## 5 Conclusions  
This study evaluated critical parameters related to the expectorant effects on PM\({}_{2.5}\)-induced sub-acute pulmonary injuries in Balb/c mice. The results exhibited that oral administration of AJ from 400 to 100 mg/kg provides significant pulmonary protective effects against PM\({}_{2.5}\)-induced damage, excessive mucus production, and respiratory acidosis. These protective effects are likely due to AJ's strong anti-inflammatory and mucolytic expectorant activities, as evidenced by elevated levels of lung ACh and substance P, along with the suppression of MUC5B and MUC5 AC mRNA expressions. The impact of AJ at 400 mg/kg was similar to that of AX (250 mg/kg) under the experimental conditions. Exposure to PM\({}_{2.5}\) resulted in increased phenol red secretions, lung congestion, elevated levels of ACh, and substance P, and the upregulation of mucus production genes, leading to inflammatory lung injuries and respiratory acidosis. However, these negative effects were significantly and dose-dependently mitigated by a 10-day AJ administration. Notably, AJ at 400 mg/kg exhibited effective inhibition of pulmonary injuries, similar to AX at 250 mg/kg, via anti-inflammatory pathways. Additionally, the AJ administration led to increased phenol red secretions, higher PAS+ cell counts, and thicker bronchial mucosa, with reduced MUC5 AC and MUC5B expression, indicating potent mucolytic expectorant activity. These findings highlight the possibility that AJ may be a promising candidate for developing alternative respiratory health products or functional food ingredients, based on its demonstrated dose-dependent pulmonary protective properties.  
The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app15010099/s1](https://www.mdpi.com/article/10.3390/app15010099/s1)]([https://www.mdpi.com/article/10.3390/app15010099/s1](https://www.mdpi.com/article/10.3390/app15010099/s1)): Table S1. Specific conditions for HPLC analysis; Table S2. Oligonucleotides used for quantitative RT-PCR; Table S3. Primary antisera and detection kits used for immunohistochemistry; Figure S1. HPLC chromatogram of atractylenolide III in _Attractylades japonica_ root extract; Figure S2. Changes in body weights of intact or PM\({}_{2.5}\)-treated mice with pulmonary injury.  
Conceptualization, Y.-S.K. and S.-K.K.; methodology, [PERSON] ([PERSON]), [PERSON] ([PERSON]), H.C., Y.-S.K. and S.-K.K.; software, S.-K.K.; validation, Y.-S.K., K.M.I.B., J.-S.C. and S.-K.K.; dermal analysis, [PERSON] ([PERSON]), S.M., Y.-S.K. and S.-K.K.; investigation, Y.-S.K. and S.-K.K.; data curation, [PERSON] ([PERSON]), S.M., J.L. ([PERSON]) and H.C.; writing--original draft preparation, [PERSON] ([PERSON]), S.M. and K.M.I.B.; writing--review and editing, S.M., Y.-S.K., K.M.I.B., J.-S.C. and S.-K.K.; visualization, Y.-S.K., K.M.I.B., J.-S.C. and S.-K.K.; supervision, Y.-S.K., J.-S.C. and S.-K.K.; project administration, S.-K.K.; funding acquisition, S.-K.K. All authors have read and agreed to the published version of the manuscript.  
This work was supported by a research grant from the Gyeongsang National University in 2024.  
The animal study was approved by the Animal Experimentation Ethics Committee of Daegu Haany University [Approval No.: DHU2022-016; Approved on: 22 February 2022].  
Not applicable.  
The original contributions presented in this study are contained within the article and Supplementary Materials; further inquiries can be directed to the corresponding authors.  
Authors [PERSON] and [PERSON] were employed by the company GAPI BIO Co., Ltd., Republic of Korea. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.