Discussion
This study marks the first application of organ-on-a-chip and organoid
technologies to create a human-derived, more physiologically relevant,
and functionally diverse model for fire-related smoke inhalation acute
lung injury (SI-ALI). Through the integration of advanced methodologies,
deeper insights into the pathophysiological mechanisms of fire-related
SI-ALI have been achieved, along with the identification of potential
therapeutic agents. This research provides a novel and robust platform
for advancing both the study and treatment of fire-related SI-ALI.
Previous research on lung injury from fire-related smoke inhalation
primarily used wood combustion smoke, which posed limitations due to the
lack of control over combustion processes and unclear smoke
concentrations, often relying on subjective estimates, thus compromising
model stability[19]. Additionally, the composition
of natural wood differs significantly from modern construction
materials, making traditional data inadequate for contemporary
fire-related SI-ALI studies[20]. By controlling
material proportions and combustion processes, a system was developed to
simulate modern non-metallic material combustion accurately (Figure 2B,
Supplementary Figure 2C and Supplementary Text 1). Fourier-transform
infrared spectroscopy confirmed the system’s stability, providing a
foundation for a reproducible SI-ALI model.
To create a more physiologically relevant SI-ALI model, organ-on-a-chip
and organoid technologies were integrated. However, traditional lung
chip models had limitations[21], such as complex
perfusion pumps prone to bubble formation and contamination, as well as
limited cell seeding capacity. To address these issues, an optimized
lung chip model was developed with a gravity-driven, pump-less system to
reduce contamination risks and improve
practicality[22]. The expanded cell seeding area
allowed for three parallel experiments within a single chip (details in
the methodology section), increasing efficiency and enabling advanced
molecular research. The chip design included a non-overlapping area
between the vascular and alveolar layers, allowing real-time observation
of capillary leakage and other pathological processes (Figures 1B and
2A).
A co-culture system of human alveolar epithelial and endothelial cells
was established on the chip, simulating the alveolar-capillary barrier
(Figure 3F). Over time, increased transepithelial resistance indicated
tight junction formation (Supplementary Figure 1B). Confocal microscopy
confirmed the bilayer structure, demonstrating successful barrier
formation (Supplementary Figures 1C-D). This model allowed for the
direct introduction of smoke into the alveolar chamber, simulating
fire-related lung damage (Figure 2C). The chip technology bypassed the
need for gases to dissolve in an aqueous solution, providing a more
accurate replication of smoke damage to the alveolar epithelium and
allowing for comprehensive analysis of intercellular interactions during
injury[23].
The chip’s response to prolonged toxic smoke exposure revealed oxidative
stress and apoptosis, consistent with clinical
findings[24-26]. Electron microscopy showed
significant subcellular abnormalities, such as dense mitochondria and
altered endoplasmic reticulum structures. Increased macrophage adhesion
and chemotaxis were observed, aligning with clinical immune cell
infiltration findings. The non-overlapping chip areas enabled real-time
observation of alveolar-capillary leakage, challenging to replicate in
traditional experiments. ELISA analysis indicated a substantial release
of inflammatory cytokines following smoke exposure, independent of
immune cell presence (Figures 3 and 4).
The advanced chip design allowed for separate extraction of endothelial
and epithelial cells for molecular analysis. Despite direct smoke
stimulation of epithelial cells, qPCR analysis showed a similar response
in endothelial cells, with upregulated inflammation and
apoptosis-related genes, highlighting intercellular signaling during
smoke exposure. This interaction, overlooked in previous single-cell
models, warrants further investigation in fire-related SI-ALI (Figure
4).
To further elucidate the mechanisms of fire-related SI-ALI, a proteomic
analysis was conducted to identify differentially expressed proteins
(DEPs) post-smoke exposure in the chip model. KEGG enrichment analysis
indicated these DEPs are involved in critical pathways such as cysteine
and methionine metabolism, amino acid biosynthesis, mineral absorption,
carbon metabolism, lysosomal function, and spliceosomal activity (Figure
5B and Supplementary Text 2). Notably, cysteine and methionine
metabolism, linked to sepsis-related lung damage, and amino acid
biosynthesis, associated with ferroptosis and drug efficacy in
sepsis-induced lung injury, were emphasized[27,
28]. The role of mineral absorption, pertinent to injuries caused by
particulate inhalation (e.g., graphene, asbestos), aligns with the
smoke’s solid particle composition[29, 30]. The
enrichment of PSAT1 in carbon metabolism suggests novel research avenues
for SI-ALI[31]. Autophagy’s role, especially in
lysosomal function, is highlighted in sepsis, COPD, and COVID-19
contexts[32-34], while the underexplored
spliceosome’s involvement is supported by its association with
phosgene-induced lung injury[35], reinforcing the
relevance of the identified pathways.
Gene Ontology Biological Process (GOBP) analysis revealed pathways
including lymphocyte aggregation, CAMKK-AMPK signaling, mitochondrial
protein processing, DNA methylation maintenance, and L-serine
biosynthesis. Lymphocyte aggregation, linked to the inflammatory cascade
in acute lung injury[36, 37]and CAMKK-AMPK
signaling, associated with smoking-related lung
cancer[38], were observed. Mitochondrial damage,
linked to ferroptosis[39], aligns with the damage
seen in smoke-exposed cells (Figure 3E), consistent with proteomic
findings. The maintenance of DNA methylation is crucial for normal lung
function, with its disruption noted in COPD[40].
Serine’s role in nucleotide synthesis and antioxidant defense was
noted[41, 42], while the hypermethylation of CpG
islands, particularly the IFN-γ promoter via the PI3K-Akt-DNMT3b
pathway, was linked to lung inflammation[43]. The
depalmitoylation pathway remains underexplored in lung injury, but our
analysis suggests its relevance, highlighting the complex pathology
mirrored in clinical presentations.
To compare in vivo and chip models, a mouse model of fire-related SI-ALI
was established. Histopathological analysis revealed significant lung
damage (Supplementary Figure 2A and 2B), consistent with oxidative
stress and cellular damage findings, aligning with the results of
previous research[44, 45] and the chip model.
Proteomic analysis showed more DEPs in the animal model, likely due to
tissue complexity. However, both models shared enrichment in carbon
metabolism and spliceosomal pathways, with common pathways including
SRP-dependent co-translational protein targeting and small
GTPase-mediated signaling, associated with metabolism, ROS, and
apoptosis. The observed differences are attributed to species-specific
variations but underscore the models’ collective reflection of SI-ALI
pathology, providing insights into underlying mechanisms (Supplementary
Text 3).
Subsequent focus was on molecules with significant fold-change in the
chip model, specifically DOCK2, TST, NIT1, RAP1GDS1, and KRR1. These
molecules were investigated for endothelial and epithelial cell
responses to smoke exposure (Figure 5D and Figure 6A). TST, involved in
enhancing mitochondrial function and reducing
ROS[46], and NIT1, responsible for hydrolyzing
deaminated glutathione, have been underexplored in lung
injury[47]. RAP1GDS1, linked to lung
carcinogenesis[48, 49], and DOCK2, with
anti-inflammatory properties, suggest potential as SI-ALI markers and
therapeutic targets[50, 51]. These findings
highlight critical molecules relevant to fire-related SI-ALI, warranting
further investigation.
In the combined proteomic analysis of animal and chip models, only COMT
consistently showed an upward expression trend, observed at both
transcriptional and translational levels in endothelial and epithelial
cells (Figure 5C-D and Figure 6A). COMT, involved in catecholamine
metabolism, may reduce oxidative stress from polycyclic aromatic
hydrocarbons (PAHs) in A549 cells[52]and regulate
lung water and sodium storage[53]. However, its
role in smoke-induced acute lung injury (SI-ALI) remains largely
unexplored. Our findings suggest that COMT could be crucial in the
pathology of fire-related SI-ALI.
To identify potential therapies, a computational analysis of COMT’s
structure was performed[54]. Screening against the
FDA-approved drug library revealed compounds like Ractopamine HCl,
Bimatoprost, and Fenoterol Hydrobromide (Figure 6B and Supplementary
Text 4). These drugs, targeting 5-HT, adrenergic, and prostaglandin
receptors, are mainly used for glaucoma and respiratory conditions, yet
their relevance to lung injury is unexamined. Our study indicates they
may offer therapeutic potential in fire-related SI-ALI, although further
validation is needed.
The chip model’s drug screening ability was further explored for SI-ALI
treatment development. Severe oxidative stress and inflammation
following smoke exposure were consistent with clinical observations. To
address these, intravenous vitamin C and nebulized budesonide were
tested, significantly reducing oxidative stress and inflammation (Figure
7).
Vitamin C is known for its antioxidant and immune-modulating effects,
with high doses shown to mitigate organ damage in
sepsis[55], suggesting it may benefit SI-ALI
treatment. However, the absence of clinical trials leaves no established
guidelines. Glucocorticoids, despite their anti-inflammatory properties,
pose risks like electrolyte imbalances and
immunosuppression[56]. Budesonide, when nebulized,
may minimize systemic side effects, offering potential for fire-related
SI-ALI[57], though clinical evidence remains
limited.
This study indicates that the combined therapy holds promise, providing
a foundation for clinical use while underscoring the need for further
research. The chip model’s advanced features enabled evaluation of
different administration routes, which traditional models struggle to
achieve. This reinforces the chip’s value in preclinical drug testing
and new treatment development for SI-ALI.
Given the superior tissue specificity of human-derived lung organoids
compared to cell lines[58], ethical approval was
obtained to culture lung organoids (Figure 8A-C). A lung organoid chip
was then developed, and smoke exposure produced similar damage responses
to those seen in cell line-derived chips (Figure 8D), confirming model
stability. Analysis of key molecules in the organoid model showed
transcriptional trends consistent with previous findings, except for
KRR1 (Figure 8E). The therapeutic effects of vitamin C and budesonide
were validated, showing mitigation of smoke-induced inflammatory gene
expression. These results further corroborate our findings using the
organoid chip model.