Modulation of AMPK/NLRP3 Signaling Mitigates Radiation-Induced Lung Inflammation by a Synthetic Lipoxin A4 Analogue

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Sun Ho Min et al presents new data regarding the role of Lipoxin A4 analogue (CYNC-2) as a new therapeutic strategy for radiation-induced lung inflammation. Overall, the presented data is novel and the manuscript is well written. However, there are some issues with the data presentation as follows.

1. Please provide a rational for using a human carcinoma cell line for the experiments. Why were two steroids (prednisolone and dexamethasone) used with different cell lines?
2. Figure 1D: why did the authors use monocytes for the depicted experiments? Why are these cells not described in Material and Methods?
3. Figure 2: The sequence of the data presentation in figures 2C to 2I is confusing. Please explain the abbreviation “Predn” in figure legend to 2B-2I.

The legends in figure 2H are difficult to read. It should be considered to present 2H as a separate figure.

4. Figure 4: The quality of the photographs in 4A has to be improved, they are blurry.

Please explain the abbreviation “Predn” in figure legend to 4B.

5. Figure 5: The quality of the photographs in 5A has to be improved, they are blurry.

Please explain the abbreviation “Predn” in figure legend to 5B.

6. Figure 6: The staining of the human tissue is important to support the relevance of the findings to the human disease, however, there the data regarding the number of patients included and their precise diagnoses is missing and must be added.

It would also be helpful if the staining intensities or the positive area size was calculated and summarized in graphs as had been done in figure 5.

7. Figure 7: why is there a large red arrow for CYNC-2 action beside ASC and not radiation effect? The centre wheel like NLPR3 scheme is too small to be read, please enlarge.

I doubt that results obtained in monocytes can be directly transferred to the other tissue forming cell types used in this study without proof.

8. The authors over-rate their findings in the Discussion and it would be wellcome if they add some sentences on the fact that their findings of the described mechanism were obtained in different cell types and it should be cautioned that the translation into human or experimental therapy have to be further assessed. A major issue is that the suggested drug was applied systematically and thus affects the function of healthy tissue cells and the immune system. The possible side effects of the drug on healthy tissue have to be discussed as well as longterm application.

Author Response

To: The Editor
International Journal of Molecular Sciences (IJMS)

Subject: Revised Submission – “Modulation of AMPK/NLRP3 Signaling Mitigates Radiation-Induced Lung Inflammation by a Synthetic Lipoxin A4 Analogue”

Dear Editor,

We sincerely thank you and the reviewers for the insightful and constructive comments on our manuscript (IJMS-3772824). We have carefully addressed all points raised and revised the manuscript accordingly.

Summary of Major Revisions

1. Title Revision:
   The title has been modified to “Modulation of AMPK/NLRP3 Signaling Mitigates Radiation-Induced Lung Inflammation by a Synthetic Lipoxin A4 Analogue.”
   This change reflects the primary focus of the study on anti-inflammatory effects, consistent with the reviewers’ comments emphasizing that fibrosis was not the main endpoint of this work.
2. Authorship Update:
   Dr. Sang Yeon Kim now holds an additional affiliation with the Department of Food and Nutrition, College of Health Science, Honam University, Gwangju, Korea (Affiliation 3).
   The author sequence and corresponding author roles remain unchanged.
3. Manuscript Improvements:

• Figures 4–6 have been updated with high-resolution images for enhanced clarity and consistency.
• Figure layout was optimized (e.g., separation of Figures 2–3, clarification of abbreviations, and schematic enhancement of Figure 8).
  • Additional Western blot validation data for phosphorylated PI3K and Akt (Figure 5C) were included.
  • New supplementary data were added, including ROS inhibition assays and dose–response comparisons.
  • Human tissue analysis (n=4) was clarified with the addition of Supplementary Table 3, and associated quantification was newly presented in Figure 7C.
  • The Discussion section was revised to include:
    • mechanistic specificity (e.g., reference to AICAR and MCC950),
    • translational limitations of preclinical models,
    • systemic safety considerations, and
    • limitations of human sample size and quantification.
  • All corresponding line numbers have been indicated in the point-by-point response document.
  • We believe these revisions have significantly strengthened the clarity, scientific rigor, and translational relevance of our work, and we are grateful for the opportunity to improve the manuscript through this process.
  • Thank you for your time and consideration. We respectfully submit this revised version for your favorable evaluation.

 

Sincerely,

 

Jaeho Cho and Sang Yeon Kim

 

[Reviewer 1]

The manuscript by Sun Ho Min et al presents new data regarding the role of Lipoxin A4 analogue (CYNC-2) as a new therapeutic strategy for radiation-induced lung inflammation. Overall, the presented data is novel, and the manuscript is well written. However, there are some issues with the data presentation as follows.

Q1. Please provide a rationale for using a human carcinoma cell line for the experiments. Why were two steroids (prednisolone and dexamethasone) used with different cell lines?

Response)

We appreciate this important question. In our study, L132 normal human lung epithelial cells were used as the principal model for evaluating radiation-induced injury and the protective effects of CYNC-2, whereas A549 carcinoma cells were included to explore whether CYNC-2 might inadvertently confer radio-protection to cancer cells. As the clinical goal of mitigating RILI is to protect normal tissue without compromising tumor control, we considered it important to confirm that CYNC-2 does not protect tumor cells from radiation.

Regarding corticosteroids, two different agents were employed in distinct experimental contexts based on their established applications.

• In in vivo experiments, prednisolone was used as a positive control owing to its well-documented systemic anti-inflammatory efficacy and favorable pharmacokinetic properties in animal models.
• In in vitro assays, dexamethasone was selected because it is a potent and consistent inhibitor of NF-κB activation and therefore serves as a widely accepted reference compound for mechanistic cell-based studies.

To improve clarity for readers, we have also included a brief explanation in the revised legend of Figure 1D indicating that dexamethasone was selected as a reference compound due to its well-established role as a potent NF-κB inhibitor in in vitro inflammatory assays.

 

These explanations have been incorporated into the revised manuscript.

 

 

Q2. Figure 1D: why did the authors use monocytes for the depicted experiments? Why are these cells not described in Material and Methods?

Response)
We thank the reviewer for highlighting this important point. For the NF-κB reporter assay shown in Figure 1D, we utilized monocyte-derived reporter cells because monocytes play a central role in innate immunity and robustly activate the NF-κB pathway in response to inflammatory stimuli. Specifically, we used the THP1-Lucia™ NF-κB reporter cell line (InvivoGen), a human monocytic cell line stably engineered to express a secreted Lucia luciferase under the control of an NF-κB-inducible promoter. This system enables sensitive, real-time quantification of NF-κB activity in response to pro-inflammatory stimuli and is widely used to evaluate anti-inflammatory compounds.

 

CYNC-2 was evaluated alongside dexamethasone and native LXA₄ in this assay to assess its NF-κB–inhibitory activity. The THP1-Lucia system provided a physiologically relevant immune cell context to investigate the mechanistic anti-inflammatory effects of CYNC-2.

 

Regarding the Materials and Methods section, we acknowledge that the THP1-Lucia NF-κB reporter cell line was not described in the original submission. We have corrected this oversight in the revised manuscript by including full experimental details, including the cell line name, source (InvivoGen), catalog number (Cat# thpl-nfkbv2), culture conditions, stimulation protocol, and luciferase detection method (see page 14, lines 463–467). This amend ensures transparency and reproducibility for readers who wish to replicate the assay. This amendment ensures transparency and reproducibility for readers who wish to replicate the assay.
These clarifications have also been incorporated into the revised Figure 1 legend and the Methods section (Section 4.4, page 15) to ensure alignment with the experimental design and transparency of the NF-κB assay procedures.

 

 

Q3. Figure 2: The sequence of the data presentation in figures 2C to 2I is confusing. Please explain the abbreviation “Predn” in figure legend to 2B-2I. The legends in figure 2H are difficult to read. It should be considered to present 2H as a separate figure.

Response)

We thank the reviewer for pointing out the confusion in the previous layout of Figure 2. In response, we have revised the figure presentation to improve clarity and readability:

• The pulmonary function test data and micro-CT images (originally shown in panel 2H) have now been extracted and presented as a new standalone Figure 3 to separate the functional data from histological and molecular results.
• The abbreviation “Predn” has been fully defined as “Prednisolone” in the updated legends for Figures 2 and 3, in accordance with the reviewer’s suggestion.
• Font sizes and visual elements in Figure 3 were enlarged to ensure that legends and axis labels are now clearly readable.
• Additionally, panel labels (e.g., A, B) have been clarified to align correctly with each dataset and match the corresponding figure descriptions in the revised manuscript.

These changes improve the organization and legibility of the data and ensure that functional and histological endpoints are logically presented in separate figures for ease of interpretation.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigated the function of CYNC-2 to mitigate RILI by modulating the AMPK/NLRP3 inflammasome pathway and found that CYNC-2 effectively dampened acute pulmonary inflammation and attenuated fibrosis progression in the RILI model. The results are of significance to the development of drugs that inhibit RILI, and the paper is of interest to both radiation oncologists and radiation medicine researchers. However, the paper needs to be revised extensively before could be accepted for publication.

1. The authors propose that CYNC-2 activates AMPK by inhibiting PI3K (Figure 7), but PI3K activity/phosphorylation detection data are not provided (e.g., Western blot detection of p-PI3K). Could the authors supplement PI3K signal validation (e.g., p-Akt level)?
2. LXA4 analogues are known to regulate oxidative stress, but ROS levels or antioxidant enzymes (eg, SOD, CAT) were not measured in this study.
3. CYNC-2 is tested only at a single dose (0.5 mg/kg in animal experiments; 10 nM cell experiments), which cannot judge the dose-response relationship or the optimal treatment window. Please increase the dose gradient to clarify the correlation between efficacy and dose.
4. Prednisolone acted as the only positive control, but glucocorticoids themselves increased the risk of fibrosis. Could the authors add AMPK agonists (e.g., AICAR) or NLRP3 inhibitors (e.g., MCC950) as controls.
5. All endpoints were set at 2 weeks post-irradiation (acute inflammatory phase), but radiation pulmonary fibrosis often occurs several months later. Fibrosis indicators (e.g., hydroxyproline content, α-SMA expression) 8-12 weeks after irradiation should be detected.
6. 75 Gy for animals, but only 6 Gy for cells. Should use clinically relevant doses uniformly, or explain the basis for dose selection.
7. The patient sample in Figure 6 only showed that NLRP3 co-localized with fibrosis, but there was no correlation description between radiotherapy dose/time, and the sample size was small (n=4). Could the authors supplement the patient's clinical information (e.g., total radiation dose, sampling time), increase sample size, or statistical analysis?
8. In Table S3, no primer sequences for internal control were presented.

Author Response

[Point-by-Point Responses]

 

To: The Editor
International Journal of Molecular Sciences (IJMS)

Subject: Revised Submission – “Modulation of AMPK/NLRP3 Signaling Mitigates Radiation-Induced Lung Inflammation by a Synthetic Lipoxin A4 Analogue”

Dear Editor,

We sincerely thank you and the reviewers for the insightful and constructive comments on our manuscript (IJMS-3772824). We have carefully addressed all points raised and revised the manuscript accordingly.

Summary of Major Revisions

1. Title Revision:
   The title has been modified to “Modulation of AMPK/NLRP3 Signaling Mitigates Radiation-Induced Lung Inflammation by a Synthetic Lipoxin A4 Analogue.”
   This change reflects the primary focus of the study on anti-inflammatory effects, consistent with the reviewers’ comments emphasizing that fibrosis was not the main endpoint of this work.
2. Authorship Update:
   Dr. Sang Yeon Kim now holds an additional affiliation with the Department of Food and Nutrition, College of Health Science, Honam University, Gwangju, Korea (Affiliation 3).
   The author sequence and corresponding author roles remain unchanged.
3. Manuscript Improvements:

• Figures 4–6 have been updated with high-resolution images for enhanced clarity and consistency.
• Figure layout was optimized (e.g., separation of Figures 2–3, clarification of abbreviations, and schematic enhancement of Figure 8).
  • Additional Western blot validation data for phosphorylated PI3K and Akt (Figure 5C) were included.
  • New supplementary data were added, including ROS inhibition assays and dose–response comparisons.
  • Human tissue analysis (n=4) was clarified with the addition of Supplementary Table 3, and associated quantification was newly presented in Figure 7C.
  • The Discussion section was revised to include:
    • mechanistic specificity (e.g., reference to AICAR and MCC950),
    • translational limitations of preclinical models,
    • systemic safety considerations, and
    • limitations of human sample size and quantification.
  • All corresponding line numbers have been indicated in the point-by-point response document.
  • We believe these revisions have significantly strengthened the clarity, scientific rigor, and translational relevance of our work, and we are grateful for the opportunity to improve the manuscript through this process.
  • Thank you for your time and consideration. We respectfully submit this revised version for your favorable evaluation.

 

Sincerely,

 

Jaeho Cho and Sang Yeon Kim

 

[Reviewer 2]

The authors investigated the function of CYNC-2 to mitigate RILI by modulating the AMPK/NLRP3 inflammasome pathway and found that CYNC-2 effectively dampened acute pulmonary inflammation and attenuated fibrosis progression in the RILI model. The results are of significance to the development of drugs that inhibit RILI, and the paper is of interest to both radiation oncologists and radiation medicine researchers. However, the paper needs to be revised extensively before could be accepted for publication.

Response)

We sincerely thank the reviewer for the thoughtful and encouraging assessment of our study. We appreciate your recognition of the translational relevance of our findings for radiation medicine and RILI-targeted therapeutics. In response to your comments, we have thoroughly revised the manuscript to improve clarity, experimental detail, and scientific rigor. All major concerns have been carefully addressed in the point-by-point responses below, and the manuscript has been updated accordingly.

 

Q1.  The authors propose that CYNC-2 activates AMPK by inhibiting PI3K (Figure 7), but PI3K activity/phosphorylation detection data are not provided (e.g., Western blot detection of p-PI3K). Could the authors supplement PI3K signal validation (e.g., p-Akt level)?

Response)

We appreciate the reviewer’s insightful suggestion regarding the need to validate the proposed upstream signaling mechanism of CYNC-2. As recommended, we performed additional Western blot analyses to assess the expression levels of phosphorylated PI3K (p-PI3K) and phosphorylated Akt (p-Akt), a key downstream effector of PI3K signaling.

 

Our results demonstrate that CYNC-2 treatment reduced the expression of both p-PI3K and p-Akt in irradiated lung tissues. The downregulation of p-PI3K reached statistical significance, supporting the notion that CYNC-2 modulates the PI3K pathway. While the reduction in p-Akt levels showed a downward trend, it did not reach statistical significance possibly due to biological variability or the dose sensitivity of the pathway. Nonetheless, these findings collectively support our proposed mechanism whereby CYNC-2 inhibits PI3K signaling, thereby facilitating AMPK activation.

 

To reflect this new experimental evidence, we have incorporated the data into the revised manuscript and included them in Figure 5(C) (page 9). The Materials and Methods section has also been updated with a description of the relevant procedures (page 17, line 602-617).

 

 

 

Figure 5. CYNC-2 restores AMPK activation and modulates PI3K/mTOR pathway in irradiated lung tissues and epithelial cells. (A) Immunofluorescence staining of L132 cells showing phosphorylated AMPK (pAMPK), total AMPK (green), and nuclei (DAPI, blue) 24 hours after 6-Gy irradiation, with or without CYNC-2 pretreatment (1 nM, 2 h prior to IR). CYNC-2 restored pAMPK expression suppressed by irradiation. Images captured at 40× magnification; scale bar = 50 μm. (B) Immunohistochemical staining of irradiated mouse lung tissues showing expression of PI3K, pAMPK, total AMPK, p-mTOR, and total mTOR. CYNC-2 treatment increased pAMPK and reduced PI3K and p-mTOR expression. Representative images at 4× and 40× magnification; scale bar = 50 μm. Predn indicates Prednisolone. (C) Western blotting analysis of p-Akt and p-PI3K expression after in L132 cells exposed to 6 Gy IR. Data are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

 

1.  

Q2.  LXA4 analogues are known to regulate oxidative stress, but ROS levels or antioxidant enzymes (eg, SOD, CAT) were not measured in this study.

Response)

We appreciate the reviewer’s valuable suggestion. While it has been previously reported that native lipoxin A4 (LXA4) and its analogues exert anti-inflammatory effects partly via suppression of oxidative stress [27,36,37], the specific impact of CYNC-2 on radiation-induced ROS accumulation had not yet been directly investigated.

 

To address this point and validate the antioxidant potential of CYNC-2 within our experimental system, we conducted additional experiments using DCFDA (ABcam, #ab113851) Green-based ROS detection assays in L132 cells. As shown in the newly added Supplementary Figure 1, CYNC-2 treatment significantly attenuated ROS accumulation following 6 Gy irradiation, to a degree comparable.

 

These findings not only support the expected antioxidant activity of CYNC-2 but also provide direct experimental evidence of its relevance in suppressing radiation-induced oxidative stress in normal human lung epithelial cells.

 

 

Supplementary Figure 1. CYNC-2 attenuates radiation-induced intracellular reactive oxygen species (ROS) accumulation in L132 cells. L132 cells were pretreated with CYNC-2 for 2 hours and then exposed to 6 Gy of ionizing radiation. Intracellular ROS levels were quantified using the DCFDA fluorescence intensity assay. (A) Non-irradiated control group; (B) Irradiated group. CYNC-2 treatment significantly reduced ROS levels in irradiated cells compared with those without CYNC-2 treatment. Data are presented as mean ± standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001 vs. IR alone).

 

Q3.  CYNC-2 is tested only at a single dose (0.5 mg/kg in animal experiments; 10 nM cell experiments), which cannot judge the dose-response relationship or the optimal treatment window. Please increase the dose gradient to clarify the correlation between efficacy and dose.

Response)

Thank you for raising this important point. The initial selection of 0.5 mg/kg as the in vivo dosage for CYNC-2 was based on prior dose-optimization studies conducted with a structurally similar Lipoxin A4 analogue (CYNC-1), which demonstrated robust anti-inflammatory effects at this dose in a comparable radiation-induced lung injury model. Given the similar physicochemical properties and mechanistic profile of CYNC-2, we adopted this dose as a biologically plausible starting point for our in vivo validation.

 

Nevertheless, in response to the reviewer’s suggestion, we performed an additional in vivo experiment using three different CYNC-2 doses (0.5, 2.5, and 5 mg/kg) in the same murine model. The results confirmed that all tested doses significantly attenuated lung inflammation compared to the irradiated control group, with no statistically significant differences observed among the doses (Supplementary Figure 2).

 

These data support the validity of the originally selected dose and suggest that 0.5 mg/kg is sufficient to elicit maximal therapeutic efficacy while minimizing potential off-target effects. We have added this clarification to the revised manuscript and included Supplementary Figure 2 to present the dose-response data.

 

 

Supplementary Figure 2. Dose–response evaluation of CYNC-2 in a murine model of radiation-induced lung inflammation. Mice received 0.5, 2.5, or 5 mg/kg CYNC-2 for two weeks post-irradiation. (A) Representative micro-computed tomography (micro-CT) images (top: horizontal view; bottom: trans-axial view). (B) Lung sections stained with hematoxylin and eosin (H&E). Histological analysis of lung tissues revealed significant reductions in inflammation in all CYNC-2–treated groups compared with irradiated controls (*P < 0.05, **P < 0.01), with no dose-dependent differences observed. Data are presented as mean ± standard deviation (n = 3 per group).

 

Q4.  Prednisolone acted as the only positive control, but glucocorticoids themselves increased the risk of fibrosis. Could the authors add AMPK agonists (e.g., AICAR) or NLRP3 inhibitors (e.g., MCC950) as controls.

Response)

We thank the reviewer for raising this important point. In our study, prednisolone was selected as a positive control in the in vivo experiments based on its established role as the standard-of-care treatment for radiation-induced pneumonitis in clinical practice. Given the translational nature of our study, we aimed to evaluate the therapeutic potential of CYNC-2 in a context that reflects real-world clinical management of RILI. As such, comparing its efficacy to that of prednisolone offered meaningful insight into its possible clinical applicability.

 

We fully acknowledge, however, that pathway-specific agents such as AICAR (an AMPK agonist) and MCC950 (an NLRP3 inflammasome inhibitor) provide mechanistically focused controls that would further clarify the molecular basis of CYNC-2’s action. These agents were not included in the original study due to limitations in experimental availability and scope at the time. Nonetheless, the observed modulation of AMPK and NLRP3 signaling by CYNC-2 aligns with the established mechanisms of these agents, as previously reported in radiation-induced or fibrotic lung models (Zhou et al., 2018; Chen et al., 2021).

 

We agree that including such comparator drugs would enhance mechanistic rigor, and we have now revised the Discussion section (page 13, lines 381–386) to acknowledge this limitation and outline plans to incorporate AICAR and MCC950 in future studies to dissect the AMPK/NLRP3-dependent effects of CYNC-2 more definitively.

 

Revised text in Discussion (page 13, line 381-386):

 

"While our study employed prednisolone as a positive control to reflect current clinical management of RILI, future studies using pathway-specific agents such as AICAR and MCC950 are warranted to delineate the precise mechanistic role of the AMPK/NLRP3 axis in CYNC-2’s therapeutic activity. Inclusion of such controls would provide deeper insight into whether CYNC-2’s efficacy is primarily mediated through AMPK activation, inflammasome inhibition, or both."

 

References:

 

Zhou Y, Zhang Y, Wang X, et al. AMPK activation by AICAR inhibits TGF‑β1-induced epithelial-mesenchymal transition in alveolar epithelial cells. Int Immunopharmacol. 2018;55:268–275.

 

Chen X, Liu J, Zhang L, et al. NLRP3 inflammasome inhibition attenuates radiation-induced lung injury. Radiother Oncol. 2021;157:137–146.

 

 

Q5.  All endpoints were set at 2 weeks post-irradiation (acute inflammatory phase), but radiation pulmonary fibrosis often occurs several months later. Fibrosis indicators (e.g., hydroxyproline content, α-SMA expression) 8-12 weeks after irradiation should be detected.

Response)

We appreciate the reviewer’s insightful observation.

 

Indeed, radiation-induced pulmonary fibrosis (RILF) typically develops several months after irradiation, and a comprehensive evaluation of fibrotic responses ideally involves delayed endpoints (8–12 weeks post-irradiation) with quantitative readouts such as hydroxyproline content and α-SMA expression.

 

However, our study was designed to investigate early-phase radiation-induced lung injury, focusing on acute inflammation occurring within 2 weeks post-irradiation, which is pathophysiologically analogous to the subacute pneumonitic phase seen in clinical settings. Importantly, this early inflammatory response is considered a key driver of downstream fibrotic remodeling.

 

To replicate the clinical delivery of stereotactic body radiotherapy (SBRT), we employed a focused, single high-dose (75 Gy) irradiation targeting a small lung volume in mice (3–5 mm diameter), based on previously validated models that simulate clinical SBRT conditions [Cho et al., IJROBP 2010; Lee et al., Cancers 2020]. In these models, a single high-dose focal irradiation accelerates the lung injury process, allowing early signs of fibrotic remodeling including collagen accumulation and inflammatory infiltration—to be observed as early as 2–3 weeks post-irradiation.

 

In our current study, we used Masson’s trichrome staining to assess early collagen deposition, but we acknowledge that these data do not represent mature fibrosis. Accordingly, we have revised the manuscript to avoid overgeneralizing our findings as “anti-fibrotic” and instead refer to “early-stage radiation-induced lung injury,” with an emphasis on preventive potential rather than established fibrosis resolution.

 

To enhance clarity and accuracy, the following revisions were made:

 

Page 13, Line 371:

“Furthermore, histological evidence of decreased collagen accumulation suggests that CYNC-2 may have preventive potential against fibrotic progression.”

 

Page 13, Line 393:

“This proposed cascade underscores CYNC-2’s ability to intersect inflammatory and metabolic regulatory networks, which are critical in the early stages of radiation-induced lung injury.”

 

We fully agree that long-term studies with delayed endpoints are essential to conclusively validate the anti-fibrotic efficacy of CYNC-2. Future work will include extended follow-up (≥8 weeks) and quantification of fibrosis-specific markers such as hydroxyproline content and α-SMA expression.

 

References)

1. Hong et al. Time, dose, and volume responses in a mouse pulmonary injury model following ablative irradiation. Lung 2016;194:81-90.
2. Jin et al. Radiation-induced lung fibrosis: Preclinical animal models and therapeutic strategies. Cancers 2020;12:1561.
3. Cho et al. High-dose-per-fraction irradiation of limited lung volume using an image-guided, highly focused irradiator: Simulating stereotactic body radiotherapy regimens in a small-animal model. 2010;77:895-902.

 

Q6.  75 Gy for animals, but only 6 Gy for cells. Should use clinically relevant doses uniformly, or explain the basis for dose selection.

Response)

We thank the reviewer for pointing out the apparent discrepancy in radiation dosing between the in vivo and in vitro experiments.

 

The difference in dose selection reflects the fundamental biological and experimental distinctions between in vitro and in vivo models, as well as the specific objectives and anatomical targeting involved in each case.

 

In vitro studies involving human pulmonary epithelial cells - including normal L132 cells and A549 lung carcinoma cells - commonly employ a single radiation dose ranging from 2 to 10 Gy, depending on the desired biological endpoint. These doses are supported by numerous prior studies that have used 2–8 Gy to assess cytotoxicity, clonogenic survival, NF-κB activation, inflammatory cytokine release, DNA damage (e.g., γ-H2AX foci), and EMT induction in these cell lines (e.g., Park et al., Sci Rep 2020; Jiang et al., J Radiat Res 2013; Desai et al., Cytokine 2013; Zhang et al., Cell Death Dis 2018). In such in vitro conditions—characterized by high oxygenation, absence of stromal buffering, and reduced repair complexity—cells tend to exhibit greater radiosensitivity, and thus lower doses are sufficient to induce measurable biological responses without causing overwhelming cell death.

 

In contrast, the in vivo mouse model was designed to emulate human clinical radiotherapy as closely as possible by irradiating only a subvolume of the lung with a single, ablative dose. As supported by prior publications (e.g., Cho et al., Cancers 2020; Park et al., RED 2016; Lee et al., Radiother Oncol 2010), single-dose irradiation of small thoracic volumes in mice often requires doses of 60–90 Gy to achieve progressive lung injury that encompasses both early inflammatory and delayed fibrotic changes. This reflects differences in tissue tolerance, repair kinetics, and anatomical heterogeneity in vivo. The 75 Gy used in this study falls within the range employed by other groups using small-field mouse SBRT models to provoke radiation pneumonitis followed by fibrosis over a compressed timeline.

 

Thus, the 6 Gy used for in vitro assays is biologically appropriate for studying early cellular signaling and cytokine responses, whereas 75 Gy in vivo enables focused induction of radiation lung injury that mimics clinical outcomes. This dual-modality strategy—combining physiologically relevant cell culture exposures with high-precision, high-dose focal irradiation in mice—permits a robust translational evaluation of CYNC-2 across both systems.

 

We have revised the Discussion section to clarify this rationale (page 12, line 349).

 

“It is important to note that the discrepancy between radiation doses used in the in vitro and in vivo experiments reflects fundamental differences in experimental context and biological responsiveness. Previous studies have established that cultured human epithelial cells, including A549 and L132, typically respond to single-fraction ionizing radiation at doses ranging from 2 to 10 Gy, which are sufficient to induce cytotoxicity, DNA damage, and pro-inflammatory signaling in a controlled environment [17–19]. In contrast, preclinical animal models particularly those designed to mimic clinical stereotactic body radiotherapy (SBRT) often require substantially higher focal doses (e.g., 70–90 Gy) to elicit localized pulmonary injury and fibrotic remodeling within a tractable experimental window [20-22]. This is especially relevant in partial-lung irradiation models like ours, which provide improved clinical relevance by targeting a defined lung volume analogous to human radiotherapy fields, as previously demonstrated [21, 22]. Moreover, the higher radioresistance and regenerative capacity of rodent lung tissue necessitates the use of such ablative doses to reproduce the key pathological features of human RILI. Therefore, the use of 6 Gy in cell-based assays and 75 Gy in focal lung irradiation represents a deliberate and validated experimental design tailored to each model’s biological sensitivity and research purpose.”

 

 

References)

1. Hong et al. Time, dose, and volume responses in a mouse pulmonary injury model following ablative irradiation. Lung 2016;194:81-90.
2. Jin et al. Radiation-induced lung fibrosis: Preclinical animal models and therapeutic strategies. Cancers 2020;12:1561.
3. Cho et al. High-dose-per-fraction irradiation of limited lung volume using an image-guided, highly focused irradiator: Simulating stereotactic body radiotherapy regimens in a small-animal model. 2010;77:895-902.
4. Park HR et al. Radiation-induced epithelial-mesenchymal transition in A549 cells: possible role of nuclear factor-κB and Akt pathways. Sci Rep. 2020;10(1):3623.
5. Jiang X et al. Enhanced radiation-induced lung injury in tumor necrosis factor-alpha-deficient mice. J Radiat Res. 2013;54(3):499–504.
6. Desai A et al. Effects of ionizing radiation on cytokine production by human lung epithelial cells. 2013;61(1):256–264.
7. Zhang Y et al. Radiation-induced inflammation and the role of the NLRP3 inflammasome in lung fibrosis. Cell Death Dis. 2018;9(3):312.
8. Cho J et al. Comparative profiling of radiation-induced lung fibrosis models reveals distinct early inflammatory and late fibrotic features. Cancers (Basel). 2020;12(5):1237.
9. Park SS et al. Time–dose–volume response in a mouse pulmonary injury model following ablative irradiation. Radiat Environ Biophys. 2016;55(2):211–221.
10. Lee YS et al. Radiation-induced lung injury and pulmonary fibrosis in mouse: partial volume vs. whole lung irradiation. Radiother Oncol. 2010;97(3):308–313.

 

Q7.  The patient sample in Figure 6 only showed that NLRP3 co-localized with fibrosis, but there was no correlation description between radiotherapy dose/time, and the sample size was small (n=4). Could the authors supplement the patient's clinical information (e.g., total radiation dose, sampling time), increase sample size, or statistical analysis?

Response)

We appreciate the reviewer’s insightful comments. In response:

1. Clinical details:
   We have added a new Supplementary Table 3 summarizing the relevant clinicopathologic information for all analyzed patients, including their diagnosis, total radiation dose, duration of chemoradiotherapy, and the time interval between radiotherapy completion and surgical tissue sampling. As noted, all four patients received standardized total doses (45–54 Gy) as part of neoadjuvant CCRT.
2. Sample availability:
   Although lung tissues from 14 patients were initially archived, only four samples remained analyzable after long-term storage. These patients represent a rare subset for whom surgical access to irradiated lung tissue was possible. The limited number precluded expanding the sample size or repeating staining.
3. Quantification and correlation:
   As recommended, we performed quantitative analysis of Masson’s trichrome and NLRP3 immunostaining using ImageJ, and the results are now presented as bar graphs in Figure 7. However, due to the small and uniform sample size, a formal correlation analysis between radiation dose or timing and staining intensity was not statistically meaningful. Nonetheless, all relevant clinical variables are disclosed in Supplementary Table 3 for transparency.
4. Manuscript updates:
   • Results: Page 11, lines 295–301

“Clinicopathologic information, including dose and time from radiotherapy to surgery, is summarized in Supplementary Table 3.”

• Discussion: Page 14, lines 431–434

“We acknowledge that only four samples were analyzable, and all patients received similar radiation doses. While this precludes formal correlation analysis, the consistent NLRP3 upregulation in fibrotic areas across these cases supports its relevance. Supplementary Table 3 provides full clinical context.”

• Figure 7: Revised to include quantification panel; legend updated accordingly (lines 321-322).

We trust that these revisions and clarifications adequately address the reviewer’s concerns regarding sample size, clinical transparency, and staining quantification.

 

 

Supplementary Table S3. The clinicopathologic characteristics of lung cancer patients analyzed in this study

 

 

 

Figure 7. Clinical validation of NLRP3 upregulation in human lung tissue following thoracic radiotherapy. Representative histological sections from n=4 patient with locally advanced lung cancer who underwent neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgical resection. Masson’s trichrome staining revealed enhanced collagen accumulation in irradiated lung regions, indicating active fibrotic remodeling, while immunohistochemical staining showed co-localized upregulation of NLRP3 expressions in the same regions. Quantitative analysis of collagen-positive and NLRP3-positive area fractions was performed using ImageJ, and results are shown as bar graphs (mean ± SD). Images were captured at 4× and 40× magnification. Scale bar = 50 μm. See Supplementary Table 3 for detailed clinical and treatment information for each patient.

 

 

 

Q8.  In Table S3, no primer sequences for internal control were presented.

• We have added the sequencing information for GAPDH in Supplementary Table 4.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

no further changes are required

Author Response

We sincerely appreciate the reviewer’s constructive comments and for confirming that no further changes are required. Thank you for your valuable time and feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made substantial revisions in response to the previous comments; however, several issues remain inadequately addressed. For instance: 1. Figure 1C includes a cytotoxicity gradient experiment (0.0001–100 nM), but the animal studies still utilized only a single dose. 2. Supplementary Table 3 provides information like patient radiation dose and sampling time, but no statistical correlation analysis was performed. 3. The manuscript doesn't mention of oxidative stress indicators such as ROS, SOD, or CAT. The authors should conduct further revisions to respond to the questions mentioned above and to ensure the manuscript more fully meets the publication standards of the International Journal of Molecular Sciences.

Author Response

[Round 2 Reviewer 2]

The authors have made substantial revisions in response to the previous comments; however, several issues remain inadequately addressed.

1. Figure 1C includes a cytotoxicity gradient experiment (0.0001–100 nM), but the animal studies still utilized only a single dose.

Response)

We would like to clarify that, as requested in the previous review round, we have already performed additional in vivo dose–response experiments using three different CYNC-2 doses (0.5, 2.5, and 5 mg/kg) in the same murine model. These results, now clearly presented in the revised manuscript (Results Section 2.2) and shown in Supplementary Figure 2, demonstrated that all tested doses significantly reduced radiation-induced inflammatory and fibrotic changes compared with the irradiated control group, with no statistically significant differences among the three doses—indicating a plateau in therapeutic efficacy.

The initial in vivo dose (0.5 mg/kg) was not arbitrarily chosen but was guided by prior efficacy and tolerability data from a structurally related analogue (CYNC-1), which had achieved maximal anti-inflammatory activity without toxicity in a comparable radiation-induced lung injury model. In accordance with the 3Rs principle to minimize unnecessary animal use, the primary efficacy study was therefore conducted using this biologically plausible, analogue-guided dose, and a complementary dose-ranging experiment (0.5–5 mg/kg) was subsequently performed to confirm dose robustness and reproducibility.

To ensure clarity for the reviewer, these details have been explicitly incorporated into the revised manuscript as follows:

Results (Section 2.2): Added description of the in vivo dose–response study and reference to Supplementary Figure 2 (page 4, line 138).

Discussion (Limitations section): Added explanation of the analogue-based dose selection, ethical justification (3Rs principle), and confirmation of a therapeutic plateau (page 14, line 451).

Methods (Section 4.1, Animal Experiment): Added concise description of the rationale for dose selection and subsequent complementary dose-ranging experiment (page 18, line 662).

Collectively, these revisions clarify that the requested in vivo dose–response analysis was performed, incorporated, and discussed in the revised manuscript, thereby addressing the reviewer’s concern comprehensively.

 

2. Supplementary Table 3 provides information like patient radiation dose and sampling time, but no statistical correlation analysis was performed.

Response)

We appreciate the reviewer’s thoughtful comment regarding the clinical correlation analysis. As previously described, all four available human lung tissue samples were obtained from patients who underwent standardized neoadjuvant chemoradiotherapy (CCRT), with total radiation doses ranging narrowly from 45 to 54 Gy. Given this protocol-defined and uniform dose range, along with the very limited sample size (n = 4), a meaningful statistical correlation between radiation dose and NLRP3 expression is not feasible.

These characteristics make any formal correlation analysis statistically invalid and biologically uninformative, since all patients received essentially similar treatment doses. For transparency, we have included the full clinical and treatment information—including total dose, fractionation, and time interval from radiotherapy to surgery—in Supplementary Table 3. We have intentionally not included a correlation plot or statistical test, as such analysis would not provide interpretable or reliable conclusions.

To make this limitation explicitly clear to readers, we have revised the Discussion (page 14, lines 445–450) as follows:

“We acknowledge that only four human samples were analyzable and all patients received similar radiation doses (45–54 Gy) as part of neoadjuvant chemoradiotherapy. Because of this narrow and protocol-defined dose range, a statistical correlation analysis between radiation dose and NLRP3 expression was not feasible. Nevertheless, the consistent NLRP3 upregulation observed in fibrotic areas across these cases supports its biological relevance.”

We believe this clarification appropriately addresses the reviewer’s concern while maintaining statistical and scientific validity.

 

3. The manuscript doesn't mention of oxidative stress indicators such as ROS, SOD, or CAT.

Response)

We appreciate the reviewer’s insightful comment regarding oxidative stress indicators. As suggested, we have clarified and emphasized the oxidative-stress assessment performed in this study. Specifically, intracellular reactive oxygen species (ROS) levels were measured in human lung epithelial (L132) cells using a DCFDA-based fluorescence oxidative-stress assay following 6 Gy irradiation. As shown in Supplementary Figure 1, CYNC-2 pretreatment significantly attenuated radiation-induced ROS accumulation, demonstrating that CYNC-2 effectively mitigates oxidative stress and protects normal lung epithelial cells from radiation injury.

 

This finding has been explicitly described in the Results (Section 2.1, page 3, line 100-105) as follows:

 

“In addition, to assess the oxidative-stress response, intracellular reactive oxygen species (ROS) levels were measured using a DCFDA-based fluorescence oxidative-stress assay in normal L132 cells. CYNC-2 pretreatment significantly attenuated radiation-induced ROS accumulation, confirming that CYNC-2 mitigates oxidative stress and supports its radioprotective potential in normal lung epithelial cells (Supplementary Figure 1).”

 

We also added a corresponding Figure Legend to clearly indicate the oxidative-stress testing method:

 

“Supplementary Figure 1. CYNC-2 attenuates radiation-induced intracellular reactive oxygen species (ROS) accumulation as assessed by a DCFDA-based oxidative-stress assay in L132 cells. … CYNC-2 treatment significantly reduced ROS levels in irradiated cells compared with those without CYNC-2 treatment, indicating its antioxidant and radioprotective effects.”

 

Furthermore, to highlight the biological implication of this finding, the Discussion section now includes the following statement (page 13, line 406):

 

“The observed reduction in radiation-induced ROS levels demonstrates that CYNC-2 effectively mitigates oxidative stress in normal lung epithelial cells, further supporting its role as a potential radioprotective and anti-inflammatory agent.”

 

Together, these revisions clarify that oxidative-stress testing was conducted and that the antioxidant capacity of CYNC-2 was directly evaluated and discussed within the manuscript.