Metallothionein ameliorates airway epithelial apoptosis upon particulate matter exposure: role of oxidative stress and ion homeostasis

Abstract

To investigate the mechanism underlying particulate matter (PM) exposure-induced oxidative stress and potential rescue strategies against pulmonary damage in this context.
A combination of omics technology and bioinformatic analysis were used to uncover mechanisms underlying cellular responses to PM exposure in human bronchial epithelia (HBE) cells and imply the potential rescue.
Our results implicated that oxidative stress, metal ion homeostasis, and apoptosis were the major cellular responses to PM exposure in HBE cells. PM exposure disrupted oxidative phosphorylation (OXPHOS)-related gene expressions in HBE cells. Rescuing the expression of these genes with supplemental coenzyme Q10 (Co Q10) inhibited reactive oxygen species (ROS) generation; however, it only partially protected HBEs against PM exposure-induced apoptosis. Further, metallothionein (MT)-encoding genes associated with metal ion homeostasis were significantly induced in HBE cells, which was transcriptionally regulated by specificity protein 1 (SP1). SP1 knock-down (KD) aggravated PM-induced apoptosis in HBE cells, suggesting it plays a role in MT induction. Subsequent studies corroborated the protective role of MT by showing that exogenous MT supplement demonstrated effective protection against PM-induced oxidative stress and apoptosis in HBE cells. Importantly, exogenous MT supplement was shown to reduce ROS generation and apoptosis in airway epithelia in both HBE cells and a PM-inhaled murine model.
This study demonstrates that the impact of MT on airway epithelia by suppressing oxidative stress and maintaining metal ion homeostasis is beneficial in attenuating damage to pulmonary cells undergoing PM exposure.

Full text

Lietal. Current Medicine (2024) 3:9
https://doi.org/10.1007/s44194-024-00036-7
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Current Medicine
Metallothionein ameliorates airway
epithelial apoptosis uponparticulate matter
exposure: role ofoxidative stress andion
homeostasis
Bin Li1†, Nannan Huang1,2†, Shengnan Wei2, Qingtao Meng1, Shenshen Wu1, Michael Aschner3*,
Xiaobo Li1,2,6* and Rui Chen1,4,5*
Abstract
Purpose To investigate the mechanism underlying particulate matter (PM) exposure-induced oxidative stress
and potential rescue strategies against pulmonary damage in this context.
Methods A combination of omics technology and bioinformatic analysis were used to uncover mechanisms under-
lying cellular responses to PM exposure in human bronchial epithelia (HBE) cells and imply the potential rescue.
Results Our results implicated that oxidative stress, metal ion homeostasis, and apoptosis were the major cellular
responses to PM exposure in HBE cells. PM exposure disrupted oxidative phosphorylation (OXPHOS)-related gene
expressions in HBE cells. Rescuing the expression of these genes with supplemental coenzyme Q10 (Co Q10) inhib-
ited reactive oxygen species (ROS) generation; however, it only partially protected HBEs against PM exposure-induced
apoptosis. Further, metallothionein (MT)-encoding genes associated with metal ion homeostasis were significantly
induced in HBE cells, which was transcriptionally regulated by specificity protein 1 (SP1). SP1 knock-down (KD) aggra-
vated PM-induced apoptosis in HBE cells, suggesting it plays a role in MT induction. Subsequent studies corroborated
the protective role of MT by showing that exogenous MT supplement demonstrated effective protection against PM-
induced oxidative stress and apoptosis in HBE cells. Importantly, exogenous MT supplement was shown to reduce
ROS generation and apoptosis in airway epithelia in both HBE cells and a PM-inhaled murine model.
Conclusion This study demonstrates that the impact of MT on airway epithelia by suppressing oxidative stress
and maintaining metal ion homeostasis is beneficial in attenuating damage to pulmonary cells undergoing PM
exposure.
Keywords Particulate matter, Oxidative stress, Ion homeostasis, Metallothionein
†Bin Li and Nannan Huang contributed equally to this work.
*Correspondence:
Michael Aschner
michael.aschner@einsteinmed.edu
Xiaobo Li
101011116@seu.edu.cn
Rui Chen
ruichen@ccmu.edu.cn
Full list of author information is available at the end of the article

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Lietal. Current Medicine (2024) 3:9
1 Introduction
Urban particulate matter (PM) in ambient air is a het-
erogeneous mixture of particle sizes and chemicals. PM
typically originates from mobile and industrial fossil fuel
combustion, and its specific constituents and sources
have not been fully elucidated. e adverse health effects
of PM exposure have been associated with respiratory-
related morbidity and mortality (Li et al. 2018; Zhang
2021; Bowe etal. 2019). Airway inflammation, systemic
inflammation, and oxidative stress have been implicated
in experimental animals or longitudinal panel studies
(Feng etal. 2023; Xu etal. 2022).
e respiratory track epithelial cells are at the interface
between the airspace and the internal milieu, acting as
a physical and biochemical barrier to ensure transition
between these distinct compartments. Airway epithe-
lia cells can be activated by a host of stimuli, including
air pollutants (Fu etal. 2019; Barbier etal. 2023). In the
context of activation, epithelial cells provide an adap-
tive response to these stimuli, which is important for the
elimination and wound healing, and the generation of a
systemic response (Crotty Alexander etal. 2018; Gang-
war etal. 2020). erefore, airway epithelia are the first
line of defense against air pollutants, as well as the pri-
mary target of inhaled PM in organisms.
Numerous studies have focused on the mechanisms
underlying PM exposure-induced adverse health effects.
Overall, these studies corroborate that oxidative stress
mediates, at least in part, PM-induced pulmonary toxic-
ity (Gangwar et al. 2020; Lan etal. 2021). Nonetheless,
gaps in knowledge delineating the precise role of ROS in
air pollution-mediated pathologies have yet to be fully
characterized (Gangwar et al. 2020). Furthermore, with
consistent and inevitable air pollution exposure, addi-
tional studies investigating novel efficient therapeutic
interventions are urgently required (Whyand etal. 2018).
In the present study, human bronchial epithelial cells
(HBE) and mice were exposed to various concentra-
tions and doses, respectively, of PM. Omics technology
and biological assays were used to explore the molecular
pathways underlying pulmonary toxicity of PM. e cel-
lular source of ROS generation and underlying mecha-
nisms upon PM exposure was delineated. Importantly,
our findings identified a putative rescue strategy against
PM-induced toxicity, shedding novel insight on interven-
tion strategies and the putative role of MT in rescue from
PM-induced pulmonary damage.
2 Material andmethods
2.1 Particulate matter (PM)
Urban Particulate Matter (Standard Reference Material
1648a) were purchased from National Institute of Stand-
ards and Technology, USA. e components of SRM
1648a were introduced by a research group from NIST
(Schantz etal. 2012).
2.2 Cell culture
e human bronchial epithelial cells (HBE, American
Type Culture Collection) were maintained in Dulbecco’s
modified Eagle’s medium (DMED) at 37°C in 5% CO2.
e culture medium was supplemented with 10% (v/v)
fetal bovine serum (FBS), penicillin (100 U/mL), strepto-
mycin (100μg/mL).
2.3 Cellular viability
Cellular viability was evaluated with a Cell Counting
Kit-8 (CCK-8, Nanjing Jiancheng Bioengineering Insti-
tute, China). According to the reconciliation between
invitro and invivo doses of PM (Li etal. 2003), PM-
induced biological effects at the dose range of 0.2–20 μg/
cm2 invitro were equal to that at the dose of 75 μg/m3
over a 24-h period which is PM2.5 limit recommended in
China. HBE cells were plated at a density of 1 × 104 per
well in a 96-well plate and then exposed to 100 μL PM
mixture (0, 12.5, 25, 50, 100, 250, 500, and 1, 000 μg/
mL) with three biological replicates for each concentra-
tion, equal to PM (0, 0.16, 0.33, 0.65, 1.3, 3.25, 6.5, 13 μg/
cm2) for 24 h. Cell viability affected by PM were moni-
tored every 24 h up to 3 days. en 10 μL CCK-8 solution
was added to each well, the cells were incubated for 4 h
at 37°C, and the absorbance was determined at 450 nm.
2.4 RNA microarray andgene expression analysis
HBE cells were seeded in 10 cm culture dishes and
exposed to 0, 100, and 500 μg/mL PM, respectively,
with three biological replicates. Total cellular RNA was
extracted after 24 h treatment with the TRIZOL rea-
gent (Invitrogen, US), according to the manufacturer’s
instructions (Agilent Technologies, Santa Clara, CA, US),
and subjected to microarray assay. e labeled cRNAs
were hybridized onto a Human LncRNA Array v3.0
(8 × 60K; Arraystar) chip, which is designed for 26, 109
coding genes. e arrays were scanned with an Agilent
G2505C scanner and the density of fluorescence was ana-
lyzed with Agilent Feature Extraction software (version
11.0.1.1). Quantile normalization and subsequent data
processing were performed with GeneSpringGx v12.0
software package (Agilent Technologies). An absolute
fold change (FC) of 1.5 or more and p = 0.05 were set as
threshold to evaluate the significance of gene expression
differences of raw data.
2.5 Functional group analysis
e DAVID 6.7 (Database for Annotation, Visualiza-
tion and Integrated Discovery, https:// david. ncifc rf.
gov/) functional annotation tool was used to denote the

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Lietal. Current Medicine (2024) 3:9
significance of gene ontology (GO) term enrichment
in the differentially expressed mRNAs (p-value was set
less than 0.1) based on a background of homo sapiens
genome.
2.6 Animal treatments
Male C57BL/6 mice (20–22 g), were purchased from
GemPharmatech Co. Ltd. (China) and housed in a spe-
cific pathogen-free (SPF) animal facility, in which the
room temperature was maintained at 22.5 ◦C with 12h
light/dark cycle. Mice were housed six per polycarbonate
cage on corncob bedding with adlibitum access to food
and water. Mice received standard rodent chow (Jiangsu
Xietong Pharmaceutical Bio-engineering Co., Ltd.,
China) sterilized by cobalt (Co) 60 radiation and auto-
claved water adlibitum. Animals were treated humanely,
maintained, and used in accordance with Guidelines of
Committee on Animal Use and Care of Southeast Uni-
versity (20190226014).
Mice were divided into six groups (n = 6/group),
namely filtered air (FA)/Vehicle, PM (300μg/m3)/Vehicle,
PM (600μg/m3)/Vehicle, FA/metallothionein (MT), PM
(300μg/m3)/MT, and PM (600μg/m3)/MT. Exposure was
carried out as previously described (Li etal. 2021; Meng
etal. 2022). In brief, mice were placed in three stainless-
steel Hinners-type whole-body inhalation chambers; two
chambers were ventilated with PM, and the remaining
one with HEPA-filtered clean air at the same flow rate as
the PM exposure chamber. Mice were exposed for 2h per
day for 30 consecutive days. MT were administrated at
a dose of 2.5mg/kg, i.p., 1h before PM exposure every
3days. Control mice received equal volume of 0.9% saline
injection.
Mice were deeply euthanized under isoflurane anesthe-
sia 1h after the end of PM exposure on the 30th day. A
portion of the left lung tissue from each mouse was fixed
in 4% paraformaldehyde (PFA), and the remainder of the
lung tissues were stored in liquid nitrogen.
2.7 RNA isolation andquantitative real‑time PCR assay
HBE cells were exposed to 0, 50, 100, 250, or 500μg/mL
PM for 24h. Next, cells were trypsinized and collected.
Total RNA was extracted with the GenEluteTM Mam-
malian Total RNA Miniprep Kit (Sigma, US). cDNA
synthesis for coding genes was performed with 1μg of
total RNA according to the manufacturer’s instruction
(Toyobo, Tokyo, Japan).
e mRNA levels for target genes were determined by
reverse transcription of total RNA followed by quantita-
tive real-time PCR analysis (qRT-PCR) on a Quant Studio
6 Flex system (Applied Biosystems, Life Technologies,
US) with SYBR PCR Master Mix reagent kits (Toyobo,
Japan). Primer sequences are shown in Table S1. All
experiments were independently performed in triplicate.
e mRNA levels for the indicated gene are relative to
cyclophilin A (CycA).
2.8 JC‑1 staining
Changes in mitochondrial membrane potential (Δψm)
in pulmonary cells were determined with a commercial
5,50′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-
carbocyanine iodide (JC-1) kit (C2003S, Beyotime, China).
HBE cells were exposed to PM for 24 h, added with JC-1
staining working solution, and incubated at 37°C for 20
min. After the incubation, HBE cells were washed twice
with JC-1 staining buffer and evaluated under a fluores-
cence microscope (Olympus, Japan) to examine green and
red fluorescence, the relative fluorescent intensities were
determined by the ratio of green/red fluorescent intensity
with Photoshop software.
2.9 ROS generation
ROS levels in HBE cells and murine lung tissues were
determined with a commercial ROS assay kit (S0033S,
Beyotime Biotechnology, China). After exposure to 0,
50, 100, 250, or 500μg/mL PM for 24h, HBE cells were
washed with PBS. en 2’, 7’-dichlorofluorescein-diace-
tate (DCFH-DA) probe was added at a final concentra-
tion of 10μmol/L and incubated at 37°C for 30min to
determine the content of intracellular ROS.
Paraffin-fixed lung tissue sections were incubated with
DCFH-DA at 37°C for 20 min. e sections were covered
with a mounting solution containing DAPI (DAPI Fluo-
romount-G, SouthernBiotech, USA), and then observed
under a fluorescence microscope (Axio Imager M2, Zeiss,
Germany). ree non-overlapping fields containing at
least one small airway at low-power field (100 ×) of each
section were randomly selected, the relative fluorescent
(See figure on next page.)
Fig. 1 PM exposure alters mRNA expression profiles in HBE cells. a HBE cellular viability was inhibited in a concentration-depended manner
following PM exposure. (n = 3/group, one-way ANOVA for each time point). b Volcano plots showed the differentially expressed genes
following 100 or 500 μg/mL PM exposure for 24 h with a cut-off as fold change (FC) < 1.5 and p-value > 0.05 (n = 3/group). c Venn diagram showed
the differentially expressed genes in 100 or 500 μg/mL PM-exposed HBE cells. d and e GO terms (biological function, BP) of differential expressed
genes of 100 or 500 μg/mL PM-exposed HBE cells. f GO terms (BP) of differential expressed genes overlapped in 100 and 500 μg/mL PM-exposed
HBE cells. g Schematic of PM exposure resulted cellular damages in HBE cells

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Lietal. Current Medicine (2024) 3:9
Fig. 1 (See legend on previous page.)

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Lietal. Current Medicine (2024) 3:9
intensities were determined by the ratio of green/DAPI
fluorescent intensity with Photoshop software.
2.10 ATP levels
ATP levels were measured with a luciferase ATP assay
kit (Beyotime, China). HBE cells were exposed to 0, 50,
100, 250, or 500μg/mL PM for 24h and added with 200
μL of lysis buffer. Cells were collected and centrifuged at
12,000rpm for 5 min at 4°C. e luminescence of the
supernatant was assayed with a luminometer (Berthold
Detection System, Pforzheim, Germany).
2.11 Cytochrome c contents
After exposure to 0, 50, 100, 250, or 500μg/mL PM for
24h, HBE cells were washed with PBS. Mitochondrial
and cytosolic proteins of HBE cells were isolated by a
Mitochondria/Cytosol Fractionation Kit (Beyotime,
China) and subsequently quantitated using the Brad-
ford method. e levels of cytochrome c were estimated
according to the ELISA kit procedures (R&D Systems,
U.S.).
2.12 Cell apoptosis analysis
PM exposure-induced cellular apoptosis was analyzed
by flow cytometry with an Annexin V-FITC Apoptosis
Detection Kit (KeyGEN BioTECH, China). Briefly, after
exposure to 0, 50, 100, 250, or 500μg/mL PM for 24h,
HBE cells were harvested and washed twice with PBS,
followed by centrifugation at 1, 000rpm for 5min. en
the pellets were resuspended in 500 μL binding buffer
and incubated with 5 μL FITC-conjugated annexin V and
5 μL PI for 15min at room temperature in the dark. e
samples were analyzed by FACS Calibur Flow Cytometry
(BD Biosciences, US).
2.13 ELISA assays
HBE cells were exposed to 0, 50, 100, 250, or 500 μg/
mL PM for 24h. Protein content was measured by the
Bradford method. HBE cells were incubated with Ac-
DEVD-pNA, Ac-IETD-pNA, Ac-LEHD-pNA, which are
substrate peptides of caspase-3, 8, and 9, respectively
(Beyotime Institute of Bio-technology, China) for 2h at
37 °C. Release of p-nitroanilide (pNA) from substrates
was measured at 405nm by a microplate reader (Molecu-
lar Devices, U.S.)
2.14 ChIP assay
ChIP was performed using the ChIP-IT™ Express Mag-
netic assay kit (Active Motif, USA). HBE cells were
exposed to 0, 100, or 500μg/mL PM for 24h, and fixed
with 4% formaldehyde for 10min. e samples were then
centrifuged at 2, 000g for 2min and washed once with
cold PBS. e ChIP reaction antibody was a normal rab-
bit IgG (NI01, EMD Chemicals, Inc., Gibbstown, NJ) and
an anti-SP1 (1:500 dilution; ab231778, Abcam, USA).
Precipitated genomic DNA was analyzed by quantitative
PCR in triplicate measurements for each sample using
appropriate primers (TableS2).
2.15 Western blot
HBE cells were exposed to 0, 100, or 500μg/mL PM for
24h, and total proteins were extracted by a total pro-
tein extraction kit (Life Technologies, USA) from cells.
A total of 20µg of protein was subjected to electropho-
resis. Blots were then incubated with primary antibodies
against SP1 (1: 1000 dilution; ab231778, Abcam, USA) for
48h, follow by incubation with HRP-conjugated second-
ary antibodies for 1h. α-tubulin (1:5000 dilution; ab7291,
Abcam, USA) was used as a loading control. e immu-
noreactive signals were visualized with a Super ECL Plus
Kit (Yeasen, China).
2.16 TUNEL staining
Apoptotic cells in lung tissues were evaluated with ter-
minal-deoxynucleoitidyl transferase mediated nick end
labeling (TUNEL) staining by a Roche InSitu Cell Death
Detection Kit (Roche, U.S.). e nuclear stained areas
(depicted in dark brown) were identified as TUNEL-pos-
itive cell. e proportion of TUNEL-positive cells of an
airway were estimated by two experienced histologists
who were blinded to experimental design. ree to five
non-overlapping airways in each section were counted in
high-power fields (HPFS, × 400 magnification) and ana-
lyzed. e airways with the maximum number of positive
cells were selected for analysis (Li etal. 2017).
Fig. 2 Low-concentration of PM exposure induces oxidative stress in HBE cells. a Venn diagram showed the differentially expressed genes
and potential cellular responses to PM exposure in HBE cells. b Chord diagram of mitochondria-related GO terms and involved genes. c Heatmap
of mitochondria-related genes in HBE cells following PM exposure for 24 h by microarray. d qRT-PCR assays of mitochondria-related genes
in HBE cells following PM exposure for 24 h (n = 9/group, one-way ANO VA). e JC-1 staining of HBE cells following PM exposure for 24 h (n = 3/
group, one-way ANOVA). f ATP production in HBE cells following PM exposure for 24 h (n = 3/group, one -way ANOVA). g Contents of cytosol
and mitochondrial cytochrome c in HBE cells following PM exposure for 24 h (n = 3/group, one-way ANOVA). h ROS generation in HBE cells
following PM exposure for 24 h (n = 3/group, one-way ANO VA). i Schematic of the cellular responses to low-dose PM exposure in HBE cells. *
p < 0.05, ** p < 0.01, *** p < 0.001, compared to the PM (0 μg/mL) group
(See figure on next page.)

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Lietal. Current Medicine (2024) 3:9
Fig. 2 (See legend on previous page.)

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2.17 Short interfering RNA (siRNA) construction andcell
transfection
To knockdown the expression of SP1 and MT1F, SP1-
or MT1F- targeting siRNA and scrambled negative
control (NC) sequences were designed and synthe-
sized by GenePharma (Shanghai, China). HBE cells
were seeded in six-well plates and transfected with siR-
NAs using Lipofectamine 2000 Transfection Reagent
Fig. 3 High-concentration PM exposure leads to apoptosis in HBE cells. a Chord diagram of apoptotic GO terms and involved genes in 500 μg/mL
PM-exposed HBE cells. b Heatmap of apoptosis-related genes in HBE cells following PM exposure for 24 h. c qRT-PCR assays of apoptosis-related
gene expression levels in HBE cells following PM exposure for 24 h (n = 9/group, one-way ANOVA). d Cellular apoptosis induced by PM exposure
in HBE cells (n = 3/group, one-way ANOVA). e Levels of caspase-3, -8, 9 in HBE cells following PM exposure (n = 3/group, one-way ANOVA). f
Schematic of the cellular responses to high-dose PM exposure in HBE cells. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the PM (0 μg/mL) group

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Lietal. Current Medicine (2024) 3:9
(Invitrogen, USA) combined with Opti-MEM (Gibco,
USA) in line with the manufacturer’s protocol. Forty-
eight hours after transfection, knockdown efficiency was
verified by qRT-PCR or WB. e sequences of siRNAs
are listed in TableS3.
2.18 Coenzyme Q10 (Co Q10) supplementation
HBE cells were treated with Co Q10 (2.5 or 5.0 μM,
C9538, Merck, Germany) for 6h, then exposed to 0, 100,
or 500μg/mL PM for 24h. Co Q10 was dissolved in etha-
nol as a stock solution. e stock solution was incubated
at 37 ℃ for 15min prior to addition to the HBE cells. Fol-
lowing evaluation of gene expression levels, a single dose
of 5.0μM Co Q10 supplement was used for ROS genera-
tion, caspase-3, -8, -9 ELISA assay, and apoptosis detec-
tions in HBE cells. e control group were supplemented
with an equal volume of ethanol as in the 5.0μM Co Q10
group.
2.19 MT supplementation
HBE cells were treated with 1μg/mL MT (rabbit liver,
ALX-202–070, ENZO, USA)) dissolved in 0.9% saline for
6 h (Nygaard etal. 2015; Leung etal. 2018), then exposed
to 0, 100, or 500 μg/mL PM for 24 h. Gene expression
levels, ROS generation, caspase-3, -8, -9 ELISA assay, and
apoptosis were evaluated in HBE cells as described above.
2.20 Histopathological analysis
Lung tissues were fixed in PFA for 24h at 4°C, embed-
ded in paraffin, serially sectioned (5μm) and mounted
on silane-covered slides. e sections selected from each
mouse were stained after dewaxing with hematoxylin
and eosin (H&E) and evaluated under a light microscope
(400 ×) to examine tissue histology. Images were scanned
with the slide scanner Panoramic SCAN (3DHISTECH,
Hungary) to obtain a whole slide image. Histology of
murine lung tissues were evaluated by an experienced
histologist who was blinded to experimental design.
2.21 Statistical analysis
Data are shown as means ± SD, unless indicated oth-
erwise. For the comparison among multiple groups,
one-way or two-way analysis of variance (ANOVA) fol-
lowed with Dunnett’s multiple comparisons was used, as
indicated in the figure legends. e results of qRT-PCR
were analyzed by 2−ΔΔCt method. Statistical significance
was set at p < 0.05.
3 Results
3.1 mRNA expression proles inHBE cells were altered
byPM exposure
e cellular viability of HBE cells was inhibited in a
concentration-dependent manner following 24, 48, or
72 h PM exposure. e significant decrease in cellular
viability (80.21 ± 7.63%, 74.62 ± 8.82%, 70.90 ± 7.61%,
and 47.65 ± 3.56%, relative to the control) occurred
in HBE cells following 100, 250, 500, and 1000 μg/mL
PM exposure for 24 h, respectively (Fig.1a). Two doses
(100 and 500 μg/mL) of PM exposure, which resulted
in cellular viability reduction of 20% and 30%, respec-
tively, relative to the control, were chosen for the mRNA
microarray assays. A total of 140 and 758 up-regulated
genes, and 42 and 927 down-regulated genes were by
100 or 500 μg/mL in PM-exposed HBE cells for 24 h,
respectively, with a cut-off of > 1.5-fold change (FC) and
p-value < 0.05 (Fig. 1b). Venn diagram demonstrated
the number of differentially expressed genes in 100 and
500 μg/mL PM-exposed groups (Fig.1c). Gene ontol-
ogy (GO) of differentially expressed genes suggested
multiple enrichments related to mitochondrial oxida-
tive phosphorylation in the 100 μg/mL PM-exposed
HBE cells (Fig.1d and TableS4); and apoptosis in 500
μg/mL PM-exposed HBE cells (Fig.1e and Table S5).
As for overlapped genes between 100 and 500 μg/
mL PM-exposed groups (including 63 differentially
expressed genes), they were highly enriched in ion
homeostasis-related terms (Fig.1f and TableS6). ere-
fore, we hypothesized that PM exposure exerted effects
on HBE cells in a concentration-dependent manner.
At the relatively low concentration, PM exposure trig-
gered oxidative stress; with increased concentration,
ion homeostasis was disrupted, eventually resulting in
apoptosis in HBE cells (Fig.1g).
Fig. 4 Disruption of ion homeostasis contributes to apoptosis in HBE cells following PM exposure. a Chord diagram of ion homeostasis GO terms
and involved genes in 500 μg/mL PM-exposed HBE cells. b Heatmap of ion homeostasis-related genes in HBE cells following PM exposure for 24
h by microarray. c qRT-PCR assays of ion homeostasis-related genes in HBE cells following PM exposure for 24 h (n = 9/group, one-way ANOVA).
d qRT-PCR assays of MT-encoding genes in HBE cells following PM exposure for 24 h (n = 9/group, one-way ANOVA). e Schematic of transcription
factor SP1 binding to the promoter regions of MT-encoding genes and Expression levels of SP1 in PM-exposed HBE cells for 24 h (n = 3/group,
one-way ANOVA). f ChIP assays verified that SP1 binding promoted MT1X, MT1F, and MT1G transcription ((n = 6/group, t-test). g SP1 knockdown
inhibited expression of MT1F (n = 9/group, one-way ANOVA). h Apoptosis of HBE cells (control or SP1 KD) following PM exposure (n = 3/group,
two-way ANOVA). i Schematic of the cellular responses to PM exposure in HBE cells. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the PM (0 μg/
mL) group
(See figure on next page.)

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Fig. 4 (See legend on previous page.)

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Lietal. Current Medicine (2024) 3:9
3.2 PM exposure triggered oxidative stress bytargeting
phosphorylation (OXPHOS) inHBE cells
To confirm our hypothesis that relatively low concentra-
tion of PM triggered oxidative stress in HBE cells (Fig.2a),
the expression levels of 5 genes involved in mitochondrial
function and OXPHOS (Fig. 2b and c) were validated.
Consistent to the microarray data (Fig. 2c), expression
levels of NDUFC1, NDUFB1, NDUFB2, and ATP5E were
inhibited following 24 h PM exposure, and the expres-
sion levels of UQCR10 were significantly increased at 50
and 100 μg/mL groups, while decreased at 500 μg/mL
group (Fig.2d) compared to the control. Mitochondrial
functions were consistently disrupted by showing signifi-
cant collapse of the mitochondrial membrane potential
(Fig.2e), decreased ATP content (Fig.2f), increased cyto-
sol cytochrome c levels, and decreased mitochondrial
cytochrome c levels (Fig.2g) in HBE cells following PM
exposure relative to the control. PM exposure also sig-
nificantly induced ROS levels in HBE cells. Among all the
groups, the maximal ROS generation was observed in the
100 μg/mL PM-treated group (Fig.2h). erefore, it was
confirmed that HBE cells responds to relatively low PM
exposure by induction of oxidative stress (Fig.2i).
3.3 PM exposure resulted inapoptosis inHBE cells
To further confirm our hypothesis that high concen-
tration of PM exposure resulted in apoptosis in HBE
cells (Fig.3a), the expression levels of apoptosis-related
genes were validated by qRT-PCR assays. Consistent
to the microarray data (Fig.3b), 8 out of 10 apoptosis-
involved genes were significantly induced in HBE cells
following 24 h 500 μg/mL PM exposure (Fig.3c). e
flowcytometric assays showed significantly increased
apoptosis in 250 and 500 μg/mL PM-exposed HBE
cells for 24 h, relative to the control (Fig.3d). Accord-
ing to previous study, Caspase-related apoptosis can
proceed in intrinsic or extrinsic pathways (Shalini etal.
2015). In intrinsic apoptosis pathway, death-inducing
stimulus, such as ROS and DNA damage, lead to acti-
vation of apoptosome following Caspase-9 cleavage
and Caspase-3 activation. As for extrinsic apoptosis,
it depends on complex formation by death-domain-
containing proteins which activated Caspase-8 leading
to apoptosis. In Fig.3e, we identified the activation of
mitochondria-dependent (Caspase-9) and non-mito-
chondria-dependent (Caspase-8) apoptosis pathways
in HBE cells following low-concentration (50 and 100
μg/mL) and high-concentration (250 and 500 μg/mL)
PM exposure respectively. Our results indicated two
different pathways of PM-induced apoptosis in a dose-
dependent manner. ese results suggested that rela-
tively high dose of PM exposure damaged HBE cells by
inducing apoptosis (Fig.3f).
3.4 MT‑encoding genes consistently responded toPM
exposure inHBE cells
Bioinformatic analysis showed that ion homeostasis-
involved enrichments played a vital role in response to
PM exposure in HBE cells (Fig.4a). Among the 63 over-
lapped differentially expressed genes between 100 and
500 PM-exposed groups, a total of 7 genes were signifi-
cantly induced with ≥ twofold change compared to the
control (Fig.4b). Four of the 7 genes, including MAGOH,
NR1I2, CAPRIN2, and SAA1, were ion homeostasis GO
enrichments-involved genes and their expression levels
were significantly higher in PM-exposed groups com-
pared to the control (Fig. 4c). e remainder 3 genes,
including MT1F, MT1G, and MT1X, were MT-encoding
genes.
MT is a well-known antioxidant and its transcrip-
tions can be rapidly induced by stimuli, such as oxidative
stress and exogenous metals exposure (Andrews 2000).
Expression levels of MT-encoding genes were induced
in HBE cells following PM exposure (Fig.4d). Next, we
investigated their up-stream drivers. SP1 was predicted
to be a transcription factor binding to the promoter
region of MT1F, MT1X, and MT1G and its expression
levels were induced in HBE cells following PM expo-
sure (Fig.4e). ChIP assays showed that binding of SP1 to
the promoter regions of MT1F, MT1X, and MT1G was
enhanced in the PM-exposed group compared to the
control (Fig.4f).
Whether increased expression levels of MT-encoding
genes in HBE cells following PM exposure have bene-
ficial or deleterious role has yet to be determined. We
(See figure on next page.)
Fig. 5 MT efficiently protects HBE cells against PM exposure-induced apoptosis. a qRT-PCR assays of UQCR10 expression in HBE cells following PM
exposure with or without Co Q10 supplement (n = 9/group, two-way ANOVA). b ROS generation in HBE cells following PM exposure with or without
Co Q10 supplement (n = 3/group, two-way ANOVA). c Apoptosis n HBE cells following PM exposure with or without Co Q10 supplement (n = 3/
group, two-way ANOVA). d Schematic of the Co Q10 rescue against PM exposure-induced oxidative stress in HBE cells. e qRT-PCR assays of MT1X
and MT1G expression in HBE cells following PM exposure with or without MT supplement (n = 9/group, two-way ANOVA). f ROS generation in HBE
cells following PM exposure with or without MT supplement (n = 3/group, two-way ANOVA). g Apoptosis n HBE cells following PM exposure
with or without MT supplement (n = 3/group, two-way ANOVA). h Schematic of the MT rescue against PM exposure-induced apoptosis in HBE cells.
* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the PM (0 μg/mL) group

Page 11 of 16
Lietal. Current Medicine (2024) 3:9
Fig. 5 (See legend on previous page.)

Page 12 of 16
Lietal. Current Medicine (2024) 3:9
constructed SP1 knock-down (KD) HBE cells and the
expression of SP1 were ablated in HBE cells following
PM exposure (Fig. S1a and Fig.4g) significantly attenu-
ated the induction of MT1F, MT1X, and MT1G expres-
sion levels in HBE cells following PM exposure (Fig.
S1b); whereas, expression levels of MAGOH, NR1I2,
CAPRIN2 were not transcriptionally regulated by SP1
in HBE cells following both 100 and 500 μg/mL PM
exposure; expression levels of CAPRIN2 and SAA1 were
significantly decreased in SP1-KD group only follow-
ing 500 μg/mL PM exposure. (Fig. S1c). Furthermore,
SP1 KD enhanced PM exposure-induced apoptosis in
HBE cells compared to the control (Fig.4h). As shown
in Fig.4d, the expression levels of MT1F were increased
in a dose-dependent manner following PM exposure,
we thus chose to knock down the expression of MT1F
(Fig. S1d) in HBE cells as an example to assess whether
MT-encoding genes act a protect role on PM-induced
apoptosis. Indeed, we observed high apoptosis level in
HBE cells following 24 h 100 and 500 μg/mL PM expo-
sure in MT1F KD group (Fig. S1e). erefore, the induc-
tion of MT-encoding genes was protective merely based
on the increase of PM-induced apoptosis caused by SP1
KD. ese results corroborated our hypothesis that with
the increased PM exposure levels, oxidative stress, ion
homeostasis disruption, and apoptosis were sequen-
tially induced in HBE cells (Fig.4i).
3.5 MT eectively protected HBE cells againstPM
exposure‑induced cellular damages
We further explored the potential rescue to PM expo-
sure-induced damage in HBE cells. CoQ10 is an elec-
tron carrier in the mitochondrial respiratory chain,
an antioxidant, and anti-apoptotic factor (Emma etal.
2016). Here, Co Q10 supplement (5.0 μM) rescued the
expression of UQCR10 to control levels in HBE cells fol-
lowing 100 μg/mL PM exposure (Fig.5a), but failed to
rescue the expression of NDUFB1, NDUFB2, NDUFC1,
and ATP5E (Fig. S2). Co Q10 supplement significantly
reduced ROS generation in 100 and 500 μg/mL PM-
exposed cells (Fig. 5b). However, Co Q10 supplement
only partially inhibited the levels of Caspase-3 and -9
in HBE cells (Fig. S3), and slightly attenuated apopto-
sis in 500 μg/mL PM-exposed HBE cells (Fig.5c). us,
though Co Q10 effectively inhibited ROS generation, it
was not an ideal rescue reagent against PM exposure
induced apoptosis in HBE cells (Fig.5d).
Since induction of MT-encoding genes effectively
inhibited PM exposure-induced apoptosis, we tested
the effects of MT supplement on HBE cells. Following
exogeneous MT supplement, the expressions of MT1F,
MT1X, and MT1G in PM-exposed HBE cells were sta-
tistically indistinguishable from control levels (Fig.5e
and Fig. S4a). The expressions of mitochondria-related
genes were not affected by MT supplement (Fig. S4b
to f). MT displayed anti-oxidative efficacy by inhibit-
ing ROS generation (Fig.5f), fully inhibited Caspase-3,
-8 and -9 induction (Fig. S5), and reduced apoptotic
proportions in PM-exposed HBE cells compared to the
control (Fig.5g and h).
3.6 MT supplement attenuated pulmonary injuries
inPM‑exposed mice
The representative image from FA/vehicle group
showed normal morphology of small airway, which
was composed of a single layer of cuboidal epithelia.
However, in the PM-exposed murine lung, hyperplasia
of the small airway epithelia was evident, which was
characterized by increased layers of surface respira-
tory epithelial cells and desquamation into the airway
lumen (L) (Fig. 6a). Images of PM/MT groups dem-
onstrated that MT supplement effectively reversed
the disruption in airway epithelium (Fig.6a). We next
determined ROS generation in murine lung tissues. As
expected, increased ROS generation in airway epithe-
lia were showed in the PM-exposed murine lungs, and
MT supplement significantly inhibited ROS generation
to statistically indistinguishable levels from the control
(Fig.6b and c). Finally, TUNEL assays were performed
to evaluate the apoptosis in airway epithelia, which
consistently corroborated ROS generation (Fig. 6d).
Taken together, our study suggested MT as a potential
rescue against PM exposure-induced pulmonary inju-
ries both invitro and invivo (Fig.6e).
4 Discussion
In the present study, we characterized the effects of PM
exposure on the bronchial epithelia. Mitochondrial dys-
function, disruption in ion homeostasis, and increased
Fig. 6 MT supplement attenuates bronchial epithelial oxidative stress and apoptosis in mice following PM exposure. a Representative images
of small airways in mice following PM exposure by H&E staining on lung tissue sections. L: lumen of airway b & c: Representative images of ROS
generation in murine lungs following PM exposure (n = 12/group, two-way ANOVA). d & e Representative images of TUNEL staining on lung tissue
sections in mice following PM exposure (n = 12/group, two-way ANOVA). f Schematic of MT as a potential rescue against PM exposure-induced
pulmonary injuries both in vitro and in vivo. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the PM (0 μg/mL) group
(See figure on next page.)

Page 13 of 16
Lietal. Current Medicine (2024) 3:9
Fig. 6 (See legend on previous page.)

Page 14 of 16
Lietal. Current Medicine (2024) 3:9
oxidative stress contributed apoptosis in response to PM
exposure. Notably, MT afforded anti-oxidative and anti-
apoptosis efficacy, effectively rescuing bronchial epithe-
lial cells from the damage induced by PM exposure in
both mammalian cells and animals.
Cumulative evidence supports a role for oxidative
stress as a critical pathway in response to PM exposure
(Yue etal. 2019; Ge etal. 2020; He etal. 2017). Endog-
enous ROS is generated from diverse sources, including
mitochondrial respiratory chain, NADPH oxidases, nitric
oxide synthases, and cytochrome P450 (Nathan and
Cunningham-Bussel 2013; Wende et al. 2016). Consist-
ent with previous studies, our results demonstrated that
PM exposure resulted in disturbance in the mitochon-
drial respiratory chains. OXPHOS dysfunction results in
two major effects, namely, reduced ATP production and
increased ROS production (Emma et al. 2016). In the
respiratory chain, the electron carrier Co Q10 shuttles
electrons from complexes I and II to complex III. Mild
Co Q10 deficiency has been associated with increased
ROS production, meanwhile, ATP production was not
significantly changed (Quinzii et al. 2010). Co Q10 is
also a well-known antioxidant and anti-apoptotic factor
(Emma etal. 2016). In the present study, CoQ10 sup-
plement partially ameliorated ROS generation, however
it failed to rescue apoptosis in HBE cells following PM
exposure. We thus concluded that CoQ10 acted as a gen-
eral anti-oxidant, but not a specific rescue to PM expo-
sure-induced cellular apoptosis.
Oxidative stress has been intensively associated with
disruption of ion homeostasis, especially the metal ion
homeostasis. Garza-Lombó et al. reviewed the role of
redox signaling played in the cellular toxicity linked to
xenobiotic metal exposure (Garza-Lombo et al. 2018).
ough most of these studies have been conducted in
the nervous system, they emphasized that disruption in
homeostasis of essential metals and exposure to xenobi-
otic metals altered the cellular redox status and signaling.
Metals are important toxic components of PM (Li etal.
2016). ese metal components caused a cellular redox
imbalance through cumulative formation of ROS, yet, via
distinct mechanisms (Goncalves etal. 2021; Valko etal.
2016; Ying etal. 2021; Skalny etal. 2021). erefore, over-
loading of metals from PM exposure is one of the major
triggers to oxidative stress invivo. Besides, literatures also
support the idea that ion homeostasis could be disturbed
by PM exposure. Water-soluble ionic components of PM
can easily dissolve in the wet alveolar wall and affect the
alveolar cells, which directly leads to imbalanced ion
homeostasis (Park etal. 2022). Air pollution, for example,
may reduce sodium excretion, which enhances the risk
of hypertension (Kwak and Kim 2023). e exposure of
PM2.5 disrupts intracellular iron leading to cell ferroptosis
in endothelial cells (Wang and Tang 2019). On the other
hand, PM-exposure induces the aberrant expression of
ion channel-related genes, which may cause disruption of
ion homeostasis, leading to arrhythmia-like cardiotoxic-
ity in zebrafish embryos (Park etal. 2023).
Previous studies have documented the contributions of
several antioxidant systems to the regulation of ROS and
maintenance of optimal redox status, including super-
oxide dismutases, catalases and the enzymes of the glu-
tathione redox cycle (Nathan and Cunningham-Bussel
2013). Compared with these well-known antioxidants,
MT have been considered a more effective antioxidant
against metal-associated oxidative stress (Carocho etal.
2018; Yu etal. 2023). e highly conserved number and
position of cysteine residues inherent to MT enable
them to bind physiological and xenobiotic heavy met-
als, as well as to reduce reactive oxygen species (Chen
etal. 2024). As a stress-induced protein, MT have been
reportedly involved in the underlying mechanisms to
maintain metal homeostasis in cells (Witkowska et al.
2021). Corroborating to our results, it has been reported
that MT are suitable candidates for exobiotic metal
exposure, for their intriguing feature of high binding
potential for various heavy metals, and their expressions
are markedly induced during exposure to environmen-
tal insults (Mehta etal. 2016). MT in humans contains
four subfamilies, among which MT1 is widely expressed
in tissue to balance homeostasis. Typically, MT1G can
scavenge ROS through enhancing activity of NRF2/
ARE signaling without changes in free iron abundance;
although MT1F and MT1X have been linked to cell pro-
liferation, cell cycle, and apoptosis, the role of MT1F
and MT1X involve in ROS was still unknown (Pavic
etal. 2019; Rodrigo etal. 2020). Our results confirmed
the protective role of MT-code gene, especially MT1F, in
PM-exposure induced oxidative stress, ion homeostasis
disruption, and apoptosis in HBE cells.
5 Conclusion
In summary, metal ion homeostasis and oxidative stress
are major up-stream triggers of apoptosis in airway
epithelia. In this context, herein, we show that induc-
tion of metal-binding antioxidants, MT, may serve as
to offset and protect airway epithelia from the adverse
effects of PM exposure.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1007/ s44194- 024- 00036-7.
Supplementary Material 1. Appendix A. Supplementary data. Supplemen-
tary figures 1 to 5, and supplementary tables 1 to 5.

Page 15 of 16
Lietal. Current Medicine (2024) 3:9
Acknowledgements
Not applicable
Authors’ contributions
Conceptualization, X.L., R.C.; Funding Acquisition, X.L., R.C.; Investigation, B.L.,
N.H., S.W., Q.M., S.W. Supervision, M.A., R.C.; Writing Original Draft, Review, and
Editing, X.L., M.A. All Authors revised and approved the final version of the
manuscript.
Funding
This work was financially supported by the National Natural Science Founda-
tion of China (81973084), Guangdong Provincial Natural Science Foundation
Team Project (2018B030312005).
Availability of data and materials
The mRNA microarray dataset supporting the conclusions of this article are
available in the GEO repository; the accession number is GSE138870.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
Author Rui Chen is a member of the Editorial Board for Current Medicine. The
paper was handled by the other Editor and has undergone rigorous peer
review process. Author Rui Chen was not involved in the journal’s review of, or
decisions related to, this manuscript.
Author details
1 School of Public Health, Capital Medical University, Beijing 100069, China.
2 Key Laboratory of Environmental Medicine Engineering, Ministry of Educa-
tion, School of Public Health, Southeast University, Nanjing, China. 3 Depart-
ment of Molecular Pharmacology, Albert Einstein College of Medicine,
Forchheimer 209, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 4 Advanced
Innovation Center for Human Brain Protection, Capital Medical University, Bei-
jing 100069, China. 5 Institute for Chemical Carcinogenesis, Guangzhou Medi-
cal University, Guangzhou 511436, China. 6 School of Public Health, Southeast
University, Nanjing 210009, China.
Received: 10 February 2024 Accepted: 27 August 2024
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