Review Reports - Adenophora divaricata Franch. & Sav. Attenuates Particulate Matter-Induced Inflammatory Responses in RAW264.7 Macrophage Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors identified a total of 30 metabolites in the negative ion mode and multiple compounds in the positive ion mode using UPLC-Q-TOF-MS; however, the manuscript does not clearly describe whether authentic standards were used to confirm compound identities. Providing validation using reference standards or MS/MS spectral matching scores would strengthen the reliability of the metabolite identification.
In the section describing the anti-inflammatory effects of AD in RAW264.7 cells, the authors evaluated TNF-α and IL-6 levels but did not measure IL-1β secretion at the cellular level, despite the study emphasizing the TXNIP/NLRP3 inflammasome pathway. Including IL-1β quantification in the cell culture experiments would better support the proposed inflammasome-related mechanism.
The manuscript reports that AD suppressed TXNIP and NLRP3 expression in PM-stimulated RAW264.7 cells; however, it remains unclear whether AD directly regulates TXNIP expression or acts indirectly through upstream oxidative stress pathways. Additional experiments examining ROS production or antioxidant signaling could help clarify this mechanism.
In the siRNA experiments, TXNIP knockdown reduced NLRP3 activation and inflammatory signaling. However, the manuscript does not report the efficiency of TXNIP knockdown (e.g., percentage reduction in protein or mRNA expression). Providing quantitative validation of knockdown efficiency would strengthen the interpretation of these results.
The study employed RAW264.7 macrophages as the in vitro model; however, particulate matter primarily affects airway epithelial cells in the respiratory tract. The authors should clarify the rationale for selecting macrophages instead of epithelial cell models such as BEAS-2B or A549 cells.
In the animal experiments, mice were exposed to PM for only seven days. Considering that particulate matter–related pulmonary diseases often involve chronic exposure, the authors should discuss whether this short-term model adequately reflects long-term inflammatory processes in the lung.
The manuscript indicates that AD reduced inflammatory cell counts in bronchoalveolar lavage fluid (BALF), but the differential cell types appear limited to macrophages, neutrophils, and “other cells.” A more detailed characterization of immune cell populations would provide better insight into the inflammatory response.
Histopathological analysis shows that AD alleviated alveolar wall thickening and inflammatory infiltration. However, the manuscript does not clearly describe the scoring system used to quantify airway inflammation. Providing detailed criteria or references for the histological scoring method would improve methodological transparency.
Although the study highlights the TXNIP/NLRP3 inflammasome pathway, the authors also examined NF-κB signaling in lung tissues. The relationship between NF-κB activation and TXNIP/NLRP3 regulation is not sufficiently discussed and should be clarified in the Discussion section.
The Discussion suggests that AD could serve as a potential therapeutic agent for PM-induced lung inflammation. However, the manuscript does not address issues such as pharmacokinetics, bioavailability, or safety of AD extracts in humans. Including a discussion of these translational limitations would improve the clinical relevance of the study.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please refer to the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the potential anti-inflammatory effects of the methanolic extract of Adenophora divaricata against particulate matter (PM)-induced pulmonary inflammation, focusing on the TXNIP/NLRP3 inflammasome pathway. However, based on the following issues, I believe the manuscript is not suitable for publication in its current form.

The major concerns are as follows:

Limited novelty
The involvement of the TXNIP/NLRP3 inflammasome in PM-induced inflammation has already been reported in several previous studies. The present study largely confirms previously known mechanisms and does not provide sufficiently novel mechanistic insights.

Insufficient mechanistic evidence
The authors conclude that AD exerts anti-inflammatory effects through suppression of the TXNIP/NLRP3 pathway. However, experiments demonstrating causality (e.g., knockdown, pathway inhibition, or rescue experiments) are lacking. Therefore, the current results only show an association rather than a clear mechanistic relationship.

Insufficient methodological description and reproducibility concerns
Important methodological details regarding the characterization of PM, exposure conditions, and the composition or standardization of the AD extract are not adequately described. These limitations raise concerns regarding experimental reproducibility.

Limited clinical relevance of the experimental models
The study relies mainly on RAW264.7 macrophage cells and a short-term mouse exposure model. These models are insufficient to represent the complexity of human respiratory diseases caused by chronic PM exposure.

Figures are difficult to interpret
The presentation of figures and quantitative data is unclear, and the key messages of the results are not well conveyed. Substantial improvement in figure organization and data presentation is necessary.

Author Response

Please refer to the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Title: Adenophora divaricata Franch. & Sav. Attenuates particulate matter-induced inflammatory responses in RAW264.7 macrophage cells.

In this paper, the authors study the anti-inflammatory effect of Adenophora in mouse lungs. After PM administration, mice exhibited a reduction in inflammatory parameters, including cytokines, and histopathological analysis revealed a marked attenuation of inflammatory responses in lung tissue. The authors conclude that TXNIP serves as a key regulatory factor and Adenophora may serve as a potential therapeutic agent for lung inflammation.

This article deserves admiration for the great work done, but I have some concerns. It could have been presented more accurately.

Abstract: First explain what particulate matter is.

“………we investigated the mechanisms associated with AD's activity against PM-induced lung inflammation…..” You must immediately say where.

".....focusing on the role of thioredoxin-interacting protein (TXNIP) and the inflammasome..." There is no connection between TXNIP and the mechanisms associated with AD.

Why was dexamethasone used orally and not intramuscularly?

How much does the dose of Adenophora used in mice correspond to that in humans?

To make this paper more interesting, it should include some molecular biology.

Adenophora may modulate the MAPK pathway and other pathways related to cellular inflammation. When kinases are phosphorylated, they induce the activation of transcription factors (AP-1, NF-κB) and the production of inflammatory mediators such as IL-1, TNF, IL-6, etc. Adenophora extracts may inhibit the MAPK cascade. In the light of these concepts, to make this paper more interesting for the readers of this important journal, the authors should expand a bit the discussion (or introduction). Below I report an interesting article that should be studied, incorporate the meaning and report it briefly in the discussion and in the list of references.

Saggini R, Pellegrino R. MAPK is implicated in sepsis, immunity, and inflammation. International Journal of Infection. 2024;8(3):100-104. (www.biolife-publisher.it).

The anti-inflammatory activity of Adenophora has also been linked to the modulation of the PI3Kβ–Akt–mTOR signaling pathway, one of the key intracellular networks regulating the immune response. Adenophora contains flavonoids, phenolic compounds, and other compounds that have regulatory effects on PI3Kβ. Again, here, we report an article which has been recently published that should be studied, incorporate the meaning and report briefly in the discussion or introduction, and in the list of the references.

Avivar-Valderas A. Inhibition of PI3Kβ and mTOR influence the immune response and the defense mechanism against pathogens. International Journal of Infection. 2023;7(2):46-49. (www.biolife-publisher.it).

I believe these suggestions are important for improving this paper. Without these corrections the paper cannot be published. So I recommend minor revision.