A Novel Sesquiterpene from Callistephus chinensis Improves Alcohol-Induced Liver Disease by Regulating the AMPK/NF-κB Signaling Pathway and Gut Flora
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe plant name should be in a script font throughout the manuscript.
Is there any ethnomedicinal justification for using this plant species as a hepatoprotective agent?
The abstract needs to justify the use of Callistephus A as a hepatoprotective agent.
The abstract needs to explain the duration of the experiment and the doses used for the plant compound.
The conclusion in the abstract should be rewritten. It does not reflect all the work that the authors performed.
Does Callistephus A have previous pharmacological activities? Is this compound found in other plant species? The introduction must state this information.
Is there a justification for using 20 and 40 mg/kg of the plant compound?
The concentrations of silbinin and Callistephus A should express the same units. I suggest using micromolar.
Figure legend 1. Which post hoc test did the authors use?
How is it linked between the transcriptome study and the Western blot analysis?
What are the limitations of this study?
The study focuses on the hepatoprotective effect of Callistephus A. Did the authors consider using a CClâ‚„-induced hepatic damage? The discussion section should mention this information.
Author Response
Comments 1：The plant name should be in a script font throughout the manuscript.
Responses：Thanks for this comment. Your careful work has enabled us to further refine the details in our manuscript. All plant names in the text have been italicized, and the revisions have been highlighted in red for your convenience.
Comments 2：Is there any ethnomedicinal justification for using this plant species as a hepatoprotective agent?
Responses：Thanks for this comment. Callistephus chinensis Nees is an herbaceous plant in the Asteraceae family, primarily distributed in northeastern and southwestern China. According to historical records, it has been used both medicinally and as a food source, traditionally valued for its benefits to eye and liver health. The relevant references are as follows:
(1) Seo, J.Y.; Lim, S.S.; Park, J.; Lim, J.S.; Kim, H.J.; Kang, H.J.; Yoon Park, J.H.; Kim, J.S. Protection by Chrysanthemum zawadskii extract from liver damage of mice caused by carbon tetrachloride is maybe mediated by modulation of QR activity. Nutrition research and practice 2010, 4, 93-98, doi:10.4162/nrp.2010.4.2.93.
(2)Tian, Z.; Jia, H.; Jin, Y.; Wang, M.; Kou, J.; Wang, C.; Rong, X.; Xie, X.; Han, G.; Pang, X. Chrysanthemum extract attenuates hepatotoxicity via inhibiting oxidative stress in vivo and in vitro. Food & nutrition research 2019, 63, doi:10.29219/fnr.v63.1667.
(3)Chen, J.; Wang, X.; Huang, K.; Yuan, M.; Li, X.; Zhang, X. A new derivative with 6/7 ring skeleton obtained from Callistephus chinensis flowers with anti-hepatic fibrosis activity. Natural product research 2025, 1-5, doi:10.1080/14786419.2025.2475514.
Comments 3：The abstract needs to justify the use of Callistephus A as a hepatoprotective agent.
Responses: Thank you for this valuable comment. The hepatoprotective effect of CA has been included in the abstract. The relevant information for the main content is based on the following references.
(1)Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
(2)Jiang, S.; Wang, M.; Jiang, Z.; Zafar, S.; Xie, Q.; Yang, Y.; Liu, Y.; Yuan, H.; Jian, Y.; Wang, W. Chemistry and Pharmacological Activity of Sesquiterpenoids from the Chrysanthemum Genus. Molecules 2021, 26, doi:10.3390/molecules26103038.
Comments 4：The abstract needs to explain the duration of the experiment and the doses used for the plant compound.
Responses：Thank you for your suggestion. The duration and doses have been added in the abstract.
Comments 5：The conclusion in the abstract should be rewritten. It does not reflect all the work that the authors performed.
Responses：Thank you for your suggestion. Following your suggestion, we have revised the ending of the abstract.
Comments 6：Does Callistephus A have previous pharmacological activities? Is this compound found in other plant species? The introduction must state this information.
Responses：Thank you for this important comment. CA is a novel sesquiterpenoid isolated from Callistephus chinensis by our group. It features a rare 6/7 bicyclic skeleton and has demonstrated significant anti-inflammatory, antioxidant, and hepatoprotective activities in our previous experiments. As requested, we have now supplemented the Introduction with a detailed background on CA, supported by the relevant literature.
(1)Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
(2)Chen, J.; Wang, X.; Huang, K.; Yuan, M.; Li, X.; Zhang, X. A new derivative with 6/7 ring skeleton obtained from Callistephus chinensis flowers with anti-hepatic fibrosis activity. Natural product research 2025, 1-5, doi:10.1080/14786419.2025.2475514.
Comments 7：Is there a justification for using 20 and 40 mg/kg of the plant compound?
Responses：We sincerely thank you for this valuable comment. In our previous laboratory studies, Callistephus A was demonstrated to have a protective effect against immune-mediated liver injury. Based on the results of previous experiments, we selected the 20 mg/kg and 40 mg/kg doses for the current investigation. The previous published article is as follows:
(1)Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
Comments 8：The concentrations of silbinin and Callistephus A should express the same units. I suggest using micromolar.
Responses：We are grateful for this constructive suggestion. After reviewing the literature, we found that the use of mg/kg for the dosage is generally reasonable.
(1)Cai, Y.; Huang, R.; Lin, T.; Yang, L.; Zhou, C.; Li, Y.; Liu, B.; Dong, S.; Jiang, Y. Combination of Plasma Pharmacochemistry, RNA-Seq, and Molecular Docking Strategies to Reveal the Mechanism of the Alkaloid Fraction of Nelumbinis folium for the Treatment of Hyperlipidemia. Molecules 2025, 30, doi:10.3390/molecules3018372
Comments 9：Figure legend 1. Which post hoc test did the authors use?
Responses：We thank the reviewer for this comment. To further examine the group differences, a one-way ANOVA was employed for the post-hoc analysis.
Comments 10：How is it linked between the transcriptome study and the Western blot analysis?
Responses：Thanks to this comment.We predicted the possible targets and pathways of CA through the results of the transcriptome, and then we used WB and other methods to verify the pathways and targets.
Comments 11：What are the limitations of this study?
Responses：We appreciate the reviewer's insightful question regarding the limitations of our study.
In this paper, we found that CA can regulate the intestinal flora, but the specific role of the intestinal flora has not been elucidated, and we can use experiments such as fecal bacterial transplantation to determine the specific role of the intestinal flora, but the current experiments have proved that CA can regulate the abundance of a variety of beneficial bacteria.
Comments 12：The study focuses on the hepatoprotective effect of Callistephus A. Did the authors consider using a CCl₄-induced hepatic damage? The discussion section should mention this information.
Responses：Thank you for your question, our laboratory is currently conducting research on the alleviating effect of CA on CCl4-induced liver injury, and we have also added a prospect on the hepatoprotective effect of CA in the discussion section.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this manuscript, the authors have shown that CA (Callistephus A) has a protective effect against hepatic steatosis and inflammation induced by alcohol consumption. They further demonstrated that CA acts by modulating the AMPK/NF-κB pathway and reduces inflammation by inhibiting TLR4/MYD88/NF-κB pathway. They also showed that CA modulates gut flora thereby ameliorating alcohol-induced intestinal barrier impairment. The manuscript is well written with meticulously designed experiments, however I have some minor comments.
Minor
- Please add the mean +SEM values for each group in the figure legends.
- Line 460 sub-heading in method section is incorrect.
- Were expressions of other sirtuins like Sirt7, which also plays an important role in lipid metabolism, checked in the EtOH and EtOH+CA groups?
- An in vitro experiment can be done to confirm the activation of AMPK by CA using AMPK inhibitor dorsomorphin.
Author Response
Comments 1:P lease add the mean +SEM values for each group in the figure legends.
Responses：Thank you for your suggestion. The mean +SEM values were added in the revised manuscript.
Comments 2: Line 460 sub-heading in method section is incorrect.
Responses：Thank you very much for your question, thank you for your careful work, we have revised the subtitle of Part 4.1, and the relevant amendments have been highlighted in the revised draft for your review.
Comments 3: Were expressions of other sirtuins like Sirt7, which also plays an important role in lipid metabolism, checked in the EtOH and EtOH+CA groups?
Responses: Thank the reviewer for this insightful comment and for highlighting the important role of Sirt7 in lipid metabolism. SIRT7 is a member of the Sirtuins family, and based on your suggestion, we supplemented the WB validation of Sirt7 with CA treatment, which significantly restored Sirt7 expression levels in the EtOH+CA group. Thank you to the reviewers for their comments that complete our work.
Comments 4: An in vitro experiment can be done to confirm the activation of AMPK by CA using AMPK inhibitor dorsomorphin.
Responses：Thank you for this valuable comment. In accordance with your proposal, we utilize AMPK-specific inhibitors in vitro to verify that CA works by activating the AMPK pathway. The results showed that CA could still activate AMPK under the action of AMPK inhibitors.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors conducted a study entitled “A novel sesquiterpene from Callistephus chinensis improves alcohol-induced liver disease by regulating the AMPK/NF-κB signaling pathway and gut flora.” The topic has clear research significance, and the workload appears substantial with a generally logical progression. However, several major problems remain. The main issue is that many transitions are extremely abrupt, making the text difficult to follow. The authors should carefully reorganize the writing for smoother logical flow and coherence.
Lines 32–54: The transition from ALD directly to the AMPK pathway is abrupt and lacks any connecting explanation. A bridging paragraph or sentence is needed.
Line 55: Callistephus chinensis is not “a perennial.” It is an annual, occasionally biennial, herb.
Lines 61–62: The statement “we investigated the role of CA in ameliorating ALD” is insufficient. The study objectives and scientific significance should be clearly defined. The introduction should include background information about CA itself.
Line 65: There is no explanation of Figure 1A in the text. Moreover, the manuscript jumps directly to “The experimental steps are shown in Figure 1B” before the reader even knows what the experiment entails.
Figure 1 D–I: The authors should clarify which statistical tests were used. For multiple-group comparisons, a one-way ANOVA followed by Tukey’s test is recommended, and differences should be labeled with different letters (a, b, c) rather than mixed symbols (### vs Con, * vs EtOH).
Lines 101–105: The reported DEG numbers (“Con vs EtOH: 1843 down, 1453 up; EtOH vs EtOH+CA: 52 down, 39 up”) appear inconsistent. Fewer than one hundred DEGs seems unlikely, please confirm accuracy and specify the logâ‚‚FC and FDR thresholds used.
Figure 2A seems to show a Pearson correlation matrix; Figure 2G appears to be a Venn-UpSet diagram of DEGs. Figures should follow the textual order, and only the essential DEG, GO, and KEGG analyses should remain in the main text; secondary plots belong in the Supplementary Materials.
Figure 3: The simultaneous presence of both a bar chart and a bubble chart is redundant. Keep only the bubble chart and describe the results academically.
Line 127: There is no transition between the enrichment analysis (KEGG/GO) and Figure 4A (WB). The authors should introduce the linking transcriptome results to subsequent protein validation.
Figure 5D: The image is extremely dark; brightness and contrast should be adjusted for visibility.
Figure 9: Many small subpanels add little value and should be moved to the Supplementary section.
Lines 430–432: These sentences on CA’s background are misplaced, they belong in the Introduction, not in the Discussion. The first part of the Discussion through line 450 reads like background material.
The Discussion mainly repeats the Results with minimal comparison to previous studies or mechanistic interpretation. Discussion should provide theoretical integration and comparison with refs rather than restating the results.
Lines 510–520: The citations for fastp and HISAT2 are missing. DESeq should be corrected to DESeq2, and the version should be verified.
Author Response
Comments 1: Lines 32–54: The transition from ALD directly to the AMPK pathway is abrupt and lacks any connecting explanation. A bridging paragraph or sentence is needed.
Responses：Thank you very much for this comment. The relevant links and instructions for ALD and AMPK are already being added in the introduction section.
Comments 2: Line 55: Callistephus chinensis is not “a perennial.” It is an annual, occasionally biennial, herb.
Responses：Thank you very much for your feedback. This was due to our oversight. The manuscript has been revised.
Comments 3: Lines 61–62: The statement “we investigated the role of CA in ameliorating ALD” is insufficient. The study objectives and scientific significance should be clearly defined. The introduction should include background information about CA itself.
Responses：Thank you very much for this comment, I have added my research objectives and scientific significance to the introduction section of the article. In addition, we have supplemented the background information of the CA.
Comments 4: Line 65: There is no explanation of Figure 1A in the text. Moreover, the manuscript jumps directly to “The experimental steps are shown in Figure 1B” before the reader even knows what the experiment entails.
Responses：Thank you for your suggestion. It is our oversight. The caption for Fig1A has been added to the text. Furthermore, transitional sentences and words have been included in 2.1 to facilitate better understanding for the readers.
Comments 5: Figure 1 D–I: The authors should clarify which statistical tests were used. For multiple-group comparisons, a one-way ANOVA followed by Tukey’s test is recommended, and differences should be labeled with different letters (a, b, c) rather than mixed symbols (### vs Con, * vs EtOH).
Responses：Thank you very much for this comment, we have used the significance use (a-e) in this article and used one-way ANOVA followed by Tukey's test.
Comments 6: Lines 101–105: The reported DEG numbers (“Con vs EtOH: 1843 down, 1453 up; EtOH vs EtOH+CA: 52 down, 39 up”) appear inconsistent. Fewer than one hundred DEGs seems unlikely, please confirm accuracy and specify the logâ‚‚FC and FDR thresholds used.
Responses：Thank you for reviewing our manuscript and providing this valuable feedback. The criteria for defining a DEG were set as |log₂Fold Change| > 1 and an FDR-adjusted p-value < 0.05. All reported numbers are based on these stringent thresholds. We confirm that the reported DEG numbers are accurate. We believe the relatively small number of DEGs in the "EtOH vs EtOH+CA" comparison reflects the targeted and specific effects of CA action. Once again, we thank you for this insightful question. We hope the above explanation adequately addresses your concern.
Comments 7: Figure 2A seems to show a Pearson correlation matrix; Figure 2G appears to be a Venn-UpSet diagram of DEGs. Figures should follow the textual order, and only the essential DEG, GO, and KEGG analyses should remain in the main text; secondary plots belong in the Supplementary Materials.
Responses：Thank you for this valuable comment. Thank you very much for this comment, I have added my research objectives and scientific significance to the introduction section of the article. In addition, we have supplemented the background information of the CA.
Comments 8: Figure 3: The simultaneous presence of both a bar chart and a bubble chart is redundant. Keep only the bubble chart and describe the results academically.
Responses：Thank you very much for this valuable comment. In Figure 3, the histogram of GO enrichment analysis and the bubble plot of KEGG pathway enrichment are retained, and the graphs with duplication significance are deleted.
Comments 9: Line 127: There is no transition between the enrichment analysis (KEGG/GO) and Figure 4A (WB). The authors should introduce the linking transcriptome results to subsequent protein validation.
Responses：Thank you for your suggestion. The relationship and transition between KEGG/GO and Figure 4 (WB) have been supplemented in the manuscript (Lines 136–141). Furthermore, the connection between transcriptomic results and protein validation has been added to the main text (Lines 144–145). We are grateful for your comments, which have enhanced the coherence of our manuscript.
Comments 10: Figure 5D: The image is extremely dark; brightness and contrast should be adjusted for visibility.
Responses：Thank you for your suggestion. The brightness and contrast of figure 5D have been adjusted.
Comments 11: Figure 9: Many small subpanels add little value and should be moved to the Supplementary section.
Responses：Thank you for your feedback. Following your suggestion, we made appropriate adjustments to the figure9 and Some of the charts with repetitive significance have been deleted.
Comments 12: Lines 430–432: These sentences on CA’s background are misplaced, they belong in the Introduction, not in the Discussion. The first part of the Discussion through line 450 reads like background material.
Responses：Thanks for this comment. we have included the background on CA in the introduction section, and we have modified the discussion section to be marked in red font.
Comments 13: The Discussion mainly repeats the Results with minimal comparison to previous studies or mechanistic interpretation. Discussion should provide theoretical integration and comparison with refs rather than restating the results.
Responses：Thank you so much for this comment. According to your suggestion.We revised the discussion section, significantly reducing the repetition of results and instead focusing on the comparative analysis with previous literature. Furthermore, we have included an in-depth exploration of the potential mechanisms underlying our findings to enhance the theoretical depth and academic value of the paper.
Comments 14: Lines 510–520: The citations for fastp and HISAT2 are missing. DESeq should be corrected to DESeq2, and the version should be verified.
Responses：Thank you for this important comment. Specific web links for FASTP and HISAT3 have been added to Lines 544-546 of the article, and DESep has been corrected to DESeq2.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe introduction and abstract sections should mention the justification of Callistephus chinensis as a hepatoprotective agent.
Does Callistephus A have previous pharmacological activities? Is this compound found in other plant species? The information provided by the authors is scarce.
Section 4 does not indicate the justification for using 20 and 40 mg/kg of the plant compound.
Figure 7 indicates different unit concentrations for CA. These units include micromolar, micromolar/ml, and microgram/ml. All compounds should be expressed with the same concentration units to compare their biological activity.
The link between the transcriptome study and the Western blot analysis is not explained in the discussion section.
The discussion section does not indicate the limitations of the study.
The study focuses on the hepatoprotective effect of Callistephus A. Did the authors consider using a CClâ‚„-induced hepatic damage? The discussion section should mention this information.
Author Response
Comments 1: The introduction and abstract sections should mention the justification of Callistephus chinensis as a hepatoprotective agent.
Responses 1: Thank you for this valuable comment. The hepatoprotective effect of CA has been included in the abstract. The relevant information for the main content is based on the following references.
(1) Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
(2) Jiang, S.; Wang, M.; Jiang, Z.; Zafar, S.; Xie, Q.; Yang, Y.; Liu, Y.; Yuan, H.; Jian, Y.; Wang, W. Chemistry and Pharmacological Activity of Sesquiterpenoids from the Chrysanthemum Genus. Molecules (Basel, Switzerland) 2021, 26, doi:10.3390/molecules26103038.
Comments 2: Does Callistephus A have previous pharmacological activities? Is this compound found in other plant species? The information provided by the authors is scarce.
Responses 2: Thank you for your question. In our previous studies, we found that Callistephus A (CA) demonstrated protective effects against both Con A-induced immune-mediated liver injury in mice and the TGF-β1-induced LX-2 cell model. Callistephus A (CA) is a sesquiterpene compound featuring a rare 6/7 ring skeleton. This compound has so far been isolated only from Callistephus chinensis Nees and has not been found in any other plant species. It has been supplemented in the introduction to the original text (Lines 79-84).
(1) Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
(2) Chen, J.; Wang, X.; Huang, K.; Yuan, M.; Li, X.; Zhang, X. A new derivative with 6/7 ring skeleton obtained from Callistephus chinensis flowers with anti-hepatic fibrosis activity. Natural product research 2025, 1-5, doi:10.1080/14786419.2025.2475514.
Comments 3: Section 4 does not indicate the justification for using 20 and 40 mg/kg of the plant compound.
Responses 3: Thank you for your question, we have added the rationale of our dosage selection in section 4.2. The literature involved is as follows:
(1) Fu, H.; Wang, X.; Yuan, M.; Wang, N.; Zhang, X. Callistephus A from Callistephus chinensis Nees alleviates concanavalin A-induced immunological liver injury in mice by inhibiting the activation of JAK/STAT1 and MAPK signaling pathways. International immunopharmacology 2025, 148, 114153, doi:10.1016/j.intimp.2025.114153.
Comments 4: Figure 7 indicates different unit concentrations for CA. These units include micromolar, micromolar/ml, and microgram/ml. All compounds should be expressed with the same concentration units to compare their biological activity.
Responses 4: In accordance with your valuable feedback, we have revised the units for Silibinin and CA in Figure 7, which are now consistently presented as μmol/mL.
Comments 5. The link between the transcriptome study and the Western blot analysis is not explained in the discussion section.
Responses 5: Thank you for your question. We have added an analysis of the correlation between transcriptome sequencing results and Western blot experiments in the discussion section.
Comments 6: The discussion section does not indicate the limitations of the study.
Responses 6: Thanks for your suggestion, We have added content about the limitations of the article in the discussion section.
Comments 7: The study focuses on the hepatoprotective effect of Callistephus A. Did the authors consider using a CClâ‚„-induced hepatic damage? The discussion section should mention this information.
Responses 7: Thank you for your question, which we have already added in the discussion section.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have answered my questions, I have no other comments.
Author Response
Thank you for your review.
