviewer 2 Report
Ugbaja et al. present this manuscript, as part of a recent series of papers published by their group, on the use of chitosan derived from crab shell wastes as a natural therapeutic option for hyperlipidemia and co-morbidities. Chitosan, a naturally occurring glucosamine derivative has been studied extensively by various groups and has shown significant promise in the treatment of various disease conditions.
In this manuscript, the authors study the effect of addition of chitosan on renal and hepatocellular dysfunction in diet induced hyperlipidemia in mice. In particular, the authors study the effects on p53 and CD43 expression using immunohistochemistry. Taken together, their data suggests that crab chitosan exerts modulatory effects on CD43 and p53, and serves to play a protective role towards kidney and liver damage induced by the high lipid diet.
The observations in the paper are definitely interesting, and together with the studies previously published by the group, as well as by other researchers, makes a strong case for studying chitosan in various disease conditions. Some of the issues that need to be addressed by the authors before the manuscript may be considered for publication include:
Line 87: How was purity of the extracted chitosan determined? Please include details in the Methods section. How were the three concentrations of chitosan in the diet selected? Is there a maximum tolerated percentage that could be given to the mice?
Line 90: It would be very helpful for the reader if an overview of the extraction process is briefly mentioned in the text, along with the references cited currently.
Line 152: Table 1. Data presented in Table 1 seems to be identical to data presented in Table 2 in an article previously published by the authors (Ogungbemi et al., 2020. Ref.20). Perplexingly, the data in the published paper was reported to have been derived from mice after 6 weeks of treatment, while in this manuscript the same data appears to have been generated after 8 weeks of treatment (see abstract, line 19).
Author Response
REVIEWER 2
Line 87: How was purity of the extracted chitosan determined? Please include details in the Methods section. How were the three concentrations of chitosan in the diet selected? Is there a maximum tolerated percentage that could be given to the mice?
RESPONSE
The degree of deacetylation and precipitation of the chitosan used was used to ascertain the purity after the chitosan was extracted. The procedure for the extraction as well as the purification has been included in the manuscript. See the methodology section. The doses chosen for this study was based on literature findings whereby inclusion between 1- 5% was suggested to be safe and produced desired biological effects. Unfortunately, this study did not sought to evaluate the maximum percentage doses that are tolerated.
Line 90: It would be very helpful for the reader if an overview of the extraction process is briefly mentioned in the text, along with the references cited currently.
RESPONSE
The overview of the extraction process has been included.
Line 152: Table 1. Data presented in Table 1 seems to be identical to data presented in Table 2 in an article previously published by the authors (Ogungbemi et al., 2020. Ref.20). Perplexingly, the data in the published paper was reported to have been derived from mice after 6 weeks of treatment, while in this manuscript the same data appears to have been generated after 8 weeks of treatment (see abstract, line 19).
RESPONSE
Thank you for this very important observation. A comprehensive explanation of what happened can be found below.
Admittedly, the data presented in table 1 of the present manuscript was meant to be body weight as well as weekly average bodyweight gain and NOT cumulative bodyweight gain as presented. Regrettably, it was due to human error that exactly the same result was presented again. The study presented in the paper by Ogungbemi et al. (2020) was indeed part of this present work, but different aspect. Therefore, the duration of the experiment was 6 weeks and NOT eight weeks (the experiment lasted for 8 weeks because of two weeks of acclimatization). To this end, we tender our unreserved apology for the discrepancy. We understand the severity and the implication of the mistake and the table 1 result has been taken down and replaced by bodyweight data and average weekly body weight gain in table 2.
Round 2
Reviewer 1 Report
Chitosan from crabs (Scylla serrata) Represses Hyperlipidemia-induced Hepato-renal Dysfunction in Rats: Modulation of CD43 and p53 Expression
Review 2
This manuscript examines an interesting topic about the effect of chitosan in high-fat diets (HFD) on the liver (ALT, AST, ALP) and kidney (ALT, AST, urea, and creatinine) biomarkers, as well as the expression of CD43 and p53 proteins in these organs, in a murine model. The objective of the study was to evaluate the effect of chitosan in rats with a hyperlipidemic diet. The results show interesting data on the effect of chitosan in HFD. The authors addressed the observations and improved the manuscript for better understanding. Only 2 observations in the abstract and in methods sections should be corrected and complemented, respectively. Other minor editing remarks can be easily corrected. Suggestions are detailed below
Abstract
Line 30. According to the authors' response and the conclusion at the end of the revised manuscript, the percentage of chitosan should be 1-3%, then the conclusion of the abstract of 3 and 5% chitosan should be reviewed and corrected.
Materials and methods
Only a few small mistakes need to be corrected such as:
Line 88: 2hrs by 2 h
Line 90: 2 hours by 2 h
Line 96: 4 hours by 4 h and 0.5 mol/dm3 by 0.5 mol.dm-3
Line 98: 1hour by 1 h
Line 100: 0.1mol.dm-3 by 0.1 mol.dm-3 and 30 minutes by 30 min
Line 106: C1 by C1
Line 107: C2 by C2 and V1 by V1
Line 108: V2 by V2 and 1cm3 by 1 cm3
Line 109: 0.1mol.dm-3 by 0.1 mol.dm-3
Line 110: Eliminate “in %” is suggested.
Line 119: 12 hours by 12 h
Lines 124-125: Indicate which macronutrient was adjusted by adding chitosan 1-,3- and 5%?
Comments for author File: Comments.pdf
Author Response
REVIEWER 1
Abstract
Line 30. According to the authors' response and the conclusion at the end of the revised manuscript, the percentage of chitosan should be 1-3%, then the conclusion of the abstract of 3 and 5% chitosan should be reviewed and corrected.
RESPONSE
Between 3-5% inclusion was effective. This has been corrected in the manuscript
Materials and methods
Only a few small mistakes need to be corrected such as:
Line 88: 2hrs by 2 h
RESPONSE
2hrs changed to 2 h
Line 90: 2 hours by 2 h
RESPONSE
2 hours changed to 2 h
Line 96: 4 hours by 4 h and 0.5 mol/dm3 by 0.5 mol.dm-3
RESPONSE
4 hours CHANGED TO 4 h and 0.5 mol/dm3 CHANGED TO 0.5 mol.dm-3
Line 98: 1hour by 1 h
RESPONSE
1hour CHANGED TO 1 h
Line 100: 0.1mol.dm-3 by 0.1 mol.dm-3 and 30 minutes by 30 min
RESPONSE
0.1mol.dm-3 CHANGED TO 0.1 mol.dm-3 and 30 minutes TO 30 min
Line 106: C1 by C1
RESPONSE
C1 replaced with C1
Line 107: C2 by C2 and V1 by V1
RESPONSE
C2 replaced by C2 and V1 by V1
Line 108: V2 by V2 and 1cm3 by 1 cm3
RESPONSE
V2 REPLACED by V2 and 1cm3 by 1 cm3
Line 109: 0.1mol.dm-3 by 0.1 mol.dm-3
RESPONSE
0.1mol.dm-3 REPLACED by 0.1 mol.dm-3
Line 110: Eliminate “in %” is suggested.
RESPONSE
% is removed as suggested.
Line 119: 12 hours by 12 h
RESPONSE
All the suugestions have been effected accordingly
Lines 124-125: Indicate which macronutrient was adjusted by adding chitosan 1-,3- and 5%?
The chitosan was mixed with the normal diet or the high-fat diet, no macronutrient was removed.
For instance, 1% supplementation was done by adding 1 g of the chitosan to 99 g of the feed, and so on. This has been explained in the manuscript file
Reviewer 2 Report
Thank you for your response. Overall, this manuscript suggests the role of chitosan in obesity or HFD in rats but the will require more efforts to understand the role of chitosan in liver metabolism clearly. The correlation between CD43 and p53 might provide some clue. Link between CD43 and p53 could help readers to understand CD43 could be a good biomarker for obesity related complications.
Author Response
REVIEWER 2
Thank you for your response. Overall, this manuscript suggests the role of chitosan in obesity or HFD in rats but the will require more efforts to understand the role of chitosan in liver metabolism clearly. The correlation between CD43 and p53 might provide some clue. Link between CD43 and p53 could help readers to understand CD43 could be a good biomarker for obesity related complications.
RESPONSE
Thank you for your observation, the link between CD43 and p53 as regards obesity id multifaceted. The CD43 overexpression correlates positively with increased inflammation, being a chemotaxis factor that aids the trafficking of inflammatory cells. On the other hand, p53 overexpression also potentiate cancer in the adipose tissue. However, the downregualation of p53 in the visceral organs might be deleterious due to accumulation of lipids in the organs. This is bearing in mind that p53, together with cellular integrity function also coordinates the energy homeostasis in the cell. Furthermore, obesity (arising fro hyperlipidemia) has been classed as a low-grade systemic inflammatory response (meta-inflammation). Therefore, exacerbation of inflammation by CD43 and abatement of p53 might potentiate the meta-inflammation which can cause organ failures. An explanation linking CD43 and p53 to obesity has been included in the discussion section where p53 and CD43 were dicussed. See the highlighted areas.
