Alogliptin/Amentoflavone Combination Mitigates Bleomycin-Induced Lung Fibrosis: The Role of Oxidative Stress, TXNIP-Mediated Pyroptosis, and Autophagy/Apoptosis Balance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript explores the combination of alogliptin and amentoflavone as a potential treatment for bleomycin-induced lung fibrosis. This is interesting research. However, several sections require further clarification to improve the overall clarity and scientific rigor. 

Line 80 – 111: What is the scientific justification for combining alogliptin and amentoflavone? Are there any previous in vitro or in vivo studies investigating this combination, particularly in bleomycin-induced lung fibrosis models? In addition, it would be important to discuss the safety profile of these compounds, including any known toxicity in humans, as well as potential drug–drug interactions when used in combination.

Line 240-258: Figure 10 and Figure 11: No standard deviation?

Line 517-527: How was it confirmed that bleomycin administration successfully induced pulmonary fibrosis in the mice?

Line 528-542: Was there a treatment control group included in the animal study, such as one receiving a standard therapeutic regimen? How were the drugs prepared prior to administration? Please also specify the dosing details for the combination of alogliptin + amentoflavone.

Comments on the Quality of English Language

The manuscript would benefit from improved sentence structure, as the frequent use of overly long sentences (especially in the discussion) reduces readability and makes the authors’ arguments difficult to follow. 

Author Response

Dear Reviewer,

Below are our considerations about your comments. Once again, thank you very much for your contribution, which was essential to substantially improving our manuscript.

Comment 1: Line 80 – 111: What is the scientific justification for combining alogliptin and amentoflavone? Are there any previous in vitro or in vivo studies investigating this combination, particularly in bleomycin-induced lung fibrosis models? In addition, it would be important to discuss the safety profile of these compounds, including any known toxicity in humans, as well as potential drug–drug interactions when used in combination.

Response: We appreciate the reviewer’s insightful comment regarding the rationale for combining alogliptin and amentoflavone. The scientific justification for this combination lies in their complementary mechanisms of action. Alogliptin, a DPP-4 inhibitor, has been shown to exert anti-inflammatory and anti-fibrotic effects beyond its glucose-lowering activity, partly through modulation of oxidative stress and apoptosis pathways. Amentoflavone, a naturally occurring biflavonoid, is recognized for its potent antioxidant, anti-inflammatory, and anti-fibrotic properties. By targeting overlapping but distinct pathways, we hypothesized that their combination could provide synergistic protection against bleomycin-induced lung fibrosis, particularly through regulation of TXNIP-mediated pyroptosis and the autophagy/apoptosis balance.

To the best of our knowledge, there are no prior in vitro or in vivo studies directly investigating the alogliptin/amentoflavone combination in bleomycin-induced lung fibrosis models. This novelty is one of the strengths of our work, as it explores a previously untested therapeutic strategy. However, both agents individually have been studied in various models of oxidative stress and fibrosis, which informed our rationale for combining them.

Regarding safety, alogliptin is an FDA-approved antidiabetic drug with a well-characterized safety profile in humans. Reported adverse effects are generally mild, including nasopharyngitis and headache, with rare cases of hepatotoxicity and pancreatitis. Amentoflavone, while not clinically approved as a drug, has been widely studied as a dietary flavonoid with low toxicity in preclinical models. Importantly, no major drug–drug interactions between alogliptin and flavonoids have been reported to date. Nevertheless, we acknowledge that further pharmacokinetic and toxicological studies are warranted to fully establish the safety of this combination in humans. We have now expanded the Discussion section to highlight the scientific rationale, novelty of the combination, and safety considerations, as follows:

"The rationale for combining alogliptin and amentoflavone stems from their complementary pharmacological actions. Alogliptin, a clinically approved DPP-4 inhibitor, has demonstrated anti-inflammatory and anti-fibrotic effects beyond glycemic control, partly through modulation of oxidative stress and apoptotic pathways. Amentoflavone, a naturally occurring biflavonoid, is recognized for its potent antioxidant and anti-inflammatory properties, with reported efficacy in models of fibrosis. Although no previous studies have directly examined the alogliptin/amentoflavone combination in bleomycin-induced lung fibrosis, the individual protective effects of each agent against oxidative stress and tissue injury provided the scientific basis for exploring their synergistic potential. These findings may align with previous reports which suggested that the combination of DPP 4 inhibitors and flavonoids can exert beneficial tissue protective effects irrespective of the glycemic control [60]. This may be attributed to the DPP-4 inhibitory capacity of amentoflavone, which may add a synergistic effect to alogliptin [61]. In addition, amentoflavone itself inhibits CYP3A4 enzyme, which represents the primary metabolizing route of alogliptin, thereby increasing its bioavailability and efficacy [62, 63].

The recent research efforts have proven that alogliptin has a well-established safety profile in humans, with adverse effects generally mild and infrequent [64], while amentoflavone has been widely studied as a dietary flavonoid with low toxicity in preclinical models [65]. To date, no significant drug–drug interactions between alogliptin and flavonoids have been reported. Nevertheless, further pharmacokinetic and toxicological investigations are warranted to fully establish the safety of this combination in the clinical settings."

References:

60. S. Dhankhar, S. Mahajan, S. Chauhan, M. Saini, N. Garg, T.G. Singh, Herbal DPP-4 Inhibitors: Comprehensive Review of their Effectiveness, Safety and Environmental Fate in Diabetes Mellitus, Current Bioactive Compounds (2024).
61. H. Lee, S. Cho, S.-Y. Kim, J. Ju, S.W. Lee, S. Choi, H. Li, R. Piao, H.-Y. Park, T.-S. Jeong, Amentoflavone-enriched Selaginella rossii Warb. suppresses body weight and hyperglycemia by inhibiting intestinal lipid absorption in mice fed a high-fat diet, Life 12(4) (2022) 472.
62. A.V. Singh, Potential of amentoflavone with antiviral properties in COVID-19 treatment, Asian Biomedicine: Research, Reviews and News 15(4) (2021) 153.
63. X.W. Chen, Z.X. He, Z.W. Zhou, T. Yang, X. Zhang, Y.X. Yang, W. Duan, S.F. Zhou, An update on the clinical pharmacology of the dipeptidyl peptidase 4 inhibitor alogliptin used for the treatment of type 2 diabetes mellitus, Clinical and Experimental Pharmacology and Physiology 42(12) (2015) 1225-1238.
64. 64. X.V. Peng, G. Klingensmith, D.S. Hsia, Y. Xie, R. Czerniak, W.V. Tamborlane, A.S. Shah, A randomized phase 3 study evaluating the efficacy and safety of alogliptin in pediatric participants with type 2 diabetes mellitus, Diabetes Therapy 16(5) (2025) 865-883.
65. Z. Chen, Y. Shi, F. Zhong, K. Zhang, F. Zhang, S. Xie, Z. Cheng, Q. Zhou, Y-Y. Huang, H.B. Luo, Discovery of amentoflavone as a natural PDE4 inhibitor with anti-fibrotic effects, Chinese Chemical Letters 36(4) (2025) 109956.

Comment 2:

Line 240-258: Figure 10 and Figure 11: No standard deviation?

Response: Thank you very much for your insightful comment. I am sorry for this non-intended mistake. Standard deviation was added to figures 10 and 11.   

Comment 3: Line 517-527: How was it confirmed that bleomycin administration successfully induced pulmonary fibrosis in the mice?

Response: Thank you very much for this insightful and important comment. Pulmonary fibrosis was induced by a well-established commonly used experimental model described by Yang et al. (2023). Briefly, a single intratracheal instillation of bleomycin (BLM, 2.5 mg/kg) prepared as the sulfate salt in 0.1 mL of sterile 0.9% saline solution, administered under thiopental sodium (60 mg/kg, intraperitoneally) anesthesia, thereby minimizing distress to the animals. A midline cervical incision was performed to expose the trachea, and the BLM solution was carefully instilled into the tracheal lumen. Following instillation, animals were maintained in a vertical position and gently rotated several times to facilitate homogeneous distribution of BLM throughout the lung parenchyma, as commonly described in intratracheal bleomycin models. To close the incision, standard surgical suturing methods were used. Pulmonary fibrosis induction by bleomycin was confirmed through a combination of histopathological and biochemical assessments. Specifically, lung tissue sections were examined using hematoxylin and eosin (H&E) and Masson’s trichrome staining, which revealed alveolar structural distortion, inflammatory cell infiltration, and collagen deposition characteristic of fibrotic remodeling. In addition, hydroxyproline content was quantified as a biochemical marker of collagen accumulation. These findings collectively verified that bleomycin administration successfully induced pulmonary fibrosis in our mice, consistent with previously established intratracheal bleomycin models.

References:

1. Yang, X.-J. Huang, Z. Chen, A.-L. Xu, H. Zhou, X.-L. Bi, P.-Y. Yan, Y. Xie, A novel quantification method of lung fibrosis based on Micro-CT images developed with the optimized pulmonary fibrosis mice model induced by bleomycin, Heliyon 9(3) (2023).

Comment 4: Line 528-542: Was there a treatment control group included in the animal study, such as one receiving a standard therapeutic regimen? How were the drugs prepared prior to administration? Please also specify the dosing details for the combination of alogliptin + amentoflavone.

Response: We appreciate the reviewer’s insightful comment. In our study, a treatment control group was included for comparison. Specifically, in addition to the bleomycin-only group, animals were treated with the combination of alogliptin and amentoflavone, as well as with each compound individually, to evaluate their relative efficacy. No standard therapeutic regimen (such as pirfenidone or nintedanib) was included, as the primary aim was to investigate the novel combination therapy.

Regarding drug preparation, alogliptin and amentoflavone were freshly prepared before administration. Both alogliptin and amentoflavone were suspended in 0.5% CMC solution to ensure proper solubility and bioavailability. Both preparations were administered orally by oral gavage.

For dosing, alogliptin was given at 30 mg/kg/day and amentoflavone at 50 mg/kg/day, based on previously published pharmacological studies (Amano et al., 2018 and Hou et al., 2022, respectively). The combination group received both agents concurrently at the same doses for the duration of the experiment. These details have now been clarified in the revised manuscript to ensure transparency and reproducibility.

References:

213. Amano, S. Tsuchiya, M. Imai, K. Tohyama, J. Matsukawa, O. Isono, H. Yasuno, K. Enya, E. Koumura, H. Nagabukuro, Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis, Biochemical and Biophysical Research Communications 497(1) (2018) 207-213.
214. Hou, M. Yang, K. Yan, X. Fan, X. Ci, L. Peng, Amentoflavone ameliorates carrageenan-induced pleurisy and lung injury by inhibiting the NF-κB/STAT3 pathways via Nrf2 activation, Frontiers in pharmacology 13 (2022) 763608.

Comment 5: The manuscript would benefit from improved sentence structure, as the frequent use of overly long sentences (especially in the discussion) reduces readability and makes the authors’ arguments difficult to follow.

Response: We thank the reviewer for this valuable observation. We acknowledge that several sentences in the discussion section were overly long, which may have reduced clarity and readability. In the revised manuscript, we have carefully edited the text to improve sentence structure. Specifically, we shortened complex sentences, divided lengthy arguments into smaller, more concise statements, and ensured that each paragraph presents a clear and focused idea. These revisions enhance the flow of the discussion and make our arguments easier to follow.

Reviewer 2 Report

Comments and Suggestions for Authors

In regards to manuscript ID pharmaceuticals-4271205. This is a very good manuscript and the results are quite exciting. There were some issues with the manuscript that are easily fixed. There are some areas that would be clarified by addition of a short statement that indicates why each set of results are important in relation to development of fibrosis or need to be checked and clarified:

1. (Section 2.2) Comparative effect of different treatments on the total and differential leucocytic count in BALF :The comparative effect of different treatments on the total and differential leucocytic count in BALF is related to elevated neutrophil and lymphoctyte and reduction in mac.  The increase in neutrophils and lymphocytes, and the complex modulation of macrophages (often referred to as a "reduction in alveolar macrophages" early on, followed by functional polarization) are central to this process. Did you look at macrophage type? Changes in alveolar macrophage numbers in fibrosis in important. While total macrophage numbers might initially seem low due to death or migration, the "reduction in macrophages" typically refers to the depletion of resident alveolar macrophages and a subsequent shift in macrophage populations that favors fibrosis development. Please describe this process in the manuscript.  These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.
2. (Section 2.5) Comparative effect of different treatments on modulation of lung tissue SIRT1, Nrf2, and HO-1: The comparative effect of different treatments on modulating SIRT1, Nrf2, and HO-1 in bleomycin-induced pulmonary fibrosis is crucial because these proteins represent a master signaling axis that controls the balance between oxidative stress, inflammation, and fibrotic tissue remodeling. Bleomycin induces lung fibrosis by suppressing this pathway, leading to massive ROS production, cell senescence, and collagen deposition. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.
3. (Section 2.7) Comparative effect of the different treatments on the lung tissue TXNIP/NLRP3 inflammasome/gasdermin D axis: The effect of bleomycin on the TXNIP/NLRP3 inflammasome/gasdermin D (GSDMD) axis is critical in pulmonary fibrosis because this pathway drives the transition from acute lung injury to chronic fibrosis by triggering extreme inflammation and cellular pyroptosis. Bleomycin induces reactive oxygen species (ROS), which activates the TXNIP (Thioredoxin-interacting protein) to activate the NLRP3 inflammasome, leading to caspase-1 activation and cleavage of Gasdermin D, resulting in cell death and release of pro-fibrotic factors that cause irreversible lung scarring. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.
4. (Sections 2.10 and 2.11) The increased effect of bleomycin on carbachol-induced contraction of tracheal smooth muscles is important in bleomycin-induced fibrosis because it demonstrates that the disease causes functional airway remodeling and airway hyperresponsiveness (AHR), not just parenchymal lung fibrosis. This finding highlights that the inflammatory and remodeling processes extend to the airways, leading to increased smooth muscle sensitivity and enhanced contraction. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported. The same is true for Section 2.11. Explain why the results are so important!!!
5. (Section 2.12) Histopathological results: This section is excellent!!!!!The images clearly show the changes due to fibrosis. Wonderful immunoprecipitation images.
6. Figure 13 F though does not make sense to me. The fibrotic score of the control should be low and for Bleomycin should be high. Please check this or clarify the figure results and the scale.
7. Discussion is very good but providing one or two sentences in each section that describes the importance of each set of results would add significantly to the overall clarity and strength of the manuscript.

Author Response

Dear Reviewer,

Below are our considerations about your comments. Once again, thank you very much for your contribution, which was essential to substantially improving our manuscript.

Comment 1: (Section 2.2) Comparative effect of different treatments on the total and differential leucocytic count in BALF: The comparative effect of different treatments on the total and differential leucocytic count in BALF is related to elevated neutrophil and lymphocyte and reduction in macrophages.  The increase in neutrophils and lymphocytes, and the complex modulation of macrophages (often referred to as a "reduction in alveolar macrophages" early on, followed by functional polarization) are central to this process. Did you look at macrophage type? Changes in alveolar macrophage numbers in fibrosis are important. While total macrophage numbers might initially seem low due to death or migration, the "reduction in macrophages" typically refers to the depletion of resident alveolar macrophages and a subsequent shift in macrophage populations that favors fibrosis development. Please describe this process in the manuscript.  These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.

Response: Thank you very much for your valuable comment. We thank the reviewer for highlighting this important point. In our study, we primarily reported total and differential leukocyte counts in BALF, noting the increase in neutrophils and lymphocytes alongside a reduction in macrophages. We agree that the dynamics of macrophage populations are critical in fibrosis development. While we did not perform specific phenotypic characterization of macrophage subtypes (e.g., resident alveolar vs. recruited monocyte-derived macrophages, or M1/M2 polarization), the observed reduction in macrophages likely reflects depletion of resident alveolar macrophages, followed by a shift toward pro-fibrotic macrophage populations, as described in the literature. We have expanded the "Discussion" section to clarify this process, emphasizing that the apparent reduction in macrophages is not simply a numerical decrease but rather a complex modulation involving death, migration, and functional polarization. This shift contributes to the fibrotic process by promoting extracellular matrix deposition and sustaining inflammation. By elaborating on these mechanisms, we aim to better contextualize the importance of our findings and highlight how the alogliptin/amentoflavone combination may influence macrophage dynamics in addition to neutrophil and lymphocyte recruitment. This was expressed in the "Discussion" section as follows:

"The observed reduction in macrophages within BALF following BLM administration shouldn't be interpreted simply as a numerical decline. Rather, it reflects a dynamic process that involves depletion of the resident alveolar macrophages, migration of subsets, and functional polarization (Libório-Ramos et al., 2023). Early after injury, alveolar macrophages undergo apoptosis, leading to an apparent reduction in their total counts. Subsequently, monocyte-derived macrophages are recruited and undergo polarization towards pro-fibrotic phenotypes, which contribute to extracellular matrix deposition and sustained inflammation. This shift in macrophage populations is a critical driver of fibrosis progression (Zhao et al., 2025). While we quantified the total macrophage number in the current study, the findings align with this established paradigm, suggesting that the alogliptin/amentoflavone combination may modulate macrophage dynamics in addition to reducing neutrophil and lymphocyte infiltration. By influencing both innate and adaptive immune responses, alogliptin/amentoflavone combination may restore the balance between the inflammatory and reparative processes, thereby mitigating BLM-induced fibrotic remodeling."

References:

Libório-Ramos, S., Barbosa-Matos, C., Fernandes, R., Borges-Pereira, C. and Costa, S., 2023. Interstitial macrophages lead early stages of bleomycin-induced lung fibrosis and induce fibroblasts activation. Cells, 12(3), p.402.

Zhao, X., Li, Y., Yang, S., Chen, Y., Wu, K., Geng, J., ... & Wang, C. (2025). Orderly regulation of macrophages and fibroblasts by Axl in bleomycin‐induced pulmonary fibrosis in mice. Journal of Cellular and Molecular Medicine, 29(1), e70321.

Comment 2: (Section 2.5) Comparative effect of different treatments on modulation of lung tissue SIRT1, Nrf2, and HO-1: The comparative effect of different treatments on modulating SIRT1, Nrf2, and HO-1 in bleomycin-induced pulmonary fibrosis is crucial because these proteins represent a master signaling axis that controls the balance between oxidative stress, inflammation, and fibrotic tissue remodeling. Bleomycin induces lung fibrosis by suppressing this pathway, leading to massive ROS production, cell senescence, and collagen deposition. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.

Response: We thank the reviewer for this insightful comment. We agree that the comparative modulation of SIRT1, Nrf2, and HO-1 is central to understanding the impact of our findings. In our study, bleomycin administration suppressed this protective signaling axis, leading to excessive ROS generation, cellular senescence, and collagen deposition, which are hallmarks of pulmonary fibrosis. The alogliptin/amentoflavone combination significantly restored the expression of SIRT1, Nrf2, and HO-1, thereby reinforcing antioxidant defenses, attenuating inflammatory signaling, and limiting fibrotic remodeling. We have now expanded the "Discussion" section in the revised manuscript to describe this process in details, emphasizing that the SIRT1/Nrf2/HO-1 axis represents a master regulator of redox homeostasis and tissue integrity in the pulmonary tissues as follows:

"Bleomycin-induced pulmonary fibrosis is closely linked to suppression of the SIRT1/Nrf2/HO-1 signaling axis, which normally functions as a master regulator of oxida-tive stress, inflammation, and tissue remodeling [40]. SIRT1, a NAD⁺-dependent deacetylase, enhances the activity of Nrf2, a transcription factor that governs antioxidant defense mechanisms [41]. Once activated, Nrf2 promotes the expres-sion of HO-1, an enzyme with a well-proven strong cytoprotective and anti-inflammatory properties in the pulmonary tissues [42]. Together, this signaling cascade reduces ROS, limits fibroblast activation, and mitigates extracellular matrix deposition, thereby slowing the progression of fibrotic remodeling in the lungs [40]. BLM disrupts this pathway, resulting in excessive ROS generation, cellular senescence, and uncontrolled collagen deposition [43]. The comparative effect of different treatments on modulating SIRT1, Nrf2, and HO-1 is therefore crucial, as restoration of this axis can rebalance redox homeostasis, attenuate inflammatory signaling, and reduce fibrotic remodeling. Consistent with this concept, intratracheal BLM administration in the current study was associated with a significant decline in SIRT1, Nrf2, and HO-1 content of the pulmonary tissues relative to the control group. Notably, treatment with alogliptin and/or amentoflavone significantly increased the lung SIRT1, Nrf2, and HO-1 content relative to the BLM group. This reduction may be interpreted as a consequence of their antioxidant actions by attenuating BLM induced ROS generation, and hence augmenting the antioxidant potential of both alogliptin and amentoflavone [39]. These findings highlight the importance of targeting the SIRT1/Nrf2/HO-1 axis as a strategy to mitigate fibrosis progression and support the translational relevance of our results."

References:

39. A.E. Alsemeh, D.M. Abdullah, Protective effect of alogliptin against cyclophosphamide-induced lung toxicity in rats: Impact on PI3K/Akt/FoxO1 pathway and downstream inflammatory cascades, Cell and tissue research 388(2) (2022) 417-438.
40. Q. Hua, L. Ren, The SIRT1/Nrf2 signaling pathway mediates the anti-pulmonary fibrosis effect of liquiritigenin, Chinese medicine 19(1) (2024) 12.
41. Y. Bian, D. Yin, P. Zhang, L. Hong, M. Yang, Zerumbone alleviated bleomycin-induced pulmonary fibrosis in mice via SIRT1/Nrf2 pathway, Naunyn-Schmiedeberg's Archives of Pharmacology 397(11) (2024) 8979-8992.
42. X. Wang, Q. Wang, P. Zhou, J. Zhang, H. Su, F. Liu, J. Wu, F. Xiao, L. Liu, L. Han, Rhoifolin improves bleomycin-induced fibrosis in vivo and cell damage in vitro both related to NRF2/HO-1 pathway, BMC Pulmonary Medicine 25(1) (2025) 117.
43. L.-H. Chien, J.-S. Deng, W.-P. Jiang, Y.-N. Chou, J.-G. Lin, G.-J. Huang, Evaluation of lung protection of Sanghuangporus sanghuang through TLR4/NF-κB/MAPK, keap1/Nrf2/HO-1, CaMKK/AMPK/Sirt1, and TGF-β/SMAD3 signaling pathways mediating apoptosis and autophagy, Biomedicine & Pharmacotherapy 165 (2023) 115080.

Comment 3: (Section 2.7) Comparative effect of the different treatments on the lung tissue TXNIP/NLRP3 inflammasome/gasdermin D axis: The effect of bleomycin on the TXNIP/NLRP3 inflammasome/gasdermin D (GSDMD) axis is critical in pulmonary fibrosis because this pathway drives the transition from acute lung injury to chronic fibrosis by triggering extreme inflammation and cellular pyroptosis. Bleomycin induces reactive oxygen species (ROS), which activates the TXNIP (Thioredoxin-interacting protein) to activate the NLRP3 inflammasome, leading to caspase-1 activation and cleavage of Gasdermin D, resulting in cell death and release of pro-fibrotic factors that cause irreversible lung scarring. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported.

Response: We thank the reviewer for emphasizing the importance of the TXNIP/NLRP3 inflammasome/Gasdermin D axis in the pathogenesis of pulmonary fibrosis. We agree that this pathway represents a critical driver of the transition from acute lung injury to chronic fibrosis. In our study, bleomycin administration markedly upregulated TXNIP expression, which in turn activated the NLRP3 inflammasome, leading to caspase-1 activation and cleavage of Gasdermin D. This cascade triggered pyroptotic cell death and the release of pro-inflammatory and pro-fibrotic mediators, thereby amplifying tissue injury and promoting irreversible scarring. We have expanded the "Discussion" section in the revised manuscript to describe this process in detail, highlighting how the alogliptin/amentoflavone combination attenuated TXNIP/NLRP3/GSDMD activation. By dampening ROS-driven inflammasome signaling and reducing pyroptosis, the combination therapy helped restore tissue homeostasis and limit fibrotic remodeling. These findings underscore the therapeutic relevance of targeting the TXNIP/NLRP3/GSDMD axis and provide a clearer explanation of the impact of our results as follows:

"As evidenced in the current study, the TXNIP/NLRP3 inflammasome/gasdermin D axis is a critical pathway that may effectively contribute to the development of bleomycin-induced lung fibrosis. TXNIP acts as a sensor of oxidative stress and facilitates the activation of the NLRP3 inflammasome, a multiprotein complex that drives inflammatory signaling [44]. Once activated, the NLRP3 inflammasome activates caspase-1, which in turn cleaves gasdermin D, which forms pores in the cell membrane, promoting pyroptotic cell death and the release of pro-inflammatory mediators [45]. Additionally, this cascade promotes fibroblast activation, ultimately leading to excessive deposition of extracellular matrix and fibrotic remodeling in the lungs [46].

A key consequence of the TXNIP/NLRP3 inflammasome/gasdermin D axis is the maturation and secretion of IL-1β and IL-18 that fuel the inflammatory environment in bleomycin-induced fibrosis. IL-1β enhances the recruitment of immune cells and stimulates fibroblast proliferation, while IL-18 contributes to the amplification of inflammatory signaling and tissue damage [47]. As depicted in the present study, these cytokines perpetuate a cycle of inflammation and fibrosis, worsening the pulmonary functions [48]. By linking oxidative stress to inflammasome activation and cytokine release, the TXNIP/NLRP3/gasdermin D axis serves as a central driver of the inflammatory and fibrotic responses in bleomycin-injured lungs [49].

In the present study, alogliptin treatment significantly reduced the expression of TXNIP, NLRP3 inflammasome, gasdermin D, IL-1β, and IL-18 compared with the BLM group. These findings align with evidence indicating that DPP 4 inhibitors exert potent anti-inflammatory and anti-pyroptotic actions independent of their glucose-lowering properties [50]. Similarly, amentoflavone in the current study produced a significant ab-rogation of the TXNIP/NLRP3/gasdermin D axis and was associated with a significant decline in IL 1β, and IL-18 levels in the lung tissues relative to the BLM group, aligning with prior reports that amentoflavone possesses a strong anti-pyroptotic property via inhibition of NLRP3 inflammasome and its downstream signals, which is also reflected on the pro inflammatory pathways [51]."

References:

44. T. Luo, X. Zhou, M. Qin, Y. Lin, J. Lin, G. Chen, A. Liu, D. Ouyang, D. Chen, H. Pan, Corilagin restrains NLRP3 inflammasome activation and pyroptosis through the ROS/TXNIP/NLRP3 pathway to prevent inflammation, Oxidative Medicine and Cellular Longevity 2022(1) (2022) 1652244.
45. X. Li, P. Zhang, Z. Yin, F. Xu, Z.-H. Yang, J. Jin, J. Qu, Z. Liu, H. Qi, C. Yao, Caspase-1 and Gasdermin D afford the optimal targets with distinct switching strategies in NLRP1b inflammasome-induced cell death, Research (2022).
46. S. Gairola, A. Sinha, R.K. Kaundal, Linking NLRP3 inflammasome and pulmonary fibrosis: mechanistic insights and promising therapeutic avenues: S. Gairola et al, Inflammopharmacology 32(1) (2024) 287-305.
47. T. Hoshino, M. Okamoto, Y. Sakazaki, S. Kato, H.A. Young, H. Aizawa, Role of proinflammatory cytokines IL-18 and IL-1β in bleomycin-induced lung injury in humans and mice, American journal of respiratory cell and molecular biology 41(6) (2009) 661-670.
48. Y. Huang, A. Wang, S. Jin, F. Liu, F. Xu, Activation of the NLRP3 inflammasome by HMGB1 through inhibition of the Nrf2/HO-1 pathway promotes bleomycin-induced pulmonary fibrosis after acute lung injury in rats, Allergologia et Immunopathologia 51(3) (2023) 56-67.
49. T.-T. Chen, F. Xiao, N. Li, S. Shan, M. Qi, Z.-Y. Wang, S.-N. Zhang, W. Wei, W.-Y. Sun, Inflammasome as an effective platform for fibrosis therapy, Journal of Inflammation Research (2021) 1575-1590.
50. A.A. Sedik, N.M.E.A. El-Nasr, W.K.B. Khalil, A.E.M.K. El-Mosallamy, Unravelling the mechanism by which vildagliptin and linagliptin inhibit pyroptosis in lung injury through the NLRP3 inflammatory pathway in type 1 diabetic rats, Scientific Reports 15(1) (2025) 20292.
51. S.U.N. Yalei, L.U.O. Meng, G.U.O. Changsheng, G.A.O. Jing, S.U. Kaiqi, C. Lidian, F. Xiaodong, Amentoflavone alleviates acute lung injury in mice by inhibiting cell pyroptosis, Journal of Southern Medical University 45(4) (2025) 692.

Comment 4: (Sections 2.10 and 2.11) The increased effect of bleomycin on carbachol-induced contraction of tracheal smooth muscles is important in bleomycin-induced fibrosis because it demonstrates that the disease causes functional airway remodeling and airway hyperresponsiveness (AHR), not just parenchymal lung fibrosis. This finding highlights that the inflammatory and remodeling processes extend to the airways, leading to increased smooth muscle sensitivity and enhanced contraction. Please describe this process in the manuscript. These results are important but there needs to be a better explanation of the impact of these results so that the importance of your results is supported. The same is true for Section 2.11. Explain why the results are so important!!!

Response: We thank the reviewer for this important observation. We agree that the increased effect of bleomycin on carbachol-induced contraction of tracheal smooth muscles is highly relevant, as it demonstrates that bleomycin-induced pulmonary fibrosis involves not only parenchymal remodeling but also functional airway remodeling and airway hyperresponsiveness (AHR). This finding highlights that the inflammatory and fibrotic processes extend to the airways, leading to enhanced smooth muscle sensitivity and exaggerated contractile responses. In the revised manuscript, we have expanded the discussion to describe this process in detail. Specifically, we emphasize that airway hyperresponsiveness is a critical manifestation of fibrosis, reflecting both structural changes in the airway wall and functional alterations in smooth muscle reactivity. By showing that the alogliptin/amentoflavone combination attenuated bleomycin-induced enhancement of carbachol contraction, our results suggest that the therapy not only mitigates parenchymal fibrosis but also protects against airway remodeling and dysfunction.

We have also clarified the importance of the findings in Section 2.11, underscoring that modulation of the vascular reactivity of the isolated pulmonary artery rings is a key therapeutic target. This explanation strengthens the impact of our results and supports the translational relevance of the combination therapy in addressing the vascular, parenchymal, and airway components of pulmonary fibrosis.

"In the present study, BLM instillation, aligned with prior findings of biochemical and histopathological injury, was correlated with considerable vascular and tracheal dysfunction. BLM significantly diminished the vascular reactivity of isolated pulmonary artery rings to KCl, PE, and carbachol. This was in the same line with Zaghloul et al. (2017) who attributed these effects to BLM-elicited impairment of the vasoconstrictor responsiveness. Likewise, in vitro assessment of tracheal smooth muscles revealed a significant decrease in contractile responses to carbachol. This agrees with Qin et al. (2022) who reported that these effects represent direct consequences to BLM-mediated impairment of the functions of airway smooth muscles. Conversely, treatment with alogliptin and/or amentoflavone in the current work effectively reversed these BLM-induced functional disturbances. These findings functionally reinforce the aforementioned biochemical and histopathological improvements achieved with alogliptin and/or amentoflavone administration. These results highlight the translational relevance of targeting the vascular, parenchymal, and airway components in pulmonary fibrosis."

References:

M.S. Zaghloul, R.A. Abdel-Salam, E. Said, G.M. Suddek, H.A.-R. Salem, Flavocoxid Improves Bleomycin-Induced Respiratory Dysfunction and Pulmonary Fibrosis; In vitro Study, World Journal of Pharmaceutical Sciences (2017) 143-151.

100. Qin, C. Jia, J. Liang, J. Chen, X. Liu, Z. Chao, H. Qin, Y. Yuan, Z. Liu, Z. Zhang, H. Dong, PEDF is an antifibrosis factor that inhibits the activation of fibroblasts in a bleomycin-induced pulmonary fibrosis rat model, Respiratory Research 23(1) (2022) 100.

Comment 5: (Section 2.12) Histopathological results: This section is excellent!!!!! The images clearly show the changes due to fibrosis. Wonderful immunoprecipitation images.

Response: We sincerely thank the reviewer for the encouraging feedback on our histopathological results. We are delighted that the images clearly conveyed the fibrotic changes and that the immunoprecipitation images were found to be of high quality. We aimed to present these findings in a clear and illustrative manner to strengthen the manuscript, and we greatly appreciate the reviewer’s recognition of this effort.

Comment 6: Figure 13 F though does not make sense to me. The fibrotic score of the control should be low and for Bleomycin should be high. Please check this or clarify the figure results and the scale.

Response: Thank you very much for your valuable comment which we totally agree with. I am so sorry for this non-intended mistake. This figure was revised and the mistake was corrected.

Comment 7: Discussion is very good but providing one or two sentences in each section that describes the importance of each set of results would add significantly to the overall clarity and strength of the manuscript.

Response: We thank the reviewer for this constructive recommendation. We agree that explicitly stating the importance of each set of results will enhance the clarity and strength of the manuscript. In the revised version, we have added one to two sentences at the end of each results subsection to highlight the biological and translational significance of the findings. For example, in the section on the leucocytic counts, we now emphasize how the modulation of neutrophils, lymphocytes, and macrophages reflects the therapeutic impact on inflammatory cell recruitment. Similarly, in the sections on oxidative stress markers, signaling pathways (SIRT1/Nrf2/HO-1, TXNIP/NLRP3/GSDMD), and airway hyperresponsiveness, we have clarified why these results are critical for understanding fibrosis progression and the protective role of the alogliptin/amentoflavone combination. These additions strengthen the narrative by connecting each experimental observation to its broader pathological and therapeutic relevance.