Anti-Neuroinflammatory Effects of Cardenolides in IL-1β-Activated SK-N-SH Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted manuscript presents the anti-neuroinflammatory activity of cardenolides in a cellular model. The evaluated compounds included digitoxigenin, digitoxigenone, and coroglaucigenin, and the evaluated parameters included inhibition of p38 MAPK, NF-κB, IL-6, and IL-8. The tested compounds were evaluated at different concentrations, such as 10 and 25 uM. The retrieved evidence significantly improves the scientific validation of the medicinal features of natural products. The manuscript is well-structured, and the evidence is solid. Still, I have the following minor comments.

1. The abstract should be revised to state the main concentrations at which the results were obtained. In addition, the authors are advised to include two concise lines highlighting future directions for this work.
2. The manuscript should be revised to avoid writing in the first person.
3. Section 2.1 must present changes in cell viability for each tested concentration. The current version just presents general features about the graph.
4. Following the previous comment, the same should be done for the remaining evaluated parameters by presenting the calculated values, such as the percentage of inhibition or the percentage of dead cells. 
5. Improve the discussion section by comparing the results with other studies that evaluated similar compounds. In this regard, the authors are advised to compare possible factors that may influence these differences.
6. The materials and methods are well described and provide the necessary information for reproducibility.
7. The conclusion section must be separated from the rest of the manuscript with the proper heading.
8. The reference list must be updated. Several references are from 2005, 2013, and 2015. The authors are advised to update them with research articles published in high-impact journals from 2023-2025.

Author Response

The submitted manuscript presents the anti-neuroinflammatory activity of cardenolides in a cellular model. The evaluated compounds included digitoxigenin, digitoxigenone, and coroglaucigenin, and the evaluated parameters included inhibition of p38 MAPK, NF-κB, IL-6, and IL-8. The tested compounds were evaluated at different concentrations, such as 10 and 25 uM. The retrieved evidence significantly improves the scientific validation of the medicinal features of natural products. The manuscript is well-structured, and the evidence is solid. Still, I have the following minor comments.

1. The abstract should be revised to state the main concentrations at which the results were obtained. In addition, the authors are advised to include two concise lines highlighting future directions for this work.

Answer: We have added the used concentrations and our thoughts to possible future directions to the abstract.

2. The manuscript should be revised to avoid writing in the first person.

Answer: We have revised all formulations in the first person.

3. Section 2.1 must present changes in cell viability for each tested concentration. The current version just presents general features about the graph.

Answer: We have added changes in percentage to the graphs.

4. Following the previous comment, the same should be done for the remaining evaluated parameters by presenting the calculated values, such as the percentage of inhibition or the percentage of dead cells.

Answer: We have added changes in percentage in comparison to IL-1b to all figures.

5. Improve the discussion section by comparing the results with other studies that evaluated similar compounds. In this regard, the authors are advised to compare possible factors that may influence these differences.

Answer: We have added additional information to the discussion.

6. The materials and methods are well described and provide the necessary information for reproducibility.

Answer: Thank you.

7. The conclusion section must be separated from the rest of the manuscript with the proper heading.

Answer: We have separated the conclusion section from the discussion and moved the section to the end under the proper heading.

8. The reference list must be updated. Several references are from 2005, 2013, and 2015. The authors are advised to update them with research articles published in high-impact journals from 2023-2025.

Answer: In the literature research we focused on articles relevant to our work. Especially for the AA/COX-2 pathway research, there is a lot of good data from the 2000s, with findings that have not been outdated yet. Therefore, we tried to use recent articles references wherever possible but used older references as well were appropriate.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Anti-Neuroinflammatory Effects of Cardenolides in IL-1β-Activated SK-N-SH Cells” investigates the potential anti-neuroinflammatory effects of three cardenolide derivatives Digitoxigenin, Digitoxigenone, and Coroglaucigenin in IL-1β-stimulated human neuroblastoma SK-N-SH cells. The study is potentially interesting; however, several issues should be addressed to strengthen the experimental rationale and interpretation of the findings.

1. The authors should consider performing cell viability assays in an appropriate normal cell line to determine the potential cytotoxicity and selectivity of the three cardenolide derivatives.
2. Previous studies have reported anticancer effects of cardenolide derivatives through inhibition of Na⁺/K⁺-ATPase and induction of tumor-cell apoptosis. However, the present study does not appear to show substantial cytotoxicity in SK-N-SH cells. The authors should discuss possible reasons for this discrepancy, including differences in cell type, concentration, treatment duration, or cellular sensitivity.
3. In Figure 1 please clarify why the IL-1β + Coroglaucigenin treatment group was not included in the cell viability experiment. If this treatment was excluded for a specific experimental reason, the authors should provide an explanation.
4. In western blot analysis for the phosphorylated NF-κB and p38 MAPK data, the authors should also assess the corresponding total NF-κB and total p38 MAPK levels. Normalizing phosphorylated proteins to their respective total protein levels would provide a more accurate assessment of pathway activation.
5. In Figures 7 and 8 the method used to calculate IL-6 and IL-8 levels as % of IL-1β is unclear. The authors should provide a detailed explanation of the calculation, normalization method, and rationale for expressing these data as “% IL-1β.”
6. The conclusion should clearly identify which of the three cardenolide derivatives showed the strongest anti-neuroinflammatory activity based on the experimental results. The authors should also discuss the possible structural or mechanistic reasons that could explain the differences in activity among Digitoxigenin, Digitoxigenone, and Coroglaucige.

Author Response

The manuscript entitled “Anti-Neuroinflammatory Effects of Cardenolides in IL-1β-Activated SK-N-SH Cells” investigates the potential anti-neuroinflammatory effects of three cardenolide derivatives Digitoxigenin, Digitoxigenone, and Coroglaucigenin in IL-1β-stimulated human neuroblastoma SK-N-SH cells. The study is potentially interesting; however, several issues should be addressed to strengthen the experimental rationale and interpretation of the findings.

1. The authors should consider performing cell viability assays in an appropriate normal cell line to determine the potential cytotoxicity and selectivity of the three cardenolide derivatives.

Answer: We have performed an MTT-assay as measure for cell viability in the used SK-N-SH cell line to assess cytotoxicity of the used compounds. Since all experiments were conducted in that cell line, we did not evaluate cytotoxic effects of the compounds in other cell lines for this study. Since the current study does not focus on differences between different cell types/lines but on inflammatory effects, selectivity is not the main focus but should be included in future studies involving mixed cell cultures, organoids or in vivo models.

2. Previous studies have reported anticancer effects of cardenolide derivatives through inhibition of Na⁺/K⁺-ATPase and induction of tumor-cell apoptosis. However, the present study does not appear to show substantial cytotoxicity in SK-N-SH cells. The authors should discuss possible reasons for this discrepancy, including differences in cell type, concentration, treatment duration, or cellular sensitivity.

Answer: We have added additional information on the cytotoxic effects of the cardenolides and possible factors responsible for observed differences in different cell lines and cardenolides. In cancer cell lines, IC50 values differ dependent on the model or cell line used and range between high nM to low µM concentrations.  

3. In Figure 1 please clarify why the IL-1β + Coroglaucigenin treatment group was not included in the cell viability experiment. If this treatment was excluded for a specific experimental reason, the authors should provide an explanation.

Answer: Thank you very much for the comment. It is a mistake, that the IL-1b + Coroglaucigenin group was not included. The figure was revised accordingly.

4. In western blot analysis for the phosphorylated NF-κB and p38 MAPK data, the authors should also assess the corresponding total NF-κB and total p38 MAPKlevels. Normalizing phosphorylated proteins to their respective total protein levels would provide a more accurate assessment of pathway activation.

Answer: We agree that it would be more accurate to assess pathway activation using the ratio of phosphorylated to total protein forms. Due to technical reasons we decided to use vinculin as an constitutive expressed protein as reference, that should still allow an accurate assessment of pathway activation especially regarding the short incubation times used in that experiments. Due to the short incubation time, relevant changes in the total protein amount seem unlikely as an confounder for the phosphorylation investigations.

5. In Figures 7 and 8 the method used to calculate IL-6 and IL-8 levels as % of IL-1β is unclear. The authors should provide a detailed explanation of the calculation, normalization method, and rationale for expressing these data as “% IL-1β.”

Answer: We have added an explanation of the normalization of the concentrations of IL-6 and IL-8 in the methods section. Since the induction of IL-1b can differ between different passages of cells, the normalization with the IL-1bpositive control as 100% rules out errors in cell counts/well or differences in cellular responses to the induction. Furthermore, we have normalized all data shown in the manuscript for easier comparison and understanding of figures presented.

6. The conclusion should clearly identify which of the three cardenolide derivatives showed the strongest anti-neuroinflammatory activity based on the experimental results. The authors should also discuss the possible structural or mechanistic reasons that could explain the differences in activity among Digitoxigenin, Digitoxigenone, and Coroglaucige.

Answer: We have added the information, that Digitoxigenin showed the strongest anti-neuroinflammatory efficacy in the conclusion. Furthermore, we have added additional information on the possible reasons for the different activities focusing on structural aspects.