Anti-Inflammatory Effects of the Algal Diterpenoid Ruguloptone A by Modulation of M2 Response in Early Diabetic Retinopathy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reports an in vitro and in vivo (rodent models) evaluation of antiinflammatory [potential of two oxidated diterpenoids from seaweed. These compounds isolated previously by the authors showed promising activities in attenuating inflammatory processes in the DR context which makes this paper interesting and suitable for publication. It is rather well-written and the results are clearly presented. The scope of experiments is broad and provides a lot of useful data.

However, there are some minor technical details that have to be explained as well as some suggestion to improve the merit of the article:

1. Line 128 (2.2).  says "iNOS and HO-1 were purchased ..." - you mean antibodies against, or the proteins?

2. In most figures reporting LPS stimulate cells response (all but Fig. 3) there is no reference drug (positive control) used to demonstrate the relative potential of the tes compounds. In the IL-6 measurement you show dexamethasone as reference while nothing in other  assays. Why so? 

3. A question, not comment: Just wondering as having less experience with this particular experimental setup - from the authors' observations - how reliable is a-tubulin as a loading control and actin in qPCR, in the context of potential cell morphology that has been observed (e.g.  ameboid vs. ramified microglia) and that could influence cytoskeleton?

4. Please, pay attention to the lines 471-475 - I think, no comment is required to understand, although I totally agree with what's written there.

5. Nonetheless, I also think that the above advise in mind, the Discussion could focus more on the specific results of this study and try to discuss more on the potential mechanisms of action related to the pharmacophore structure and why actually such compounds exert the observed effects.  E.g. are they mimicking some endogenous regulatory molecules or are directly interacting with genes or proteins. It is a big question but why not to try to approach it from the medicinal chemistry point of view? Also, the discussion could more quantitatively consider the observed potential comparing to other similar studies.

Author Response

Please see the atachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigated the effects and mechanisms of rugukadiol A (RK) and ruguloptone A (RL) on M1/M2 responses in early inflammatory events of DR using LPS‑stimulated microglial (Bv.2) and macrophage (RAW264.7) cells and an ex vivo physiological model of DR. It provides a reference for the therapeutic potential of diterpenoids in DR, particularly for treatment strategies of DR. Minor revision is recommended before acceptance.

1. In the preliminary experiments, the effects of the two compounds were tested in two cell models, while the results were inconsistent, making it difficult for readers to follow. Could a simple table be provided to summarize the effects of the two compounds?
2. In Figure 7, please verify whether the first column represents the merged images of the subsequent two pictures. For example, in the LPS+RL 60' group, the merged image appears to be questionable.
3. In line 393, please briefly clarify the relationship of the Nos2 and Hmox1with inflammation.
4. In Figure 6, please provide an explanation for why treatment with RL alone also caused a significant increase in the phosphorylation level of p38α-MAPK in microglial cells.
5. In line 343, the authors state that “p38α-MAPK is critical for the ability of immune cells to respond to various pro-inflammatory stimuli including LPS.” Why is the term “non‑canonical activation of p38α-MAPK” referred in the manuscript? What is the “classical activation” ?
6. In lines 471–474, please verify the relevant content.
7. In the references, the format of the hyperlinked DOI content should be consistent.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attachment

Comments for author File: Comments.pdf

Author Response

Please see the atachment

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attachment

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully evaluated the revised version of the manuscript entitled “Anti-inflammatory
effects of the algal diterpenoid ruguloptone A by modulation of M2 response in early diabetic
retinopathy.”

The authors have made substantial improvements in response to the reviewer’s comments. The
manuscript is now clearer, with better methodological transparency, inclusion of statistical
details, clarification of experimental procedures (e.g., qPCR normalization, blinding), and
improved discussion of biological relevance. The addition of preliminary dose–response data
represents a meaningful step forward.

Author Response

Please see the attachment

Author Response File: Author Response.pdf