Inhalable carrier-free self-assembled leonurine-ursolic acid nanoaggregates ameliorate acute lung injury by suppressing TLR4/MyD88-NET axis

Figures

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  Fig. 1. Schematic illustration of self-assembled LUNP synergistically ameliorating ALI via regulation of the TLR4/MyD88-NET axis.

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  Fig. 2. Characterization of LUNP. These results confirm the successful self-assembly of LUNP, demonstrating its favorable morphology, uniform size distribution, and high physiological stability. (A) Schematic diagram of the self-assembly nonoaggregates of LUNP; (B) SEM and (C) TEM images of the LUNP; (D) Size distribution and (E) Zeta potential of LUNP measured by DLS. (F) UV-vis absorption spectrum, (G) Infrared spectroscopy, (H) PXRD, and (I) 1H NMR spectra of LN, LUNP, and UA; (J) Photographs and absorbance measurements at 595 nm of LN, UA, and LUNP after 2-h penetration through the AM layer; (K) LN release curves of the LUNP at different pH (7.4 and 5.0).

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  Fig. 3. Molecular dynamic simulation of LUNP. The simulation results elucidate the underlying intermolecular interactions and stability driving the self-assembly process of the LUNP system. (A) The conformations and detailed interactions of LN and UA at various time points throughout the 100 ns simulation; (B) RMSD plot of LN and UA displaying structural stability of LUNP; (C) The average distance and (D) the number of hydrogen bonds formed between LN and UA during the simulation period. (E) Optimized geometric structure and coordination configuration of the LN cation—UA anion complex.

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  Fig. 4. Cellular uptake of LUNP and evaluation of its anti-permeability and anti-migration properties. These findings demonstrate that LUNP is efficiently internalized by the target cells and significantly preserves barrier integrity while inhibiting inflammatory cell migration. (A) Representative immunofluorescence images of 16HBE cells treated with LUNP-C6 or C6 for 30 min, 1 h, and 2 h; (B) Cellular uptake profiles of LUNP analyzed by FCM at 30 min, 1 h, and 2 h; (C) Corresponding histogram of LUNP-C6 uptake in 16HBE cells. (D) Representative images showing FITC-dextran permeability in 16HBE cells; (E) Fluorescence intensity of FITC-dextran in 16HBE cells across different groups; (F) Representative images of 16HBE cell migration after LPS stimulation and LUNP treatment. (G) Quantification of cell migration expressed as migration area percentage. (H) Schematic illustration of LUNP absorption and its activation process. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Fig. 5. Anti-ALI effects of LUNP and its regulation of the TLR4/MyD88/NF-κB pathway in vivo. Collectively, the in vivo data indicate that LUNP profoundly alleviates lung tissue injury by effectively blocking the TLR4-mediated inflammatory cascade. (A) Schematic illustration of the in vivo anti-ALI experimental procedure; (B) BALF total cell counts in different groups (n = 5); (C) Quantitative levels of TNF-α, IL-6, IL-1β, and IFN-γ in BALF across treatment groups (n = 5); (D) ROS levels in BALF cells analyzed by FCM (n = 3); (E) Representative H&E-stained images of mouse lung tissues (n = 3); (F-I) Expression levels of TLR4, MyD88, p65, and p-p65 in lung tissues from different groups (n = 3); (J) Analysis of the most stable molecular conformation of the TLR4/MD2-LN complex; (K) Per-residue decomposition energy analysis and (L) RMSD analyses of the TLR4/MD2-LN complex during a 50-ns simulation; (M) Analysis of the most stable molecular conformation of the TLR4/MD2-UA complex and (N) Per-residue decomposition energy analysis; (O) RMSD analyses of the TLR4-UA complex during a 50-ns simulation. Data are expressed as mean ± SD. The statistical significance was calculated using a one-way…

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  Fig. 6. Inhibition of NET Formation by LUNP and Biodistribution Profile. These results suggest that LUNP effectively suppresses NET release and exhibits excellent targeted accumulation within the lung tissues. (A) Schematic illustration of NET regulation; (B) Relative MPO-DNA complex levels and (C) cfDNA content in BALF (n = 5); (D) Immunofluorescence staining of lung tissues for CitH3 (blue) and MPO (red), and (E) corresponding spatial distribution analyses across arrows (n = 3); (F) Quantification of MPO/DAPI fluorescence intensity (n = 3); (G-I) Western blot analysis of NOX4 and PAD4 expression in lung tissues (n = 4); (J) Molecular conformation of the NOX4-LN complex, and (K) per-residue decomposition energy analysis with (L) RMSD trajectory over a 50-ns MD simulation; (M) Representative conformation of the NOX4-UA complex, (N) per-residue energy decomposition, and (O) RMSD analysis; (P) In vivo fluorescence imaging and (Q) corresponding lung fluorescence intensity at various time points following inhalation of LUNP-IR780 or free IR780; Statistical analysis was performed using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) and Students t-test for pairwise…

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  Fig. 7. RNA-seq analysis of lung tissues treated with LPS + PBS or LPS + LUNP. Transcriptomic profiling reveals that LUNP treatment systematically regulates pro-inflammatory gene networks and extensively restores pulmonary immune homeostasis. (A) Volcano plot showing the distribution of DEGs (gray: non-significant; blue: downregulated genes; purple: upregulated genes); (B) Heatmap of DEGs; (C) KEGG pathway enrichment analysis of DEGs; (D) Target–pathway network analysis; (E) GO enrichment analysis of DEGs; (F) GO chord function analysis; (G) Schematic illustration of the regulatory processes induced by LUNP. (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 8. Biosafety evaluation of LUNP in vitro and in vivo. Overall, comprehensive toxicological assessments confirm that LUNP possesses excellent biocompatibility with negligible systemic toxicity. (A) Schematic illustration of the biosafety assessment protocol; (B) Cell viability of 16HBE cells and (C) hemolysis ratio of erythrocytes after treatment with LUNP at various concentrations (n = 3); (D) Hematological and biochemical parameters (WBC, RBC, Mid, Lymph, Gran, PLT, and HGB) in the blood of healthy mice 24.5 h after inhalation of PBS or LUNP (n = 5); (E) H&E staining of heart, liver, spleen, lung, and kidney tissues in each group (n = 3).