Anti-Adhesive Properties of Calcium Alginate from Sargassum fusiforme against Particulate Matter-Induced Inflammation
Round 1
Reviewer 1 Report
The manuscript titled “Anti-fine dust effect of Calcium alginate from Sargassum fusiforme against particulate matter induced inflammation” by Hua Fu and others demonstrates the Calcium alginate from Sargassum fusiforme (SFCA) could attenuate the fine dust induced inflammation via reducing the inflammatory and pro inflammatory protein expressions in cell culture modal. Furthermore, authors demonstrate the in vivo anti fine dust effect of SFCA using a zebrafish modal. Collectively the reviewer certainly considers this study is interesting and insightful. However below points should address to improve the manuscript.
Comments
- Manuscript contains number of topographical and language errors. Ex: line number 50, 150, 153, 177.etc. Therefore, authors should read and correct the mistakes very carefully.
- Authors should check all figures. Ex. Fig 5a the alignment, font and size should be carefully check and correct.
- In the figure 4 authors show the anti inflammatory effect of SFCA in cell culture modal. I would suggest to show the data as secretary amounts of cytokines rather showing the level as a percentage.
- Anti inflammatory function of SFCA which acts against the fine dust induced inflammation is discussed in figure 4 and figure 5. It is important to show the mRNA transcription levels of chemokines and inflammatory cytokines in in vivo and cell culture modals.
- What was the reason for selecting specific SFCA concentration in in vivo experiments? Please add justification or preliminary study data.
- Discussion should consist of a data justification and knowledge from previous publications.
Author Response
Point 1: Manuscript contains number of topographical and language errors. Ex: line number 50, 150, 153, 177.etc. Therefore, authors should read and correct the mistakes very carefully.
Response 1: Thank you for your professional reminder. We have marked up using the “Track Changes” function. Errors in Line number 50, 150, 153, 177 and other sections were revised in the manuscript.
Line 52: Due to high contents of inorganic arsenic (iAs) found in marine algae compared to other foods [13], our previous study decreased heavy metal concentrations in S. fusiforme through acid-washing [14].
Line 163: Zebrafish embryo assay
Line 166: Adult zebrafish were obtained from Nanjing EzeRinka Biotechnology Co., Ltd. (Nanjing, China), and embryos were harvested by natural spawning with a 14/10 h light/dark cycle in 28.5oC.
Point 2: Authors should check all figures. Ex. Fig 5a the alignment, font and size should be carefully check and correct.
Response 2: Yes. We revised the alignment, font and size of all figures.
Point 3: In the figure 4 authors show the anti-inflammatory effect of SFCA in cell culture modal. I would suggest to show the data as secretary amounts of cytokines rather showing the level as a percentage.
Response 3: Yes. Regarding to the level of cytokines, there are two ways to express cytokines, secretary amounts and percentage. In our study, we showed the percentage of cytokines followed the previous publications (Environmental toxicology and pharmacology, 2017, 55: 37-43 and International journal of biological macromolecules, 2017, 104: 1185-1193).
Point 4: Anti-inflammatory function of SFCA which acts against the fine dust induced inflammation is discussed in figure 4 and figure 5. It is important to show the mRNA transcription levels of chemokines and inflammatory cytokines in vivo and cell culture modals.
Response 4: Chemokines are a large family of small, secreted proteins and inflammatory chemokines function mainly as chemoattractant for leukocytes, recruiting monocytes and other effector cells from tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response or promote wound healing. However, in our study, ROS level, Hoechst 33342 staining, Western blotting and zebrafish in vivo assays were performed to anti-inflammatory investigation of SFCA. The evidences indirectly prove the changes of chemokines. Thus, the level of chemokines will be carried out for further study.
Point 5: What was the reason for selecting specific SFCA concentration in vivo experiments? Please add justification or preliminary study data.
Response 5: In our lab previous study, the method of fine dust induced-zebrafish in vivo assay was established and published related articles (Algae, 2017, 32(3): 261-273. and Toxicology Reports, 2021, 8: 349-358.). The justification or preliminary study data were shown in the previous. The concentration of in vivo assay is same with that of in vitro assay. Hence, the SFCA concentrations in vivo were selected as 25, 50 and 100 μg mL-1.
Point 6: Discussion should consist of a data justification and knowledge from previous publications.
Response 6: We gratefully appreciated for your valuable suggestion. We have added the data justification and knowledge from previous publications.
Line 293-330:
Generally, there are two main factors contributing to air pollution: anthropogenic activities and natural events. Anthropogenic activities that release emissions to the atmosphere include industrial expansion [24], coal or plant burning [25], vehicle con-sumption [26], and mineral particles [27]. Most of the air contamination in natural events is characterized by visible changes, such as hazy weather and sand storms from the Gobi desert [28]. PM particles, especially PM 2.5 (particulate matter less than 2.5 μm in size), cause severe damage to the respiratory and cardiovascular systems [29], and are the main contributors to air pollution in East Asia [30]. Although PM has be-come a serious problem in most developing countries, there is limited information on PM chemical composition and mobility trends. Some studies have investigated the ambient ultra-fine particles during the fine dust season in Gwangju, Korea and Stuttgart, Germany [31,32]. Accordingly, polluted air in China from 2011 to 2013 has been found to contain particles composed of Ca2+, Mg2+, K+, Na+, Cl-, SO2, SO42-, NO3-, and NH4+ ions [33]. Another study investigated the markers OC2, EC1, and NO3-/SO42- ratio in PM 2.5 emissions in Xi'an in the winters of 2006, 2008, and 2010 [34]. The composition of the dust (Fe, Co, Mg, Al, Ca, Ni, Zn, Sb, V, Cr, As, Se, Cd, Mn, Pb) was analyzed by inductively coupled plasma mass spectrometry [35]. An interesting study describes the pathway of PM-induced inflammation and oxidative stress in macrophages [36]. The evidence supports the theory that dust particles can activate IL-1β, IL-16, NF-κB, and COX-2 expression in human myeloid leukemia cells, indicating strong inflammatory responses [37]. Although reports showed the adverse effects of PM on single organ systems, this work investigated the transfer inflammatory response between skin cells and macrophages. In our results, SFCA was strongly reduced metal ions with increasing concentrations. Hence, it implies SFCA could significantly reduce harmful metals exiting in air pollution.
The keratinocyte model is widely used in dermatological studies to investigate results from the outside layers of the skin. Keratinocytes protect the inner skin cells. Once those stimulated by PM, apoptosis was triggered that likely manifests as skin irritation and damage [38], which further produces secondary mediators that upregulate the expression of IL-1β and IL-6, resulting in an inflammatory response in HaCaT cells [39]. Macrophages regulate inflammation via phagocytosis and antigen presentation, and result in the production of many mediators and cytokines [40]. Macrophage activation is an important strategy to prevent infection, and it is stimulated by cytokines such as interferon γ, IL-1β, and TNF-α, or by certain bacterial extracellular components such as LPS, and external chemicals [41]. The SFCA isolated from acid-processed S. fusiforme were assessed by the in vitro transfer inflammatory response system. The results found that SFCA lowered the contents of PGE2, TNF-α, IL-1β and IL-6 overexpressed by PM stimulation in both skin cells and macrophages. Therefore, the evidence of reductive ability of SFCA in PM-induced inflammation was found.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript the authors studied the protective effect of calcium alginate from Sargassum fusiforme against particulate matter induced inflammation. The results of the study have a potential to provide useful information for utilization of anti-inflammatory substances originating from marine biomasses. The manuscript contains information not been documented previously, the investigation is done at a reasonable level and the results are generally well presented.
Nevertheless, there are some errors and problems.
1) The manuscript contains various spelling mistakes: ‘Find’ à ‘Fine’ (line 13), ‘mental’ à ‘metal’ (line 50), ‘was powder’ à ‘powder was’ (line 79), ‘Scheme 1’ à ‘Table 1’ (line 180), ‘find dust’ à ‘fine dust’ (line 206), etc.
2) Numbers in chemical formulas should be subscripted thorough the manuscript.
3) Title of the Table 2 should be corrected as it now reads: ‘This is a table. Tables should be placed in the main text near to the first time they are cited’.
4) The caption of the Figure 1 is incomplete.
5) Legend is missing for the bars presented in Figures 3a and 4a.
6) The title seems to be confusing. It could be expected that the study deals with anti-adhesive properties and the alginate prevents the attachment of harmful dust particles on cells. But it seems to be just a study about anti-inflammatory properties of alginate.
7) Line 91. The method used to measure the molecular weight of alginate is very unusual. Dextran sulphates are branched polysaccharides and thus are not a good markers for estimating the molecular weight of an alginate (which is a linear polysaccharide and not sulphated at all). It is doubtful that the molecular weight could be reasonably estimated from a plot presented in Figure 1.
8) The manuscripts lacks the structural details for the alginate studied (e.g. G-M, G-G and M-M ratios). The structural features are among the most important properties that determine the biological activity of the polysaccharide.
Author Response
Point 1: The manuscript contains various spelling mistakes: ‘Find’ à ‘Fine’ (line 13), ‘mental’ à ‘metal’ (line 50), ‘was powder’ à ‘powder was’ (line 79), ‘Scheme 1’ à ‘Table 1’ (line 180), ‘find dust’ à ‘fine dust’ (line 206), etc.
Response 1: Thank you for your carefully and professional comments. We have marked up using the “Track Changes” function.
Line 15: Fine dust generated by particulate matter (PM) pollution is a serious ecological issue in industrialized countries and causes disorders of the respiratory system and skin in humans.
Line 52: Due to high contents of inorganic arsenic (iAs) found in marine algae compared to other foods [13], our previous study decreased heavy metal concentrations in S. fusiforme through acid-washing [14].
Line 83: The dialyzed and lyophilized powder was considered as calcium alginate from S. fusiforme (SFCA).
Line 196: Table 1. Chemical composition of SFCA.
Line 223: This result suggests SFCA might effect in anti-adhesive for human skin.
Point 2: Numbers in chemical formulas should be subscripted thorough the manuscript.
Response 2: Thank you for your valuable comment. All numbers in chemical formulas has subscripted thorough the manuscript.
Point 3: Title of the Table 2 should be corrected as it now reads: ‘This is a table. Tables should be placed in the main text near to the first time they are cited’.
Response 3: These problems caused by our carelessness have been corrected. ‘This is a table. Tables should be placed in the main text near to the first time they are cited’ has been replaced by ‘Changes of elemental metal compositions in PM-induced keratinocytes with SFCA during 24 h’.
Point 4: The caption of the Figure 1 is incomplete.
Response 4: Thank you for pointing out our problems. In this manuscript, ‘FT-IR spectroscopic analysis of structural features of SFCA, SFCA-processing: calcium alginate gels isolated from citric acid washed S. fusiforme; SFCA-unprocessing: calcium alginate gels isolated from original S. fusiforme.’ has been added.
Point 5: The caption of the Figure 1 is incomplete.
Response 5: Thank you for your valuable comments. The legend of Fig. 3a and 4a have been revised in the Figure caption.
Point 6: The title seems to be confusing. It could be expected that the study deals with anti-adhesive properties and the alginate prevents the attachment of harmful dust particles on cells. But it seems to be just a study about anti-inflammatory properties of alginate.
Response 6: Yes. According to the valuable comments, we changed the title as ‘Anti-adhesive properties of Calcium Alginate from Sargassum fusiforme Against Particulate Matter-Induced Inflammation’.
Point 7: Line 91. The method used to measure the molecular weight of alginate is very unusual. Dextran sulphates are branched polysaccharides and thus are not a good markers for estimating the molecular weight of an alginate (which is a linear polysaccharide and not sulphated at all). It is doubtful that the molecular weight could be reasonably estimated from a plot presented in Figure 1
Response 7: The determination of molecular weight was followed by the previous method (Ecotoxicology and environmental safety, 2018, 160: 24-31.). According to the reviewer’s comment, we deleted the figure of molecular weight.
Point 8: The manuscripts lacks the structural details for the alginate studied (e.g. G-M, G-G and M-M ratios). The structural features are among the most important properties that determine the biological activity of the polysaccharide.
Response 8: Yes. The structural detail of polysaccharide such as M/G ratio is very important for its bioactivities. We added the M/G ratio in the revised manuscript.
Line 99-103: Proton nuclear magnetic resonance (1H NMR) spectroscopy was acquired on SFCA solution (1% w/v) in D2O, with recordings at 80 oC using an AVANCE III HD 400 (Bruker Scientific Instruments, USA) spectrometer operating at a frequency of 400 MHz. The mannuronic acid to guluronic acid (M/G) ratio was analyzed according to the previous method.
Line 185-186: SFCA was characterized by proton nuclear magnetic resonance spectroscopy, resulting M/G ratio was 1.25.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript brings an evaluation of the protective effect of calcium alginate from Sargassum fusiforme against inflammation to statute the possible anti-fine dust effects for appropriate applications.
Authors used for the bioassays and the anti-inflammatory effects of the macroalgae cells samples of RAW 264.7 macrophage and HaCaT keratinocytes.
In all, the manuscript is interesting. Recommendations are cited on the “comments for authors” section to improve the provided version of the article.
Recommendations
- For the evaluation of the anti-fine dust effects, authors used calcium alginate from Sargassum fusiforme at several concentrations. However, they did not indicate the origin of the macroalgae used for calcium alginates preparation (sampling period and site, harvest, …).
- Throughout the manuscript authors must revise the citation of chemical formula and units:
* Na2CO3 instead of Na2CO3
* 4°C not 4oC…
- In material and methods section: Paragraph 2.3 and 2.4 and Lines 128-130 and Line 153 …, author listed referenced methods without providing any details about how the experiments were conducted. I suggest to add brief description of each analysis cited.
- Authors must justify the PM concentration used for the conducted tests (125 µg.mL-1 of PM).
- Authors provided in the results section Figure 5 related to the in vivo anti-fine dust effect of SFCA, with 10 µg.mL-1 of PM. Authors must indicate the reason of modifying the PM concentration (in comparison with the other tests).
- The quality of all figures cited in the article must be improved to be more visible (size, format, …).
- Authors should extend the conclusion part to bring out the main and important results obtained through the experiments of the provided study.
Author Response
Point 1: For the evaluation of the anti-fine dust effects, authors used calcium alginate from Sargassum fusiforme at several concentrations. However, they did not indicate the origin of the macroalgae used for calcium alginates preparation (sampling period and site, harvest, …).
Response 1: Yes. According to the valuable comments, we added the origin of the Sargassum fusiforme used for calcium alginates preparation.
Line 68-69: S. fusiforme was harvested from Jeju, Korea in May 2019 and collected position is 33°25'24.1"N/ 126°55'22.7"E. Repository was deposited in Jeju National University (Jeju, Korea).
Point 2: Throughout the manuscript authors must revise the citation of chemical formula and units:
* Na2CO3 instead of Na2CO3
* 4°C not 4oC…
Response 2: All the citation of chemical formula and units has revised thorough the manuscript.
Point 3: In material and methods section: Paragraph 2.3 and 2.4 and Lines 128-130 and Line 153 …, author listed referenced methods without providing any details about how the experiments were conducted. I suggest to add brief description of each analysis cited.
Response 3: We added briefly experimental information in section 2.3 and 2.4.
In line 128-130 and line 153, the methods of MTT assay, DCFH-DA assay, ICP-OES, cell death, ROS, and NO production were mentioned. In section 2.5 and 2.8 of our manuscript, the above methods were all shown their details.
Line 86-92: The proximate composition of SFCA was determined by the following methods. The metal content was analyzed according the standard method. The muffle furnace was employed to analyze ash content with the temperature at 600°C for 6 h [17]. The protein content of SFCA was measured using the Kjeldahl method with bovine serum albumin as the standard; the total carbohydrate content of SFCA was measured by the phenol sulfuric acid method using glucose as the standard; the Folin-Ciocalteu method was applied to analyze polyphenol content with gallic acid as the standard [18].
Point 4: Authors must justify the PM concentration used for the conducted tests (125 µg. mL-1 of PM).
Response 4: The optimal PM concentration and exposure time followed the previous study for cell viability and inflammation induction. Literature has been cited in the article line 190. (Toxicology Reports 2021, 8, 349-358.)
Point 5: Authors provided in the results section Figure 5 related to the in vivo anti-fine dust effect of SFCA, with 10 µg. mL-1 of PM. Authors must indicate the reason of modifying the PM concentration (in comparison with the other tests).
Response 5: In our lab previous study, the method of fine dust induced-zebrafish in vivo assay was established and published related articles (Algae, 2017, 32(3): 261-273. and Toxicology Reports, 2021, 8: 349-358.). The justification or preliminary study data were shown in the previous. The optimal PM concentration in vitro is not suit for zebrafish. The concentration was strong toxicity to zebrafish. Hence, the PM concentrations in vivo was selected as 10 μg mL-1.
Point 6: The quality of all figures cited in the article must be improved to be more visible (size, format, …).
Response 6: The quality and resolution of all figures were improved.
Point 7: Authors should extend the conclusion part to bring out the main and important results obtained through the experiments of the provided study.
Response 7: Thank you for your valuable comment. We have extended the conclusion part.
Line 343-349: SFCA, the calcium alginate, isolated from acid-processed S. fusiforme exhibited strong anti-adhesive activities via promoting cell growth and reducing inflammatory factors in PM-treated macrophages and keratinocytes in vitro. Additionally, SFCA showed the protective effects against PM-induced damages in vivo 72 hpf zebrafish models. SFCA could be a candidate agent in anti-adhesive effect and cosmeceutical formulations. Further, the present study could extend the appropriate application of algae in the management of fine dust particles.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The scientific and presentation quality of the manuscript has significantly improved with last revision. Therefore I would like to accept the manuscript for publication with minor revisions.
01. Topographical error in the text and poor presentation figurs (ex. Table 2, labeling, Figure 4a, Figure 4c)
Author Response
Thank you very much for assessing our manuscript. We appreciate your detailed review and comments.
Author Response File: Author Response.docx
Reviewer 2 Report
The authors have considered the comments of the reviewers and have made the respective changes in the manuscript. I think the manuscript could be accepted for publication.
Author Response
Thank you very much for assessing our manuscript. We appreciate your detailed review and comments.
Reviewer 3 Report
The revised version of the manuscript includes all remarks and modifications indicated. In my opinion the provided version is now suitable for publication
Author Response
Thank you very much for assessing our manuscript. We appreciate your detailed review and comments.
