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RESEARCH ARTICLE
Negative air ions through the action of
antioxidation, anti-inflammation, anti-
apoptosis and angiogenesis ameliorate
lipopolysaccharide induced acute lung injury
and promote diabetic wound healing in rat
Yu-Hsuan Cheng
1☯
, Hung-Keng Li
1,2☯
, Chien-An Yao
3☯
, Jing-Ying Huang
1
, Yi-Ting Sung
1
,
Shiu-Dong Chung
2,4,5
*, Chiang-Ting ChienID
1
*
1Department of Life Science, School of Life Science, College of Science, National Taiwan Normal University,
Taipei, Taiwan, 2Division of Urology, Department of Surgery, Far-Eastern Memorial Hospital, New Taipei
City, Taiwan, 3Department of Family Medicine, National Taiwan University Hospital, Taipei, Taiwan,
4Department of Nursing, College of Healthcare & Management, Asia Eastern University of Science and
Technology, New Taipei City, Taiwan, 5General Education Center, Asia Eastern University of Science and
Technology, New Taipei City, Taiwan
☯These authors contributed equally to this work.
*ctchien@ntnu.edu.tw (CTC); chungshiudong@gmail.com (SDC)
Abstract
Negative air ions (NAIs) being bioactive and negative charged molecules may confer antiox-
idant and anti-inflammatory activity. We assessed the effect of NAIs on two inflammatory
diseases in animal models including lipopolysaccharide (LPS) induced acute lung injury
(ALI) and wound healing in diabetic rats. We used intra-tracheal infusion of LPS to induce
ALI and made a full-thickness cutaneous wound in streptozotocin-induced diabetic female
Wistar rats. We evaluated NAIs effects on reactive oxygen species amount, leukocyte infil-
tration, wound healing rate, western blot, and immunohistochemistry in the lungs of ALI and
skin sections of wounds. Our data found NAIs exposed saline displayed higher antioxidant
activity vs. non-exposed saline. NAIs exposure did not significantly affect arterial blood pres-
sure and respiratory frequency in control and LPS treated groups. LPS increased leukocyte
infiltration, caspase 3/Poly-ADP-ribose-polymerase-mediated apoptosis formation and
decreased Beclin-1/LC3-II-mediated autophagy in lungs. NAIs exposure conferred pulmo-
nary protection by depressed leukocyte infiltration and caspase 3/Poly-ADP-ribose-poly-
merase mediated apoptosis and enhanced LC3-II-mediated autophagy in LPS induced ALI.
NAIs treatment resulted in a significantly accelerated wound closure rate, decreased eryth-
rocyte accumulation and leukocyte infiltration mediated oxidative stress and inflammation,
and upregulated expression of skin collagen, vascular endothelial growth factor receptor-2
(VEGFR-2) and factor transforming growth factor-beta 1 (TGF-β1) vs non-treated group.
Based on these results, it is suggested that NAIs conferred a protection through the upregu-
lating LC3-II-dependent autophagy mechanism and downregulating leukocyte infiltration
mediated inflammation and caspase 3/Poly-ADP-ribose-polymerase signaling in the LPS-
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OPEN ACCESS
Citation: Cheng Y-H, Li H-K, Yao C-A, Huang J-Y,
Sung Y-T, Chung S-D, et al. (2022) Negative air
ions through the action of antioxidation, anti-
inflammation, anti-apoptosis and angiogenesis
ameliorate lipopolysaccharide induced acute lung
injury and promote diabetic wound healing in rat.
PLoS ONE 17(10): e0275748. https://doi.org/
10.1371/journal.pone.0275748
Editor: Y. Peter Di, University of Pittsburgh,
UNITED STATES
Received: May 5, 2022
Accepted: September 22, 2022
Published: October 26, 2022
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0275748
Copyright: ©2022 Cheng et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
treated ALI and promoted diabetic wound healing through the enhancing skin collagen syn-
thesis, VEGFR-2 and TGF-β1 pathways.
1. Introduction
Air ions are electrically charged molecules or atoms present in the atmosphere. Negative air
ions (NAIs) are negatively charged molecules that obtain electrons in the air [1,10], whereas
positive air ions (PAIs) lose electrons. NAIs are bioactive molecules and have been reported to
affect the cardiovascular and respiratory systems. There were several studies to demonstrate
the beneficial or therapeutic effects of NAIs exposure on lung function, metabolic measures,
and asthmatic symptoms [2,3]. The biological effects of air ions on indoor air quality as well
as the various health advantages have been reported by Grinshpun et al. [4]. By use of mass
spectrometers analysis, the major components of NAIs are CO
3−
, O
−
, O
3−
, and NO
3−
[5,6].
Several methods can be applied to produce NAIs including radiation lines or cosmic rays in
the air, ultraviolet rays in sunlight, natural and anthropogenic corona discharge, shearing
forces of water/Lenard effect, and plant-based sources of energy [7]. Among indoor air purifi-
ers of different types, ionic emitters have gained increasing attention and are presently used
for removing dust particles, aeroallergens and airborne microorganisms from indoor air [4].
In the present study, we established the NAIs generator using artificial corona discharge in
combination with electronic electrostatic precipitate collection.
NAIs have been found to inhibit growth of bacteria and fungi on solid media, to exert a
lethal effect on vegetative forms of bacteria suspended in water, and to decrease the viable
count of bacterial aerosols [8]. Particulate matter (PM) is an air pollutant that endangers
human health [9]. Related researches have demonstrated that NAIs were effective in decreas-
ing PM2.5 amount [10]. In biomedical application, Krueger et al. [11] indicated NAIs acceler-
ate tracheal activity in mammals in vitro and in vivo and found that the hemoglobin of animals
killed by carbon dioxide recovered its bright red color more quickly in NAIs than in ordinary
air. Sirota et al. [12] showed that inhaling NAIs for 60 min at the dosage of 320–350 000 ions/
cm
2
in animals activated the respiratory organs by acting directly on mucosal and blood, but
their mechanism is unknown. Recently, the outbreak of coronavirus disease 2019 (COVID-19)
caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) has spread widely in the
world [13]. SARS-CoV and SARS-CoV-2 could induce acute lung injury (ALI) and inflamma-
tion, which is mimicked by lipopolysaccharide (LPS), a bacterial endotoxin present on the
outer membrane of Gram-negative bacteria [14], implicating SARS-CoV-2 and LPS through
similar pathways of binding toll-like receptor-4 (TLR4) to activate angiotensin converting
enzyme 2 (ACE2), to induce ALI and hyperinflammation [15,16]. There has been an increase
in the understanding of the mechanisms for induction of ALI, however, there have been no
alternative treatments to reduce mortality rates in ALI patients [17]. Autophagy is a biological
process that occurs in eukaryotes and maintains cell homeostasis and viability through phos-
phatidylinositol-3-kinase (PI3K)/AKT/the mammalian target of rapamycin (mTOR) signaling
to activate protective Beclin-1/Atg5-Atg12/LC3-II signaling [16,18]. Given the urgency for the
protection of COVID-19 induced ALI, NAIs may be adapted to ameliorate LPS induced ALI.
On the other hand, chronic refractory wounds are a multifactorial comorbidity of diabetes
mellitus (DM) with the characteristic of impaired vascular networks and increased oxidative
stress and inflammation. Rat skin has been approached to evaluate the possible effect of PAIs
or NAIs on wound healing. The data informed PAIs interfered with wound healing, while
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Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This source of funding was partly
supported by Ministry of Science and Technology
of ROC with the funding number: MOST-107-
2218-E-003-001 (Chiang-Ting Chien). The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
NAIs accelerated wound healing [3]. However, the mechanism was not clarified. Kim et al.
[19] demonstrated that NAIs treatment inhibited intracellular reactive oxygen species produc-
tion, p38 mitochondrial protein initiator kinase activation, and activation protein 1 (c-Fos and
c-Jun) activation in human keratinocyte cell line HaCaT implicating antioxidant and anti-
inflammatory effects. Based on these information, this study was designed to explore the NAIs
effect on two inflammatory animal models: 1) LPS induced ALI and inflammation in rats, and
2) wound healing of skins in DM rats.
2. Methods and materials
2.1 NAIs generation setup
NAIs generation was produced and supplied by the Orito Ecological Negative Ion Machine B
(A-168 OLiDE Oriday Technology Co., Ltd., Banqiao District, New Taipei City, Taiwan). The
principle for producing NAIs was demonstrated in Fig 1A.
2.2 Animal model of ALI
Female Wistar rats (200–250 g) were purchased from BioLASCO Taiwan Co. Ltd. (I-Lan, Tai-
wan) and housed at the Experimental Animal Center, National Taiwan Normal University, at
a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 o’clock).
These animals were fed a standard chow (Laboratory Rodent Diet 5001 containing 0.4%
sodium) and tap water ad libitum. All surgical and experimental procedures were approved by
Institutional Animal Care and Use Committee of National Taiwan Normal University
(Approval number 110012 on the date of 2021 August 5) and were in accordance with the
guidelines of the National Science Council of Republic of China. All animal experiments were
performed under anesthesia, all efforts were made to minimize suffering and finally sacrificed
with overdose of anesthesia. These messages were described in detail in following experiments.
The animals were grouped as follows: n = 9 animals per group: Control (Con) group, LPS
treated (LPS) group, Control group + NAIs treated (Con + NI) group and LPS treatment
+ NAIs treated (LPS + NI) group. Rats were anesthetized with subcutaneous urethane (1.2 g/
kg, Sigma-Aldrich, St. Louis, MO). Femoral venous catheter (PE-50; Clay Adams, Parsippany,
NJ) was inserted to facilitate blood sampling and medication management. Vascular patency
was maintained by intravenous infusion pump (Infors, Bottmingen, Switzerland) at a rate of
1.2 ml/min. A femoral artery was catheterized to measure arterial blood pressure by a poly-
graph (ML845 Power Lab 4/25 T System, ADInstruments, Sydney, Australia). During the
preparation process, all rats recorded breathing frequency and arterial blood pressure with a
polygraph. During the stabilization period of 30 min after anesthesia, 200 μg/kg of LPS stimu-
lation was immediately infused into trachea at 200 μl for 210 min. Physiological functions
were continuously recorded for 30 min of baseline period and 210 min of experimental period,
and arterial blood gases and respiratory mechanics were recorded at the end of the experiment.
The NAIs treatment to the rats by 10-cm distance of NAIs exposure were performed for 210
min. Control rats were also placed indoors, but are only exposed to the air in the environmen-
tal chamber. We determined the respiratory rate, flow rate and breathing depth in these ani-
mals in total 240 min. After experiments, the animals were sacrificed with overdose of
urethane.
At different times after LPS stimulation (previously specified), the lungs were removed and
immobilized, through the trachea at a pressure of 30 cm H
2
O, in 24 h via the trachea in the
form of 10% buffer. The lungs are then dehydrated by a series of ethanol solutions, cleared
with methyl salicylate, and embedded in paraffin. Parts of the lung tissue (5 microns) were
mounted on a glass slide and stained with hematoxylin and eosin (H&E) for light microscopy.
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For immunochemical analysis, these sections were first treated with a blocking buffer for 15
min to block non-specific background binding. After phosphate buffer saline (PBS) flushing,
the section is treated with A Zymed element (Zymed Laboratory in South San Francisco, CA).
This included secondary biotin goat anti-rabbit serum, followed by marsh peroxide-tagged
streptococcal and subsequently developed diamine amphetamine substrate solutions that pro-
duce a permanent color.
2.3 Hydrogen peroxide (H
2
O
2
) scavenging capacity of NAIs
In this part of study, the H
2
O
2
scavenging activity of the saline with or without NAIs treatment
was measured by a luminol-amplification ultrasensitive chemiluminescence detection tech-
nique as described in our laboratory [20]. In brief, 200 μL of saline with or without NAIs treat-
ment was mixed with 0.5 mL of 0.1 mmol/L luminol (5-amino-2,3-dihydro-
1,4-phthalazinedione, Sigma, Chemical Co., St. Louis, MO, USA) and 0.1 mL of H
2
O
2
(0.03%)
Fig 1. A: the principle of NAIs production in this study. B: three original data from an ultrasensitive chemiluminescence analyzer. C:
statistical difference between saline and NAIs treated saline (Saline + Ni). Data were expressed as mean ±SEM (n = 6). �P<0.05 between
two groups comparision.
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and was analyzed with a chemiluminescence analyzing system (CLA-ID3, Tohoku Electronic
Inc. Co., Sendai, Japan). The chemiluminescence signals emitted from the mixture of saline,
H
2
O
2
and luminol, which represented the hydrogen peroxide content in the mixture, were
recorded. The increased chemiluminescent signals from the sample–luminol–H
2
O
2
mixture
were determined for 300 s. The total chemiluminescent counts were calculated by the area
under the curve and were displayed as counts/10 s.
2.4 Bronchoalveolar lavage for neutrophils and reactive oxygen species
(ROS) amount
We obtained bronchoalveolar lavage (BAL) of rats to detect oxidative stress and leukocytes as
described previously in our laboratory [21]. In brief, after LPS stimulation, the chest cavity was
opened, the left main-stem bronchus was ligated, and the right lung was lavaged with 1 aliquot
of Ca
2+
/Mg
2+
-free phosphate buffer saline (pH 7.4). The aliquot volume was estimated to 35
ml/kg of body weight (approximately 90% of total lung capacity). The aliquot was instilled into
the lungs 3 times before final collection. The BAL were also performed with a chemilumines-
cence analyzer to quantify ROS amount as described above. A cell counting analyzer (Sysmex
TM K-1000; TOA Medical Electronics Co. Ltd., Kobe, Japan) was applied to measure the num-
ber of neutrophils present in the BAL samples.
2.5 Western blot of lung tissue
In this study, we used immunoblotting techniques to measure autophagy-related Beclin-1 and
LC3-II, apoptosis-related caspase 3 and poly-(ADP-ribose)-polymerase (PARP) signaling
pathway in the lung tissue. The methods have been well established and described previously
in our laboratory [22]. In brief, twenty μg of proteins were used in 10% polyacrylamide gels
with electronphoretically transferred to nitrocellulose membranes (Amersham Biosciences,
Buckingham, England, UK). The membrane was blocked and incubated at 4˚C. The primary
antibodies were applied to identify Beclin-1 (Cell Signaling Technology, Inc., Danvers, MA,
USA), LC3-II (Cell Signaling Technology), the activation fragments (17 kDa of cleaved prod-
uct) of caspase 3 (CPP32/Yama/Apopain, Upstate Biotechnology, Lake Placid, NY), PARP
(Cell Signaling Technology), and β-actin (Sigma). The density of protein bands and the appro-
priate molecular mass were semi-quantitatively analyzed using an image analysis system
(Alpha Innotech, San Leandro, CA, USA).
2.6 In situ detection of apoptosis and autophagy formation in the lung
We assessed the degree of apoptosis by evaluating the expression of caspase 3 and PARP and
terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay (DNA
Fragmentation Kit; BioVision, Milpitas, California, USA) and autophagy by Beclin-1 (Cell Sig-
naling Technology, Inc.) and LC3-II (Cell Signaling Technology, Inc.) staining in the rat lungs.
The determined techniques have been followed as reported previously [22]. In brief, tissue sec-
tions from 10% formalin fixation and paraffin embedding were deparaffinized, rehydrated,
and stained with H&E or immunohistochemically. For autophagy or apoptosis related proteins
staining, the 5-μm tissue sections (Leica RM 2145, Nussloch, Germany) were incubated over-
night at 4˚C with mouse anti-rat Beclin-1 antibody (BD Biosciences, San Jose, CA, 1:100) or
LC3-II (Cell Signaling Technology, Inc., 1:100), Caspase 3 (CPP32/Yama/Apopain, Upstate
Biotechnology, Lake Placid, NY, 1:100), PARP (Cell Signaling Technology, Inc., 1:100). Subse-
quently, biotinylated secondary antibodies (Dako, Botany, NSW, Australia) were applied, fol-
lowed by incubation with streptavidin-conjugated horseradish-peroxidase (Dako). The
chromogen used in this study was Dako Liquid diaminobenzidine. Twenty high-power (×400)
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fields were randomly selected from each gastric section, and the value of brown deposits/total
section area for Beclin-1, LC3-II, caspase-3 or PARP stain was analyzed with Adobe Photoshop
7.0.1 image software. TUNEL was performed according to a previously described method [16].
Briefly, 5-μm-thick sections of gastric tissues were prepared, deparaffinized, and stained using
the TUNEL assay kit. Twenty high-power (×400) fields were randomly selected for each sec-
tion, and the level of each oxidative stress was analysed with a Sonix Image Setup (Sonix Tech-
nology Co., Ltd., Hinschu, Taiwan, ROC).
2.7 Type I diabetes induction
The protocol for induction of diabetes was demonstrated previously [23]. We used intraperito-
neal streptozotocin (STZ at 65 mg/kg; Sigma, Missouri, USA) to induce type I DM. We deter-
mined the body weight and fasted blood glucose before and after STZ treatment every week.
After one week of STZ injection, the fasted (8 h) blood glucose level was examined by using
Contour plus blood glucose monitoring system (Bayer, Leverkusen, Germany). The fasted
blood glucose >250 mg/dL was identified as successful diabetes induction. Under 2% isoflur-
ane anesthesia, the skin hair on the rat back was shaved off and cleaned with iodine wine, and
the post-disinfection surgical scissors created a square full cortex wound of 3 cm ×3 cm in size
(cortex thickness<3 mm) in the center of the rat’s back. The rats were given standard feed
and povidone iodine to prevent wound infection. For analgesia, 20 mg/kg dose paracetamol
was added to daily drinking waters of the rats in all groups. Wound closure was measured at 0,
3, 7, 10, 14, and 21 days after injury. The animals were euthanized with overdose of urethane
on days 7, 14 and 21 and wound samples from adjacent skins were collected, fixed in 10%
paraformaldehyde for histological examination or quick-frozen in liquid nitrogen and stored
at -80˚C for further analysis.
2.8 Grouping for wound healing
Experimental rats were divided into: (1) Control (Con) group: normal rats that only created
full cortex wounds on the back and did not receive any treatment; (2) Diabetes mellitus group
(DM group): diabetic rats induced by STZ were made a full-skin wound on the back; (3) Con-
trol rats with NAIs Group (Con + NI group): healthy rats treated with NAIs and made a full
cortex wound on the back and (4) Diabetes mellitus with NAIs treatment group (DM + NI
group): diabetic rats treated with NAIs after manufacturing a full cortex wound on the back.
Total 24 rats were used and each group was 6 animals.
Rats in the NAIs treated group were placed under the Oridge Ecological Negative Ion
Machine B on a standard 45.5 ×23 ×20.5 cm
3
experimental cage, and blowed the wound with
NAIs for one hour every day. The rats in the sham surgery group were kept in another cham-
ber to avoid interference from NAIs. On the 0th, 3rd, 7th, 11th, 14th and 21st days after
wound manufacturing, the wound healing situation was recorded.
2.9 Histopathology of rat skins
Rat skins were prepared with H&E for morphologic analysis, and with Masson’s Trichrome
and Sirius red stains to visualize collagen organization. The protocols of histopathology were
described previously in our laboratory [16]. In brief, the tissues were collected, treated with
formaldehyde (10%), and embedded in paraffin wax. The samples were then cut into sections
with a thickness of 5 μm (Leica RM 2145, Nussloch, Germany). After the tissue sections had
been waxed and hydrated in the usual way, the sections were stained with Masson’s Trichrome
staining kit (HT15, Sigma, USA). With this staining, the collagen was dyed blue and the kera-
tin was dyed in red color. Other slides were subjected to Sirius Red 80 (Sigma Aldrich; 0.1% of
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Sirius Red in saturated aqueous picric acid) staining. The stained samples were then examined
using a light microscope.
2.10 Immunohistochemistry (IHC) for TGF-β1 and VEGFR-2
Skin is capable of spontaneous self-repair following injury. These functions are mediated by
numerous pleiotrophic growth factors, including the vascular endothelial growth factor-2
(VEGFR-2) and transforming growth factor-β1 (TGF-β1) families [24,33]. We assessed the
effect of NAIs on the expression of the VEGFR-2 and TGF-β1 in skin wound healing of rats.
The ligands of VEGFR-2 were secreted by keratinocytes and macrophages and increased
VEGFR-2 performance, increased angiogenesis, provided more nutrients and oxygen to the
wound, and promoted wound healing [24]. The latter two were secreted in large quantities by
macrophages, which have the effects of angiogenesis and wound healing via collagen recon-
struction. In addition, according to previous studies, VEGFR-2 can increase the wound heal-
ing area, and slow down the infiltration of immune cells between tissues and the formation of
scars [25]. In the present study, the skin sections of different groups were determined by per-
forming TGF-β1 and VEGFR-2 IHC. In brief, tissue sections from 10% formalin fixation and
paraffin embedding were deparaffinized, rehydrated, and stained with VEGFR-2 or TGF-β1.
For VEGFR-2 or TGF-β1 staining, the 5-μm tissue sections were incubated overnight at 4˚C
with mouse anti-rat VEGFR-2 antibody (VEGF #9698 Cell Signaling Technology, Danvers,
MA, USA, 1:100) or TGF-β1 (sc-146 Santa Cruz, Dallas, Texas, USA, 1:100). Next, biotinylated
secondary antibodies (Dako, Botany, NSW, Australia) were applied, followed by incubation
with streptavidin-conjugated horseradish-peroxidase (Dako). The chromogen used in this
study was Dako Liquid diaminobenzidine. Twenty high-power (×400) fields were randomly
selected from each section, and the value of brown deposits/total section area for VEGFR-2 or
TGF-β1 stain was analyzed with Adobe Photoshop 7.0.1 image software as described above.
2.11 Wound preparation
Under averin anesthesia, the skin hairs on the back was shaved off and cleaned with iodine
wine. By using the surgical scissors, the rat’s back in the center was created a square full cortex
wound of 3 cm ×3 cm in size (cortex thickness <3 mm). The degree of wound closure was
determined at 0, 3, 7, 11, 14, and 21 days after injury. Animals were euthanized on days 7, 14
and 21 and wound samples and adjacent normal skin were collected, fixed in 10% paraformal-
dehyde for histological examination or quick-frozen in liquid nitrogen and stored at -80˚C for
further analysis. Wound healing rate was calculated the percentage of wound closure by the
initial and final areas plotted on the slide during the experiment. Wound shrinkage is calcu-
lated as follows [26]:
A0 was the original wound area and At was the wound area at the time of biopsy on days 0,
3, 7, 11, 14 and 21, respectively.
2.12 Statistical analysis
All values are expressed as means ±SEM unless stated otherwise. Between-group comparisons
were performed by using unpaired ttests or analysis of variance with Bonferroni method as
post hoc analysis; within-group comparisons were performed by using paired t-tests or
repeated-measures analysis of variance with Bonferroni method as post hoc analysis. A value
of P<0.05 indicated statistical significance. All computations were performed with SPSS for
WINDOWS software (version 13.0; SPSS Inc, Chicago, IL).
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3. Results
The principle of NAIs generation was described in Fig 1A.In vitro experiment to determine
NAIs effect on the antioxidant activity, the saline was exposed to NAIs for one hour. Our
results demonstrated the original data from the chemiluminescent analyzer in Fig 1B and
found a markedly depressed ROS response in the NAIs treated saline as compared to non-
treated saline. The statistical data indicated that NAIs exposed saline displayed a significant
decrease in ROS level vs. the non-treated saline (Fig 1C).
We first explored the NAIs effect on mean arterial blood pressure and respiratory frequency
in response to LPS-induced ALI for 210 min. As shown in Fig 2A, the representative graphs of
physiological parameters were displayed in four groups of rats. The statistical data of the
changes of mean arterial blood pressure (Fig 2B) and respiratory frequency (Fig 2C) in four
groups of rats treated with LPS with or without NAIs exposure did not show any very signifi-
cant differences of cardiorespiratory parameters in 210 min of LPS stimulation. These data
implicate that NAIs have no effect on cardiorespiratory parameters during our acute
challenge.
We determined the effect of NAIs on autophagy and apoptosis in LPS-induced ALI by eval-
uating the protein levels of Beclin-1/LC3-II, and caspase 3/PARP signaling with Western blot-
ting and IHC. As shown in Fig 3A of the original Western blot, the expression of caspase 3 was
significantly enhanced in the LPS group vs. Con group, whereas cleavage caspase 3 was
Fig 2. The physiologic parameters including mean blood pressure (MAP) and respiratoryrate in response to LPS or NAIs in four
groups of rats. A: the original data from four groups of rats. B: the statistical data of MAP in four groups of rats. C: the statistical data of
respiratory rate in four groups of rats. Data were expressed as mean ±SEM (n = 9). �P<0.05 vs. Saline groups.
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decreased in the LPS + NI group vs. LPS group (Fig 3C). In IHC analysis, LPS stimulation
could increase the level of caspase 3 expression in our LPS-induced ALI model (Fig 3B),
whereas NAIs exposure significantly inhibited cleavage caspase 3 in LPS-induced ALI (Fig
3D). As shown in Fig 4A of the original Western blot, the expression of PARP was significantly
Fig 3. Effect of 60-min NAIs treatment on c-caspase 3 expression in LPS induced ALI for 210 min of four groups of
rats. A: original western blot of caspase 3. B: typical caspase 3 immunohistochemic graphs of four groups of rat lungs. C:
Statistical data of caspase 3 western blot. D: Statistical data of pulmonary immunohistochemic caspase 3 stains. Data are
expressed as mean ±SEM (n = 9). �P<0.05 vs. Saline group. # P<0.05 vs. LPS group.
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enhanced in the LPS group vs. Con group, whereas PARP expression was decreased in the LPS
+NI group vs. LPS group (Fig 4C). In IHC analysis, LPS stimulation could also increase the
level of PARP expression in our LPS-induced ALI model (Fig 4B), whereas NAIs exposure sig-
nificantly inhibited PARP expression in LPS-induced ALI (Fig 4D). We further determined
the leukocyte infiltration and TUNEL stain in the four groups of rat lungs with ALI. In Fig 5A,
Fig 4. Effect of 60-min NAIs treatment on PARP expression in LPS induced ALI for 210 min of four groups of rats. A:
original western blot of PARP. B: typical PARP immunohistochemic graphs of four groups of rat lungs. C: Statistical data
of PARP western blot. D: Statistical data of pulmonary immunohistochemic PARP stains. Data are expressed as
mean ±SEM (n = 9). �P<0.05 vs. Saline group. # P<0.05 vs. LPS group.
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H&E analysis showed that the marked increase of leukocytes infiltration was found in the LPS
treated lung, whereas other three groups of lungs displayed a decrease of leukocyte infiltration.
The statistical data demonstrated that the degree of leukocyte infiltration was significantly
increased in the LPS group, whereas NAIs treatment significantly decreased LPS-enhanced
leukocyte infiltration (Fig 5B). In Fig 5C, TUNEL stains showed that the marked increase of
TUNEL positive stained cells was noted in the LPS treated lung, whereas other three groups of
lungs displayed a decrease of TUNEL positive cells in the lung. The statistical data showed that
the degree of TUNEL-apoptosis was significantly increased in the LPS group, whereas NAIs
Fig 5. Effect of 60-min NAIs treatment on leukocyte infiltration, TUNEL expression, larvage neutrophils and larvage ROS in LPS
induced ALI for 210 min of four groups of rats. A: H&E stained lung sections of four groups of rats. B: statistical data of leukocyte
infiltration of four groups of rat lungs. C: original graphs of TUNEL stains of four groups of rat lungs. D: Statistical data of pulmonary
TUNEL stains of four groups of rats. E: Larvage neutrophils number and F: Larvage ROS amount in four groups of rats. Data are expressed
as mean ±SEM (n = 9). �P<0.05 vs. Saline group. # P<0.05 vs. LPS group.
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treatment significantly decreased LPS-increased TUNEL apoptosis (Fig 5D). Larvage neutro-
phils number in LPS group was significantly increased as compared to saline control group
(Fig 5E), whereas NAIs treatment significantly decreased LPS-increased larvage neutrophils
number in LPS + NI group. In addition, larvage ROS amount in LPS group was also signifi-
cantly increased as compared to saline control group (Fig 5F), whereas NAIs treatment signifi-
cantly decreased LPS-increased larvage ROS amount in LPS + NI group.
As shown in Fig 6A of the original Western blot, the expression of Beclin-1 was signifi-
cantly suppressed in the LPS group vs. Con group, whereas Beclin-1 expression was partly
recovered in the LPS + NI group vs. LPS group (Fig 6C). In IHC analysis, LPS stimulation
could also decrease the level of Becin-1 expression in our LPS-induced ALI model (Fig 6B),
whereas NAIs exposure significantly increased Beclin-1 expression in LPS-induced ALI (Fig
6D). We further determined the LC3-II Western blot and IHC in four groups of rat lungs with
ALI. As shown in Fig 7A of the original Western blot, the expression of LC3II was significantly
suppressed in the LPS group vs. Con group, whereas LC3II expression was partially recovered
in the LPS +NI group vs. LPS group (Fig 7C). In IHC analysis, LPS stimulation consistently
decreased the level of LC3II expression in our LPS-induced ALI model (Fig 7B), whereas
NAIs exposure significantly increased LC3II expression in LPS-induced ALI (Fig 7D). Our
results informed that NAIs enhanced autophagy-related protective mechanisms in LPS-
induced ALI mouse model.
The blood glucose levels of DM rats were above >250 mg/dL three days after injection and
remained stable throughout the experiment. A total of 2 rats died during the model-establish-
ment process and the induction success rate was 95%. The body weight of rats was measured
on 0, 3, 7, 11, 14 and 21 days, and the weight of all four groups fell between 300–450 g. Rat
weight, which was measured in all rats at set time points, initially increased slowly and then
showed negative growth three weeks after injection (unpresented data). All the rats showed
obvious symptoms of polydipsia, accompanied by polyuria, polyphagia and weight loss.
Representative images of wound area in each group at 0, 3, 7, 11, 14 and 21 days after sur-
gery were shown in Fig 8A. Full-thickness skin wounds on the backs of diabetic rats were cre-
ated and treated with NAIs or non-NAIs air. On day 3 after wound injury, there was no
significant differences in wound healing rate among the groups. However, the wound area was
significantly smaller in the Con + NI and DM + NI groups than in the DM group on days 7,
11, 14 and 21 (P<0.05) (Fig 8B and 8C). On the days 7 after the manufacture of the full cortex
wound on the back, it can be seen that the wound area of the Con + NI group and the DM
+ NI group was smaller than that of the untreated Con and DM group (Fig 8A). The difference
in wound healing between the treatment group and the untreated group was more obvious at
days 11, the wound shape of the untreated Con and DM groups had obvious observable
unevenness, and the wound in the Con group was still covered with thick scabs. The scab con-
dition of the wound in the DM group was still unstable, and the tissue fluid exuded continu-
ously, while the wound area of Con + NI group and the DM + NI group was significantly
smaller than that in the DM group on Day 11, the wound edge was flat and the thick scab shed-
ding was perfect and the wound remains dry continuously, with no exudation of tissue fluid.
At the days 21, the wounds in the Con, DM and DM + NI groups still had unheated scabs
(considered to be unhealed wounds), but the unhealed wound area in the Con and DM groups
was larger than that in the DM + NI group in the treatment group, and the wounds in the Con
+ NI group in the other treatment group were completely healed (Fig 8A). If the unhealed
wound area of each group within 0, 3, 7, 11, 14, 21 days is superimposed into a graph (Fig 8B)
presented, red represents the extent of the initial wound manufacturing, yellow, green, light
blue, dark blue, pink in order represents the remaining unhealed range of the wound at 3, 7,
11, 14 and 21 days, it can be more obvious that the degree of wound healing in different groups
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at different days. According to Fig 8C, the remaining unhealed wound area is small, and the
NAIs treated group has the largest healing rate in the Con + NI group at days 21. Only <1%
of the wounds in the Con + NI group are still scabs (regarded as unhealed). The wound healing
Fig 6. Effect of 60-min NAIs treatment on Beclin-1 expression in LPS induced ALI for210 min of four groups of rats.
A: original western blot of Beclin-1. B: typical Beclin-1 immunohistochemic graphs of four groups of rat lungs. C: Statistical
data of Beclin-1 western blot. D: Statistical data of pulmonary immunohistochemic Beclin-1 stains. Data are expressed as
mean ±SEM (n = 9). �P<0.05 vs. Saline group. # P<0.05 vs. LPS group.
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rate was higher in DM + NI group than DM group (Fig 8C). This finding suggests that the
NAIs confer beneficial effects in increasing wound healing rate.
The histological structure of the regenerated dermis was analyzed on day 21. As shown in
Fig 9A and 9D, the epidermis of the new granulation tissue was intact and thick in all groups.
In the Con + NI and DM + NI groups, new hair follicle formation was evident in the center of
Fig 7. Effect of NAIs treatment on LC3-II expression in LPS induced ALI of four groups of rats. A: original western
blot of LC3-II. B: typical immunohistochemic graphs of four groups of rat lungs. C: Statistical data of LC3-II western blot.
D: Statistical data of pulmonary immunohistochemic LC3-II stains. Data are expressedas mean ±SEM (n = 9). �P<0.05
vs. Saline group. # P<0.05 vs. LPS group.
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the wound surface, and collagen deposition by Masson’s stain was sufficient and orderly. In
the DM group, the significant increase of RBC accumulation (Fig 9A and 9B) and inflamma-
tory cell infiltration (Fig 9A and 9C) were observed at day 21. However, the degree of RBC
accumulation (Fig 9B) and leukocyte infiltration (Fig 9C) was significantly depressed in the
NI + NI and DM + NI groups.
The collagen deposition of four groups determined by Masson Trichrome’s stain was
shown in Fig 9D. A marked decrease in blue collagen deposition was found in DM group vs.
other three groups. NAIs treatment in DM + NI group or Con + NI group displayed a signifi-
cant blue collagen deposition compared to DM group (Fig 9E). The collagen deposition of
four groups determined by Sirius red stain was also displayed in Fig 10A. A marked decrease
in red collagen deposition was found in DM group vs. other three groups. NAIs treatment in
DM + NI group or Con + NI group displayed a significant red collagen deposition as com-
pared to DM group (Fig 10B).
The IHC results of the vascular TGF-β1 showed that the TGF-β1 exhibited in the Con + NI
and DM + NI vascular interstitiums after NAIs treatment was higher than in the untreated
Fig 8. Effect of NAIs exposure on the wound healing rate of rat skin of four groups of rats.A: Representative images of the wound surface in
each group on days 0, 3, 7, 11, 14 and 21. B: The merged images of different color correlated different time frame of wound area in four groups of
rats. At day 21, the pink colored area is markedly reduced in the Con + NI group and DM + NI group. C: Statistical data of % unclosed wound area
over time of four groups of rats. Data are expressed as mean ±SEM (n = 6). �P<0.05 vs. Con group. # P<0.05 vs. DM group.
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Fig 9. Effect of NAIs exposure on H&E and Masson’s trichrome stain in rat skin in the wound healing of four groups of rats. A:
Representative images of H&E histology on wound sections at day 21 (black arrow: WBC; red arrow: RBC). B: Analyses of RBC
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Con and DM groups (Fig 11A and 11B), and the TGF-β1 expression was statistically signifi-
cantly less from the DM group compared with the Con group (P<0.05). In contrast, there
was no statistically significant difference in the vascular interstitial manifestations of TGF-β1
in the Con + NI and DM + NI groups after NAIs treatment indicating that the TGF-β1 expres-
sion after NAIs treatment could be restored to similar to that of the original Con group (Fig
11A and 11B). The IHC results of vascular VEGFR-2 expression showed that after NAIs treat-
ment, the VEGFR-2 expression of the vascular beds in the Con + NI and DM + NI groups was
higher than that in the untreated Con and DM groups (Fig 11A and 11C), and the vascular
VEGFR-2 expression in the Con + NI and DM + NI groups had a statistically higher
(P<0.05) vs. DM group (Fig 11C).
4. Discussion
Ionic air purifiers have become widely and increasingly popular for removing dust particles,
aeroallergens and airborne microorganisms from indoor air in various settings. In addition to
the indoor air cleaning effect, the effect of NAIs has been determined by several investigators,
however, there are still controversial results (favorable and unfavorable) about the perfor-
mance of commercially available ionic air purifiers [4]. One paper showed that NAIs created
by water shearing method improve aerobic metabolism only during a 1-h exposure, which
may be caused by improvement of erythrocyte deformability, but NAIs created by corona dis-
charge have no effects [27]. A potential mechanism is that NAIs enter the circulating blood via
the lungs and electrons of these ions are delivered to the plasma protein [27]. The possible
compositions of NAIs could be identified as CO
3−
and O
−
, O
3−
, NO
3−
, etc by using mass spec-
trometers [5,6]. The antioxidant activity of erythrocyte cytosolic superoxide dismutase from
rat, bovine, man and duck was increased when measured after NAIs exposure [28]. The pri-
mary physicochemical mechanism of biological action of NAIs is suggested to be related to the
increase of superoxide dismutase activity by a low level of micromolar concentrations of H
2
O
2
stimulation [28]. Our data using NAIs treated saline at a 10-cm distance with NAIs
amount >1.6×10
8
/cm
3
displayed a more efficient scavenging H
2
O
2
activity vs. non-treated
saline (Fig 1C) implicating the direct antioxidant activity by NAIs. These information con-
ferred the hypothesis that the markedly antioxidant effect may have beneficial and biological
effects on the inflammatory related diseases.
Until now, there was no strong evidence to clearly support a beneficial role of NAIs expo-
sure on respiratory function or asthmatic symptom alleviation [29]. Liu et al. [30] observed no
significant changes in cardiorespiratory biomarkers following the use of NAIs for 1 week.
However, one previous research has reported that exercise-induced asthma was significantly
attenuated by exposure to NAIs [2] implicating the NAIs’ beneficial effect in respiratory sys-
tem. Liu et al. [31] demonstrated the molecular linkages between indoor NAIs, PM reduction
and cardiorespiratory function among children. They found that the increased NAIs associ-
ated with the decreased PM improved respiratory function mainly by eight pathways including
promoting energy production, anti-inflammation and anti-oxidation capacity [31]. NAIs were
also observed to be an energy-efficient air purification intervention that can effectively reduce
the small airway particle exposure when a sufficient NAIs concentration is obtained [32]. ALI
is a syndrome of pulmonary inflammation and permeability characterized by gas exchange
accumulation of various treatment groups on day 21 (n = 3). C: Analyses of leukocyte infiltration of various treatment groups on day 21
(n = 3). D: Representative images of Masson’s trichrome stain (blue color) on wound sections at day 21. E: Analyses of dermal collagen
expression by Masson’s stain of various treatment groups on day 21 (n = 3). Data are expressed as mean ±SEM (n = 3). �P<0.05 vs.
Con group. # P<0.05 vs. DM group.
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dysfunction associated with pathological features like pulmonary capillary endothelial cell
damage, alveolar epithelial cell injury, extensive aqualung, tiny pulmonary atelectasis, micro-
thrombi and microcirculation disturbance [10]. Our ALI model by LPS was consistently dis-
played these pathological damages as described by Zhao et al. [10]. Importantly, our present
data showed that NAIs exposure in Con or LPS-induced ALI rat model did not affect the basic
mean arterial blood pressure and respiratory frequency and also indicated their safety in
cardiorespiratory function. To explore the NAIs effect on LPS-induced ALI, our results dem-
onstrated that LPS increased inflammatory leukocytes infiltration into lung, and enhanced cas-
pase 3/PARP/TUNEL-mediated apoptosis signaling in the damaged lung. However, NAIs
exposure decreased the degree of inflammatory leukocytes infiltration and caspase 3/PARP/
TUNEL-mediated apoptosis expression in the LPS induced ALI. Our data also evidenced that
LPS depressed the possible protective signaling pathway of Beclin-1/LC3-II mediated autop-
hagy in the lung. Interestingly, NAIs exposure significantly preserved LPS decreased Beclin-1/
LC3-II mediated autophagy signaling implicating NAIs’ protective effect through the action of
increased autophagy signaling. Recently, effective antiviral therapeutics are urgently required
to fight SARS and SARS-CoV. Our recent report found that catechins significantly reduced
LPS-induced cytokine storm and oxidative stress and ALI by inhibiting PI3K/AKT/mTOR sig-
naling to upregulate Beclin-1/Atg5-Atg12/LC3-II-mediated autophagy mechanism [16]. Liu
et al. [33] demonstrated that the NAIs-related metabolic changes reflect the reduction of
Fig 10. A: Representative histological photographs of wounded skin neighbor tissue stained with Sirius red staining are shown (Scale
bars: 20 μm). B: Quantitative histological evaluation of wounded skin neighbor skin collagen fiber expression at day 21 after wound
creation. Data are expressed as mean ±SEM (n = 6). �P<0.05 vs. Con group. # P<0.05 vs. DM group.
https://doi.org/10.1371/journal.pone.0275748.g010
Fig 11. Effect of NAIs exposure on rat skin TGFβ1 and VEGFR-2 expression in the wound healing of four groups of rats. A:
Representative images of TGFβ1 and VEGFR-2 immunohistochemistry on wound sections at day 21. B: Analyses of TGFβ1 stain of various
treatment groups on day 21 (n = 5). C: Analyses of VEGFR-2 stain of various treatment groups on day 21 (n = 5). Data are expressed as
mean ±SEM (n = 5). �P<0.05 vs. Con group. # P<0.05 vs. DM group.
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inflammation and oxidative stress, because short-term exposure to NAIs is beneficial to heart
rate variability, especially to the parasympathetic nerve activity, by successively disturbing dif-
ferent metabolic pathways which mainly reflect the increased anti-inflammation and the
reduced inflammation. NAIs with antioxidant and anti-inflammatory activity also can attenu-
ate LPS induced leukocyte infiltration mediated inflammation and apoptosis production possi-
bly through the protective action of autophagy activation. Recently, in the report of Zhang
et al. [34], NAIs can 100% inhibit the aerosol transmission of SARS-CoV-2 and influenza A
virus. In addition, the safety of 4-week NAIs exposure for Balb/c mice showed no abnormali-
ties in body weight, blood routine analysis, and lung pathology [34]. All these studies demon-
strate that NAIs can be used as a safe and effective means of blocking respiratory virus
transmission and contribute to pandemic prevention and control.
Wound healing is a complex biological process that includes blood clotting, inflammation,
fibrosis, collagen deposition, and wound contraction [35]. Poorer healing in diabetics can also
be observed in animal models of DM, where poor wound healing is found in mice with heredi-
tary DM and in streptozotocin-induced diabetic rats [36]. In diabetes in the human and animal
models, the research has identified a large number of changes associated with diabetes at the
molecular level in delayed wound healing and to a lesser extent in chronic diabetic ulcers [37].
Oxidative stress is believed to play an important role in the development of diabetic complica-
tions [38]. Increased reactive oxygen species level could be generated in diabetes through
advanced glycation end-product formation by glycation, together with glucose autoxidation
and stimulation of the polyol pathway [37]. Disruption of the redox balance and alterations to
the level or activity of reducing enzymes could contribute to poor healing and may even lead
to damage to DNA within cells involved in the normal healing process. On the other hand,
excess oxidative stress may result in tissue injury and lead to tissue in a hypoxic state. Hypoxia
is an important activator of the endothelial cells in the injured vasculature [39]. During the
hypoxic environment, the neutrophils, the first responders, infiltrate to the site of injury in the
acute phase of inflammation [40]. Our present results from H&E stains also demonstrated that
large amount of leukocytes infiltration in the DM wound skin implicating the inflammatory
response in DM. The increased leukocytes infiltration also evidenced the enhanced oxidative
stress in the injured tissue.
Importantly, wound monitoring should achieve rapid wound closure with functional tissue
and minimal scarring. Compared with DM group with less wound healing rate, the NAIs
exposure conferred significantly better effects on wound contraction. In the NAIs-treated
wounds, obviously reduced the number of leukocytes infiltration and the degree of erythrocyte
extravasation were observed on days 21. This indicated accelerated wound healing via the anti-
inflammatory and antioxidant activities of NAIs. A recent report has indicated NAIs exerted
anti-inflammatory and antioxidant effects in HaCaT cells exposed to PM implicating its appli-
cation to the prevention and treatment of inflammatory skin diseases [19]. The use of antioxi-
dant and anti-inflammatory strategies through the reduction of neutrophil adhesion and
leukocyte recruitment, or inhibition of proinflammatory cytokines release could promote
wound healing [25]. Although the mechanism remains uncertain how NAIs reduce inflamma-
tion and promote wound healing, our study evidenced that the NAIs exposure confers antioxi-
dant capability, reduces RBC accumulation and leukocyte infiltration number (inflammatory
index), accelerates angiogenesis from the results of increased TGF-β1 and VEGFR-2 expres-
sion in vascular beds (angiogenesis index) and collagen deposition by increased Masson’s stain
and Sirius red stain in the increased granular tissue, resulting in enhanced re-epithelialization.
Collagen acts as a structural support for skin and helps in functioning the cell migration, main-
taining the cell shape and inducing protein synthesis. One previous report stated that in the
proliferative phase, fibroblasts produce collagen which replaces the fibronectin-fibrin matrix
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that produce structure to the wound and in the remodeling phase, myofibroblasts deposit the
collagen by cross linking in the wound which helps in wound contraction [41]. The increased
collagen secretion by NAIs exposure may inhibit collagenase activity and elevate the deposi-
tion of the extracellular matrix proteins with increased fibroblast proliferation [42]. The possi-
ble mechanisms could be due to the increased collagen deposition to enhance wound
contraction, and the enhanced proliferation of the various cells comprising the granular tissue
by angiogenesis [25,43]. Several growth factors like TGF-β1 and VEGF released from blood
plasma or exudate in the wound and enhanced cell proliferation, migration and angiogenesis
[24,44]. TGF-β1 and VEGFR-2 have been involved in the improvement of wound repair via
increases in fibroblast repopulation and angiogenesis [25]. NAIs exposure also enhanced the
expression of TGF-β1 and VEGFR-2 around the vessels of skins, which appeared to mediate
by VEGF regulation. Our data supported the hypothesis that NAIs exposure is able to promote
TGF-β1 and VEGF dependent angiogenesis and proliferation pathways in the wound healing
process. However, the exact mechanism requires further study to determine.
5. Conclusions
NAIs with antioxidant and anti-inflammatory effects could attenuate LPS induced ALI possi-
bly through the enhancement of LC3-II-mediated autophagy signaling and the inhibition of
caspase 3/PARP/TUNEL/apoptosis signaling in the damaged lung. NAIs also promoted
wound healing possibly through the enhancement of skin collagen synthesis, vascular VEGFR-
2 and TGFβ-1 mediated angiogenesis pathways.
Supporting information
S1 Raw images.
(PDF)
Acknowledgments
We thank the Ministry of Science and Technology of ROC (MOST-107-2218-E-003-001) to
support this work.
Institutional Review Board Statement:
All surgical and experimental procedures were all approved by the Institutional Animal
Care and Use Committee of National Taiwan Normal University, with Approval Number
110012 (on the date of 2021 August 5).
Author Contributions
Conceptualization: Chiang-Ting Chien.
Data curation: Yu-Hsuan Cheng, Hung-Keng Li, Chien-An Yao, Jing-Ying Huang, Yi-Ting
Sung, Shiu-Dong Chung.
Formal analysis: Yu-Hsuan Cheng, Hung-Keng Li, Chien-An Yao, Jing-Ying Huang, Yi-Ting
Sung, Shiu-Dong Chung.
Funding acquisition: Shiu-Dong Chung, Chiang-Ting Chien.
Investigation: Yu-Hsuan Cheng, Hung-Keng Li, Chien-An Yao, Shiu-Dong Chung.
Methodology: Yu-Hsuan Cheng, Hung-Keng Li, Chien-An Yao, Jing-Ying Huang, Yi-Ting
Sung.
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Project administration: Shiu-Dong Chung, Chiang-Ting Chien.
Resources: Shiu-Dong Chung, Chiang-Ting Chien.
Software: Hung-Keng Li, Chien-An Yao.
Supervision: Chiang-Ting Chien.
Validation: Shiu-Dong Chung.
Writing – original draft: Yu-Hsuan Cheng, Hung-Keng Li, Chien-An Yao, Shiu-Dong
Chung.
Writing – review & editing: Chiang-Ting Chien.
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