Tremella fuciformis Crude Polysaccharides Attenuates Steatosis and Suppresses Inflammation in Diet-Induced NAFLD Mice
Round 1
Reviewer 1 Report
Editorial work is needed.
Abbreviations should be clearly explained before they are used, e.g. line 45 – TLR4, line 50 – HCC, line 53- LPS, ……
“HNF4α, NR2A1) is a superfamily of the nuclear receptors” – it should be “is a member of the superfamily …”
The Materials and Methods section must be improved and important data/information should be provided.
Tremella fuciformis crude polysaccharides should described/characterized to make readers understand the difference between TF polysaccharides and others. The composition of the extract used should be presented in more detail. Is the extract composition stable when different preparations are compared ?
The composition of the faecal supernatant used in the FMT-TFCP treated mice. The data on the changes in the composition of the faecal supernatant from different separations should be also provided. It is of great importance to understand what kind of treatment mixture was used as changes in different parameters under treatment were observed.
Information on MCD diet should be also presented.
All figure legends need improvement. In general, the figure legend should provide all information to easy understand data presented on this figure without sending the reader to the text to find important explanations.
Fig.2 – C- it seems that concentrations of HDL-cholesterol are presented. Are the presented differences statistically significant ?
Section 2.5 and Fig.5 . IL1β expression was not reduces in FMT-TFCP group. The effect of treatment on HNF4α in FMT-TFCP group as compared to MCD group is not statistically significant as well as in this group no changes in TNF-α expression was observed.
Section 2.6 and Fig.6. Observed changes in MDA and SOD are not significant. In addition presented data of AST clearly indicate high SEM in the MDA-group – it means that significant differences between animals in this group occurs.
In general – the Results section needs significant improvement. The Discussion section should be changed accordingly.
Author Response
Comments and Suggestions for Authors
Editorial work is needed.
Abbreviations should be clearly explained before they are used, e.g. line 45 – TLR4, line 50 – HCC, line 53- LPS, ……
“HNF4α, NR2A1) is a superfamily of the nuclear receptors” – it should be “is a member of the superfamily …
Ans: Many thanks for your valuable comments. As per your instructions, I have added the full forms of the terms given in the text.
The Materials and Methods section must be improved and important data/information should be provided.
Tremella fuciformis crude polysaccharides should described/characterized to make readers understand the difference between TF polysaccharides and others. The composition of the extract used should be presented in more detail. Is the extract composition stable when different preparations are compared ?
Ans: Yes, the activity of polysaccharides is closely related to molecular weight, higher conformation, and branching degree (Ref. Jin M, Shi J, Zhu W, Yao H, Wang DA. Polysaccharide-Based Biomaterials in Tissue Engineering: A Review. Tissue Eng Part B Rev. 2021 Dec;27(6):604-626.). The Tremella fuciformis crude polysaccharides (TFCP) are heteropolysaccharides, which can currently be standarded for molecular weight and a triple helix structure. The structure of polysaccharides is influenced by the source and preparation method, so it is necessary to clarify the source of raw materials and the preparation method, which can ensure the repeatability of the structure. The TFCP used in this study were extracted by a stable and established method with a peak molecular weight of 1100 kDa. Currently, we are examining the characteristics of TFCP including the compositions and infrared absorption spectrum. These studies will help us to further understand TFCP.
The composition of the faecal supernatant used in the FMT-TFCP treated mice. The data on the changes in the composition of the faecal supernatant from different separations should be also provided. It is of great importance to understand what kind of treatment mixture was used as changes in different parameters under treatment were observed.
Ans: As per your comments, we have added the things in the methodology and tried to improve it to make it clear for the readers. However, the fecal supernatant contains the microbiota of the host, and the composition is not possible to evaluate without Next-Generation sequencing (NGS). In this study, our prime focus was to study the role of Tremella fuciformis in mitigating the NAFLD. The purpose to add the FMT group is to get an idea about the role of microbiota in the treatment of NAFLD, on a broader scale.
Information on MCD diet should be also presented.
Ans: Thanks for this comment and we have added the diet composition in the text.
All figure legends need improvement. In general, the figure legend should provide all information to easy understand data presented on this figure without sending the reader to the text to find important explanations.
Ans: As per your instruction, we have changed the figure legend, to get a clear understanding for the readers.
Fig.2 – C- it seems that concentrations of HDL-cholesterol are presented. Are the presented differences statistically significant ?
Ans: The presented differences were not statistically significant, but as a group, it’s considerable that’s why we added this data.
Section 2.5 and Fig.5 . IL1β expression was not reduces in FMT-TFCP group. The effect of treatment on HNF4α in FMT-TFCP group as compared to MCD group is not statistically significant as well as in this group no changes in TNF-α expression was observed.
Ans: We completely agree with you, that the difference was not statistically significant in the FMT-TFCP group, for the different genes. Actually, this group was not given TFCP but administered the fecal supernatant samples taken from the MCD-TFCP group (which were on TFCP treatment). This predicts the effect of TFCP on microbiota, and how the microbiota may alter the disease progression.
The effect of microbiota from MCD-TFCP group may be influenced by multifactors such as administration dosage and treatment days. This may be the reason that the treatment effect of FMT-TFCP group was not as significant as that of the TFCP group.
Section 2.6 and Fig.6. Observed changes in MDA and SOD are not significant. In addition presented data of AST clearly indicate high SEM in the MDA-group – it means that significant differences between animals in this group occurs.
Ans: Yes, we agree that the data was not statistically significant.
In general – the Results section needs significant improvement. The Discussion section should be changed accordingly.
Ans: We have removed the grammatical errors and improved the results, legends, and discussion, altogether.
Reviewer 2 Report
The authors have made an interesting manuscript, but one that has some deficiencies to address, which requires further revision.
The introduction is adequate, but the results already show some deficiencies that must be corrected.
For example, in figure 1 A-D, the authors only put a photograph of liver staining, but did not indicate the values obtained for each condition studied with mean and standard deviation, and the corresponding statistical analysis. For example, in Figure 1E, the authors indicate the standard deviation and its statistical analysis, but in no case indicate the n of each condition, and it is an important piece of information that should appear in tables and graphs, and that can be complemented with images such as have made, that they are representative of each experimental condition. Perhaps the value of n can be omitted, but material and methods should appear in the manuscript before results, and the values of n should be indicated. The absence of the value of n is repeated in figure 2, 3, ...
There are also errors in the naming of some genes, such as SREBP that appears four times as SERBP, as for example in figure 4.
On the other hand, there are too many figures, and perhaps figure 6, in which no significant difference appears, should go to supplementary material, and in this way facilitate the reading of the manuscript, since from my point of view that information is not relevant. , in addition to being incomplete due to the absence of the value of n. Perhaps the ALT value may have significant differences and it may be an omission of the authors that is difficult to specify, but there is a very large standard deviation, which with low n values may lack statistical significance, such as n = 5 that they use the authors.
Regarding the discussion, it should be elaborated in more detail, and if it is indicated that there are values that correlate, they can show a point cloud graph to check if the individual values really correlate or not.
For all this, the manuscript must be critically rewritten by the authors in order to be sent to this journal again.
Author Response
Comments and Suggestions for Authors
The authors have made an interesting manuscript, but one that has some deficiencies to address, which requires further revision.
The introduction is adequate, but the results already show some deficiencies that must be corrected.
For example, in figure 1 A-D, the authors only put a photograph of liver staining, but did not indicate the values obtained for each condition studied with mean and standard deviation, and the corresponding statistical analysis. For example, in Figure 1E, the authors indicate the standard deviation and its statistical analysis, but in no case indicate the n of each condition, and it is an important piece of information that should appear in tables and graphs, and that can be complemented with images such as have made, that they are representative of each experimental condition. Perhaps the value of n can be omitted, but material and methods should appear in the manuscript before results, and the values of n should be indicated. The absence of the value of n is repeated in figure 2, 3, ...
Ans: As per your comment, we have modified the legend and added the n in the material and methods.
There are also errors in the naming of some genes, such as SREBP which appears four times as SERBP, as for example in figure 4.
Ans: Thanks for the comment, we have removed the discrepancy in the abbreviation.
On the other hand, there are too many figures, and perhaps figure 6, in which no significant difference appears, should go to supplementary material, and in this way facilitate the reading of the manuscript, since from my point of view that information is not relevant. , in addition to being incomplete due to the absence of the value of n. Perhaps the ALT value may have significant differences and it may be an omission of the authors that is difficult to specify, but there is a very large standard deviation, which with low n values may lack statistical significance, such as n = 5 that they use the authors.
Ans: We have moved Figure 6 to supplementary material accordingly.
Regarding the discussion, it should be elaborated in more detail, and if it is indicated that there are values that correlate, they can show a point cloud graph to check if the individual values really correlate or not.
For all this, the manuscript must be critically rewritten by the authors in order to be sent to this journal again.
Ans: As per your comment, the results are elaborated in detail and the discussion also.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Still editorial work is needed and the manuscript should be corrected by a native speaker familiar with biological/medical sciences.
Tremella fuciformis crude polysaccharides should better characterized to make readers understand the difference between TF polysaccharides and polysaccharides from other sources. The stability of extract composition should be underlined even if extract was used also by another group.
It is not clear what does it mean that “TF was reported to protect the immune organs”.
Data on faecal extract are scientifically very weak. No data on faecal microbiota are presented. There is also no data on differences between microbiota of control mice and mice treated with TF-polysaccharides. We are only informed that faecal supernatant was used. Therefore, the data on FMT-TFCP-mice group should be removed. Authors should concentrate on biological effects of TF-polysaccharides and do not mix potential biological effects of TF-polysaccharides and effects of gut microbiota.
Statistical significance should be always marked on a figure if p is indicated under this figure legend.
Fig. 2 – how HLD levels were determined ? It seems that HDL levels were assessed based on measurement of HDL cholesterol concentrations. If so, HDL-cholesterol not just HDL should be presented
2.3 – it is indicated that expression of genes encoding lipid transporters is presented. However, HMG-CoA reductase is not a lipid transporter. Therefore, improvement is needed.
Author Response
Comments and Suggestions for Authors
Still editorial work is needed and the manuscript should be corrected by a native speaker familiar with biological/medical sciences.
Tremella fuciformis crude polysaccharides should better characterized to make readers understand the difference between TF polysaccharides and polysaccharides from other sources. The stability of extract composition should be underlined even if extract was used also by another group.
Ans: Accordingly, we have added the information in “4.1 TFCP preparation and characterization” as the following.
Tremella fuciformis crude polysaccharides (TFCP), provided by Dr Xiaofei Xu, were obtained from the fruit body of Tremella fuciformis by boiling water extraction. Dry T. fu-ciformis fruiting body were collected from Gutian, Fujian province, China. The T. fu-ciformis fruiting body were crushed and sifted through 40 mesh for use. TFCPs were ex-tracted using hot water extraction method (1:60 w/v) at 95 °C for 6 h. Then, the solution was filtrated and concentrated to quarter original weight under vaccum condition at 70 °C. Deproteination was performed by adjusting pH of the solution to 4.0 with citric acid and kept at 4 °C for 2 days by isoelectric precipitation. Collecting supernatant and three equiv-alent volumes of 95% ethanol were added to precipitate polysaccharides at 4 °C overnight. The TFCPs fraction was obtained by centrifugation and lyophilization. The TFCPs powder was white in colour. The peak molecular weight of TFCP was about 1100 kDa as meas-ured using high-performance gel permeation chromatography (HPGPC). The saccharide content of TFCP was 82%, as detected by phenol sulfuric acid analysis with mannose as a standard. Protein content was measured by the Bradford method. Protein content was 2.4% in TFCPs fraction. Fourier transform infrared (FT-IR) spectroscopy of TFCPs samples was measured in the range of 4000−400 cm-1 by KBr-disk method using an FT-IR spectrometer (Nicolet IS50-Nicolet Continuum, Thermo Fisher Scientific). OMNIC software was used for spectroscopy analysis. As shown in following figure, characteristic peaks of FT-IR spectra of TFCPs fraction at approximately 3412, 2935, 1721, 1606, 1424, 1135, 1071, 915, and 803 cm-1 are presented. The absorbance at 3412, 2935 cm-1 are due to the stretching of O-H and -CH2 groups, respectively. The peaks at 1721, 1606 cm-1 demonstrate the presence of uronic acid group. High absorbance in the region of 1200-1000 cm-1 are typical for poly-saccharides molecule by the stretching of C-O, C-C, and C-OH. The peak at 915 cm-1 is due to the presence of D-glucopyranosyl and the peak at 803 cm-1 suggests the presence of α-glycosidic bonds in molecular structure of TFCPs.
In this study, we focus on the function of TFCPs on diet-induced NAFLD mice and the further researches on the TFCPs will be carried after the function of TFCPs is determined.
It is not clear what does it mean that “TF was reported to protect the immune organs”.
Ans: Many thanks for your valuable comments that help me re-analyze this manuscript. To avoid the confusion, we have removed the TF and mentioned Tremella fuciformis, and cleared the statement “TF was reported to protect the immune organs”.
Data on faecal extract are scientifically very weak. No data on faecal microbiota are presented. There is also no data on differences between microbiota of control mice and mice treated with TF-polysaccharides. We are only informed that faecal supernatant was used. Therefore, the data on FMT-TFCP-mice group should be removed. Authors should concentrate on biological effects of TF-polysaccharides and do not mix potential biological effects of TF-polysaccharides and effects of gut microbiota.
Ans: As per your instructions, we have removed the FMT group from the manuscript.
Statistical significance should be always marked on a figure if p is indicated under this figure legend.
Ans: Based on your comment, we have added the Statistical significance under the figure legend.
Fig. 2 – how HLD levels were determined? It seems that HDL levels were assessed based on the measurement of HDL cholesterol concentrations. If so, HDL-cholesterol not just HDL should be presented.
Ans: Many thanks for highlighting this point. The HDL level was determined using the kit. However, I have changed HDL with HDL-C in the text.
2.3 – it is indicated that the expression of genes encoding lipid transporters is presented. However, HMG-CoA reductase is not a lipid transporter. Therefore, improvement is needed.
Ans: Based on your instruction, we have made the correction in the heading and in the text.
Reviewer 2 Report
Congratulations to the authors for this new version that must be accepted in Current Issues in Molecular Biology
Author Response
Many thanks for your comments and recognition of merits of our manuscript.
