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Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification

  • Authors:
    • Quan Li
    • Lili Ding
    • Haijun Sun
    • Weichao Ding
    • Mengmeng Wang
    • Zhaorui Sun
    • Congxue Ma
  • View Affiliations / Copyright

    Affiliations: Intensive Care Unit, Suqian First Hospital, Suqian, Jiangsu 223800, P.R. China, Department of Emergency Medicine, The Affiliated Hospital of Xuzhou University, Xuzhou, Jiangsu 221002, P.R. China, Department of Emergency Medicine, Medical School of Nanjing University Affiliated Jinling Hospital, Nanjing, Jiangsu 210002, P.R. China, Department of Anesthesiology, Siyang Hospital, Suqian, Jiangsu 223700, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 83
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    Published online on: January 28, 2026
       https://doi.org/10.3892/etm.2026.13077
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Abstract

In the present study, a method combining network pharmacology prediction and experimental verification was used to clarify the protective mechanisms and therapeutic benefits of carnosic acid (CA) against acute respiratory distress syndrome (ARDS). Network pharmacology analysis was initially carried out to identify key targets and pathways for CA in ARDS. In vitro studies were performed using mouse alveolar macrophages (MH‑S cells) to examine the impact of CA on pyroptosis and oxidative stress triggered by lipopolysaccharide (LPS)/ATP. Pyroptosis was evaluated through Annexin V/PI staining, measurement of IL‑1β and IL‑18 levels, and expression analysis of pyroptosis‑related genes. Assessment of oxidative stress involved measuring malondialdehyde, myeloperoxidase and superoxide dismutase levels, as well as intracellular reactive oxygen species (ROS) levels. Western blotting and immunofluorescence analysis were employed to investigate nuclear factor erythroid 2‑related factor 2 (Nrf2) expression and nuclear translocation. Additionally, an in vivo ARDS mouse model was developed to further validate the therapeutic efficacy of CA through assessment of lung injury, inflammation and oxidative stress markers. Network pharmacology profiling revealed Nrf2 as the pivotal molecular target of CA, with pathway enrichment analysis highlighting its involvement in ROS homeostasis and programmed cell death pathways. Molecular docking analysis demonstrated a stable binding affinity between CA and Nrf2. In vitro experimental analysis revealed that CA notably reduced LPS/ATP‑induced pyroptosis and oxidative stress, therefore reducing apoptosis, downregulating pyroptosis‑related gene expression, and enhancing Nrf2 expression and nuclear translocation in MH‑S cells. In vivo validation in the murine ARDS model demonstrated that CA treatment effectively mitigated pulmonary pathological damage, suppressed pyroptotic signaling pathways and reduced oxidative stress biomarkers. The present integrative study demonstrated that CA may protect against ARDS by targeting Nrf2, suppressing oxidative stress and pyroptosis. These findings provide a mechanistic understanding of CA and warrant further translational research for clinical application in ARDS management.

View Figures

Figure 1

Network pharmacological analysis of
CA on ARDS. (A) Two-dimensional structure of CA retrieved from the
PubChem database. (B) Venn diagram exhibiting common targets
between CA and ARDS. (C) Functional enrichment analysis of target
genes through Gene Ontology analysis, encompassing BP, CC and MF.
(D) Kyoto Encyclopedia of Genes and Genomes pathway analysis of
target genes. (E) Protein-protein interaction network diagram of
overlapping target genes. In the PPI network, individual nodes
symbolize proteins, while the connecting edges represent the
relationships between these proteins. CA, carnosic acid; ARDS,
acute respiratory distress syndrome; BP, biological processes; MF,
molecular functions; CC, cellular components.

Figure 2

Molecular docking and molecular
dynamics simulation of CA and Nrf2. (A) RMSD calculation of the
complex. (B) Rg of the complex. (C) RMSF calculation of the protein
in the complex. (D) Distance between the docking site of the
protein and the small molecule ligand (dock site-ligand). (E)
Buried SASA between the small molecule and the protein. (F)
Superposition of the simulated conformations. (G) VDW and ELE
binding energies between the small molecule and the protein. (H)
Contribution of amino acid binding energy. (I) Hbond number. (J)
Interaction between the protein and the small molecule. RMSD, root
mean square deviation; Rg, radius of gyration; RMSF, root mean
square fluctuation; SASA, solvent-accessible surface area; ELE,
electrostatic energy; VDW, van der Waals.

Figure 3

CA inhibits pyroptosis in
LPS/ATP-stimulated MH-S cells. (A) Cytotoxicity of CA evaluated by
a Cell Counting Kit-8 assay. (B) LDH release in the supernatant
following CA treatment. (C) Percentage of PI-positive cells
following CA treatment. (D) Annexin V-FITC (green)
double-fluorescent staining representing apoptosis levels and PI
(red) double-fluorescent staining representing cell necrosis
levels. Scale bar, 100 µm. PI-positive cell rate was calculated
using ImageJ software. (E) Representative bands and (F) analysis of
protein expression of NLRP3, ASC, CASP1 and GSDMD detected by
western blotting. (G) mRNA expression levels of NLRP3, ASC, CASP1
and GSDMD detected by reverse transcription-quantitative PCR. (H)
Production of IL-18 and IL-1β in the supernatant measured by ELISA.
Data are presented as the mean ± SD of three independent
experiments. ##P<0.01 vs. control;
**P<0.01, ****P<0.0001 vs. LPS/ATP;
▲▲P<0.01 vs. CA (10 µM). CA, carnosic acid; LPS,
lipopolysaccharide; LDH, lactate dehydrogenase; NLRP3, NLR family
pyrin domain containing 3; ASC, apoptosis-associated speck-like
protein containing a CARD; CASP1, caspase-1; GSDMD, gasdermin D;
OD, optical density.

Figure 4

CA activates Nrf2 and improves the
oxidative stress in LPS/ATP-stimulated MH-S cells. Levels of (A)
MDA, (B) MPO and (C) SOD in the supernatant measured by ELISA. (D
and E) ROS generation of MH-S cells detected by
2',7'-dichlorodihydrofluorescein diacetate cellular ROS detection
assay, with fluorescence intensity calculated using ImageJ
software. Scale bar, 100 µm. (F) Representative bands and (G)
analysis of protein expression of Nrf2 and HO-1 detected by western
blotting. (H) Nuclear translocation of the Nrf2 subunit determined
by an immunofluorescence assay. Scale bar, 20 µm. Data are
presented as the mean ± SD of three independent experiments.
##P<0.01, ####P<0.0001 vs. control;
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001 vs. LPS/ATP
group; ▲P<0.05, ▲▲P<0.01 vs. CA (10
µM). CA, carnosic acid; Nrf2, nuclear factor erythroid 2-related
factor 2; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MDA,
malondialdehyde; MPO, myeloperoxidase; SOD, superoxide dismutase;
ROS, reactive oxygen species; OD, optical density.

Figure 5

CA attenuates LPS-induced acute
respiratory distress syndrome in mice. (A) PaO2 and (B)
PaCO2 data of each group. (C)
PaO2/FiO2 ratio of each group. (D)
Histopathological variation in lung tissues determined using
H&E staining. Scale bar, 50 µm. (E) Lung injury score. (F) Lung
W/D ratio assessed using histological sections. (G) Survival rate
for mice observed twice daily for 7 days. Data are presented as the
mean ± SD of three independent experiments. ##P<0.01
vs. control; *P<0.05, **P<0.01 vs. LPS;
▲P<0.05, ▲▲P<0.01 vs. CA (40 mg/kg).
CA, carnosic acid; LPS, lipopolysaccharide; ns, not significant;
PaO2, partial pressure of oxygen; PaCO2,
partial pressure of carbon dioxide; FiO2, fraction of
inspired oxygen; W/D, wet dry.

Figure 6

CA inhibits NLRP3
inflammasome-mediated pyroptosis in LPS-induced acute respiratory
distress syndrome in mice. (A) Representative bands and (B)
analysis of protein expression of NLRP3, ASC, CASP1 and GSDMD
detected by western blotting. (C) mRNA expression levels of NLRP3,
ASC, CASP1 and GSDMD detected by reverse transcription-quantitative
PCR. (D) Analysis and (E) immunohistochemical staining showing the
positive rate of GSDMD protein expression in lung tissues of mice
in each group. Scale bar, 50 µm. Production of (F) IL-18 and (G)
IL-1β in the BALF measured by ELISA. (H) LDH release in the BALF.
Data are presented as the mean ± SD of three independent
experiments. ##P<0.01 vs. control;
*P<0.05, **P<0.01 vs. LPS;
▲P<0.05, ▲▲P<0.01 vs. CA (40 mg/kg).
CA, carnosic acid; NLRP3, NLR family pyrin domain containing 3;
LPS, lipopolysaccharide; ASC, apoptosis-associated speck-like
protein containing a CARD; CASP1, caspase-1; GSDMD, gasdermin D;
LDH, lactate dehydrogenase; BALF, bronchoalveolar lavage fluid; ns,
not significant; OD, optical density.

Figure 7

CA exerts an anti-oxidative effect in
LPS-induced acute respiratory distress syndrome. Levels of (A) MDA,
(B) MPO and (C) SOD in the bronchoalveolar lavage fluid measured by
ELISA. (D) Representative bands and (E) analysis of protein
expression of Nrf2 and HO-1 detected by western blotting. (F) mRNA
expression of Nrf2 and HO-1 detected by reverse
transcription-quantitative PCR. Data are presented as the mean ± SD
of three independent experiments. ##P<0.01 vs.
control; *P<0.05, **P<0.01 vs. LPS;
▲▲P<0.01 vs. CA (40 mg/kg). CA, carnosic acid; LPS,
lipopolysaccharide; MDA, malondialdehyde; MPO, myeloperoxidase;
SOD, superoxide dismutase; Nrf2, nuclear factor erythroid 2-related
factor 2; HO-1, heme oxygenase-1; ns, not significant; OD, optical
density.

Figure 8

CA protects against acute respiratory
distress syndrome by targeting Nrf2, suppressing oxidative stress
and pyroptosis. LPS activates the NLRP3/CASP1/GSDMD signaling
pathway to induce pyroptosis. CA inhibits oxidative stress and
pyroptosis by activating Nrf2 to reduce lung injury. CA, carnosic
acid; LPS, lipopolysaccharide; NLRP3, NLR family pyrin domain
containing 3; ASC, apoptosis-associated speck-like protein
containing a CARD; CASP1, caspase-1; HO-1, heme oxygenase-1, MPO,
myeloperoxidase; MDA, malondialdehyde; SOD, superoxide dismutase;
GSDMD, gasdermin D; Nrf2, nuclear factor erythroid 2-related factor
2.
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Copy and paste a formatted citation
Spandidos Publications style
Li Q, Ding L, Sun H, Ding W, Wang M, Sun Z and Ma C: <p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>. Exp Ther Med 31: 83, 2026.
APA
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., & Ma, C. (2026). <p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>. Experimental and Therapeutic Medicine, 31, 83. https://doi.org/10.3892/etm.2026.13077
MLA
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., Ma, C."<p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>". Experimental and Therapeutic Medicine 31.3 (2026): 83.
Chicago
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., Ma, C."<p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>". Experimental and Therapeutic Medicine 31, no. 3 (2026): 83. https://doi.org/10.3892/etm.2026.13077
Copy and paste a formatted citation
x
Spandidos Publications style
Li Q, Ding L, Sun H, Ding W, Wang M, Sun Z and Ma C: <p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>. Exp Ther Med 31: 83, 2026.
APA
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., & Ma, C. (2026). <p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>. Experimental and Therapeutic Medicine, 31, 83. https://doi.org/10.3892/etm.2026.13077
MLA
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., Ma, C."<p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>". Experimental and Therapeutic Medicine 31.3 (2026): 83.
Chicago
Li, Q., Ding, L., Sun, H., Ding, W., Wang, M., Sun, Z., Ma, C."<p>Exploring the protective mechanisms and therapeutic potential of carnosic acid against acute respiratory distress syndrome through molecular docking, molecular dynamics and experimental verification</p>". Experimental and Therapeutic Medicine 31, no. 3 (2026): 83. https://doi.org/10.3892/etm.2026.13077
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