Melatonin Alleviates PM2.5-Induced Hepatic Steatosis and Metabolic-Associated Fatty Liver Disease in ApoE-/- Mice

Abstract

Background:
Exposure to fine particulate matter (PM2.5) is associated with the risk of developing metabolic-associated fatty liver disease (MAFLD). Melatonin is the main secreted product of the pineal gland and has been reported to prevent hepatic lipid metabolism disorders. However, it remains uncertain whether melatonin could protect against PM2.5-induced MAFLD.

Methods and results:
The purpose of our study was to investigate the mitigating effects of melatonin on hepatic fatty degeneration accelerated by PM2.5 in vivo and in vitro. Histopathological analysis and ultrastructural images showed that PM2.5 induced hepatic steatosis and lipid vacuolation in ApoE-/- mice, which could be effectively alleviated by melatonin administration. Increased ROS production and decreased expression of antioxidant enzymes were detected in the PM2.5-treated group, whereas melatonin showed recovery effects after PM2.5-induced oxidative damage in both the liver and L02 cells. Further investigation revealed that PM2.5 induced oxidative stress to activate PTP1B, which in turn had a positive feedback regulation effect on ROS release. When a PTP1B inhibitor or melatonin was administered, SP1/SREBP-1 signalling was effectively suppressed, while Nrf2/Keap1 signalling was activated in the PM2.5-treated groups.

Conclusion:
Our study is the first to show that melatonin alleviates the disturbance of PM2.5-triggered hepatic steatosis and liver damage by regulating the ROS-mediated PTP1B and Nrf2 signalling pathways in ApoE-/- mice. These results suggest that melatonin administration might be a prospective therapy for the prevention and treatment of MAFLD associated with air pollution.

Full text

Research Article
Melatonin Alleviates PM
2.5
-Induced Hepatic Steatosis and
Metabolic-Associated Fatty Liver Disease in ApoE
-/-
Mice
Zhou Du,
1,2
Shuang Liang,
1,2
Yang Li,
1,2
Jingyi Zhang,
1,2
Yang Yu,
1,2
Qing Xu,
3
Zhiwei Sun ,
1,2
and Junchao Duan
1,2
1
Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China
2
Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China
3
Core Facilities for Electrophysiology, Core Facilities Center, Capital Medical University, Beijing 100069, China
Correspondence should be addressed to Zhiwei Sun; zwsun@ccmu.edu.cn and Junchao Duan; jcduan@ccmu.edu.cn
Received 6 March 2022; Accepted 6 May 2022; Published 8 June 2022
Academic Editor: Reiko Matsui
Copyright © 2022 Zhou Du et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Exposure to fine particulate matter (PM
2.5
) is associated with the risk of developing metabolic-associated fatty liver
disease (MAFLD). Melatonin is the main secreted product of the pineal gland and has been reported to prevent hepatic lipid
metabolism disorders. However, it remains uncertain whether melatonin could protect against PM
2.5
-induced MAFLD.
Methods and Results. The purpose of our study was to investigate the mitigating effects of melatonin on hepatic fatty
degeneration accelerated by PM
2.5
in vivo and in vitro. Histopathological analysis and ultrastructural images showed that
PM
2.5
induced hepatic steatosis and lipid vacuolation in ApoE
-/-
mice, which could be effectively alleviated by melatonin
administration. Increased ROS production and decreased expression of antioxidant enzymes were detected in the PM
2.5
-treated
group, whereas melatonin showed recovery effects after PM
2.5
-induced oxidative damage in both the liver and L02 cells.
Further investigation revealed that PM
2.5
induced oxidative stress to activate PTP1B, which in turn had a positive feedback
regulation effect on ROS release. When a PTP1B inhibitor or melatonin was administered, SP1/SREBP-1 signalling was
effectively suppressed, while Nrf2/Keap1 signalling was activated in the PM
2.5
-treated groups. Conclusion. Our study is the first
to show that melatonin alleviates the disturbance of PM
2.5
-triggered hepatic steatosis and liver damage by regulating the ROS-
mediated PTP1B and Nrf2 signalling pathways in ApoE
-/-
mice. These results suggest that melatonin administration might be a
prospective therapy for the prevention and treatment of MAFLD associated with air pollution.
1. Introduction
Health risks associated with particulate air pollution have
become a major focus of global concern due to rapid popu-
lation growth, industrialization, and urbanization. Fine par-
ticulate matter at a size of ≤2.5 μm (PM
2.5
) has been
considered as a strong potential threat to public health that
it can penetrate through the alveoli of lungs into the sys-
temic circulation and accumulate in the liver, kidney, or
brain [1–3]. Recently, a precise imaging technique was
developed to visualize the deposition of PM
2.5
particles in
the liver through inhalation, providing solid evidence that
the PM
2.5
particles can enter the extrapulmonary organs
[4]. Toxicological studies have demonstrated that the toxic-
ity of PM
2.5
not only induces respiratory and cardiovascular
morbidities but also contributes to other unfavourable out-
comes, such as systemic metabolic disorder, obesity, and
the pathogenesis of metabolic-associated fatty liver disease
(MAFLD) [2, 5], eventually resulting in liver dysfunction
and damage. Consistent with evidence from animal studies,
a prospective cohort study showed that people living in areas
with higher PM
2.5
concentrations had a 34% higher inci-
dence of MAFLD than those living in areas with lower
PM
2.5
concentrations. The hazard ratio (HR) of MAFLD
was 1.06 for every 1 μg/m
3
increase in PM
2.5
[6]. MAFLD
covers a broad spectrum of liver abnormalities from hepatic
steatosis to inflammation and has become one of the main
cause of cirrhosis and liver cancer. Its prevalence continues
to progress universally, keeping pace with the obesity epi-
demic, reaching 20%-30% of the total population, 80–90%
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2022, Article ID 8688643, 24 pages
https://doi.org/10.1155/2022/8688643

of obese individuals, and even more subjects with type 2 dia-
betes mellitus (T2DM) [7].
The mechanism for the pathophysiology of MAFLD was
initially explained by the “two-hit”hypothesis. The first hit is
that insulin resistance leads to enhanced hepatic de novo
lipogenesis and decreased lipolysis. Mice exposed to PM
2.5
have been demonstrated to develop MAFLD, characterized
by changes in liver appearance, extensive distribution of
lipid vacuoles, and balloon-like degeneration within the lob-
ular structure [5, 8]. The accumulation of free fatty acid flux
in hepatic cells further triggers a “second hit”involving oxi-
dative stress and lipid peroxidation [9]. It has also been
reported that PM
2.5
exposure induces excessive oxygen spe-
cies (ROS) production and redox homeostasis disorder [10,
11]. In brief, oxidative stress appears to be an integral mech-
anism that conveys hepatic injury in MAFLD and plays a
well-described role in mediating the toxicity of PM
2.5
[12].
However, the specific mechanism by which PM
2.5
exposure
promotes the risk of oxidative stress-driven MAFLD
remains incompletely understood.
A growing body of evidence in the cellular and molecular
biology of lipid metabolism have shown that protein tyro-
sine phosphatase 1B (PTP1B) is a new activator in the pro-
cess of MAFLD that regulates lipogenesis in the liver [13,
14]. Total PTP1B protein levels were generally upregulated
in liver biopsies from patients with MAFLD [15]. Function-
ally, PTP1B deficiency prevents the adverse metabolic effects
of a high-fat diet, including weight gain, increased liver
lipids, and reduced glucose tolerance [16]. PTP1B
-/-
mice
also exhibited downregulation of genes involved in fat pro-
duction, including sterol regulatory element-binding pro-
teins (SREBPs) [17]. Furthermore, SREBP-1 could
extensively affect multiple metabolic steps in the liver and
extranet, thereby regulating the progression of MAFLD
[18]. However, it is still not clear whether PM
2.5
has a tar-
geted regulatory effect on PTP1B.
Melatonin is well-known for its ability to neutralize ROS
and reduce oxidative stress [19]. It upregulates nuclear factor
erythroid 2-related factor 2 (Nrf2) through inhibition of
Kelch—like ECH-associated protein (Keap1) to suppress
oxidative stress in the liver [20]. Investigations have noted
that melatonin critically participates in lipid metabolism
and potentially contributes to the onset and progression of
MAFLD [21, 22]. However, it remains uncertain whether
melatonin could protect against PM
2.5
-induced oxidative
stress in the liver and ameliorate MAFLD.
Compared with the general population, people with obe-
sity, hyperlipidaemia, or abnormal lipid metabolism are
more sensitive to PM
2.5
and have a higher risk of developing
MAFLD [23, 24]. According to a cohort study of full-exome
association of alanine aminotransferase, ApoE was found
closely linked with fatty liver [25], and allele-specific variants
of ApoE were associated with an increased incidence of
MAFLD and obesity [26]. Thus, intense efforts have been
made to investigate MAFLD based on ApoE
-/-
mice
[27–30]. Hua et al. demonstrated that naringin administra-
tion improved metabolic parameters in ApoE
-/-
mice, inhib-
ited hepatic steatosis, and reduced hepatic fibrosis [31].
Stachowicz et al. found that high fat diet resulted in more
exacerbated hepatic steatosis in ApoE
-/-
mice [32]. In this
study, ApoE
-/-
mice were chosen as an animal model to
explore the molecular mechanism of the melatonin-
mediated protective effects against PM
2.5
-induced MAFLD.
We speculated that melatonin may ameliorate PM
2.5
-
induced MAFLD. Our findings supported this hypothesis
and further indicated that melatonin alleviated the distur-
bance of PM
2.5
-triggered hepatic steatosis and liver damage
by regulating the ROS-mediated PTP1B and Nrf2 signalling
pathways. These results not only provide novel insight into
the underlying molecular mechanism by which PM
2.5
con-
tributes to the pathogenesis of MAFLD but also suggest the
use of melatonin as a potential treatment.
2. Materials and Methods
2.1. Collection and Extraction of PM
2.5
.PM
2.5
was collected
on quartz fibre filters with a special sampler (TH-1000C,
Wuhan Tianhong, China) from Capital Medical University
Table 1: The primer lists of real‐time PCR.
Primer Forward primer (5′-3′) Reverse primer (5′-3′)
mus-SOD AAGGGAGATGTTACAACTCAGG GCTCAGGTTTGTCCAGAAAATG
hsa-SOD CCCGACCTGCCCTACGACTAC AACGCCTCCTGGTACTTCTCCTC
mus-Keap1 GACTGGGTCAAATACGACTGC GAATATCTGCACCAGGTAGTCC
hsa-Keap1 ATTCAGCTGAGTGTTACTACCC CAGCATAGATACAGTTGTGCAG
mus-Nrf2 CAGCCATGACTGATTTAAGCAG CAGCTGCTTGTTTTCGGTATTA
hsa-Nrf2 TCCAAGTCCAGAAGCCAAACTGAC GGAGAGGATGCTGCTGAAGGAATC
mus-SREBP1 GCTACCGGTCTTCTATCAATGA CGCAAGACAGCAGATTTATTCA
hsa-SREBP1 CTGTGTGACCTGCTTCTTGT CTCATGTAGGAACACCCTCC
mus-SP1 GAAGCAGCAGCACAGGCAGTAG GCCAGCAGAGCCAAAGGAGATG
hsa-SP1 TCACTCCATGGATGAAATGACA CAGAGGAGGAAGAGATGATCTG
mus-PP2A AGTTACACTGCTTGTAGCTCTT AACCCATAAACCTGTGTGATCT
hsa-PP2A CGAAGGTGTGAAGGGGAAGAAGC CAGCGTGTTGAGAAGAGCGACTAG
mus-PTP1B GAGAGATCCTGCATTTCCACTA TACTTTCTTGATGTCCACGGAA
hsa-PTP1B CCATTTACCAGGATATCCGACA TGACGTCTCTGTACCTATTTCG
2 Oxidative Medicine and Cellular Longevity

Con
(a)
(b)
(c)
(d)
1.0
(e)
(f) (g) (h)
0.8
0.6
Ratio of liver size to body weight
Oil red O staining area (%)
T-CHO (mmol/g protein)
TAGs (mmol/g protein)
0.4
0.2
0.0
100
80
60
40
20
0
0.25 3
2
1
0
0.20
0.15
0.10
0.05
0.00
Sag
Tra ns
Con
Mel
PM
2.5
PM
2.5
+Mel
Con
Mel
PM
2.5
PM
2.5
+Mel
Con
Mel
PM
2.5
PM
2.5
+Mel
Mel PM2.5 PM2.5+Mel
Con
Mel
PM2.5
PM2.5+Mel
⁎⁎
⁎⁎⁎⁎⁎⁎⁎
Figure 1: Melatonin improved the increased lipid content and steatosis in the liver induced by PM
2.5
. (a) Ultrasound examination of
liver—comparison of liver echo and kidney echo. (b) The ultrastructure of liver tissues via electron microscopy (magnification, 200; scale
bar, 2 μm). (c) Liver sections with haematoxylin and eosin (H&E) staining (magnification, 200 and 400; scale bar, 60 μm and 30 μm). (d)
Liver steatosis assessed by Oil Red O staining (magnification, 200 and 400; scale bar, 60 μm and 30 μm). (e) Liver sag (anterior-posterior
diameter) and liver trans (left-right diameter) measurement to mice weight ratio. (f) The ratio of the Oil Red O-stained area to the total
tissue area. (g) Hepatic total cholesterol lipid levels (mmol/g). (h) Hepatic triacylglycerol lipid levels (mmol/g). Con: animals were treated
with saline; Mel: animals were treated with melatonin; PM
2.5
: animals were treated with PM
2.5
;PM
2.5
+Mel: animals were treated with
melatonin and PM
2.5
. Data are shown as means ± SD.n=6−12 mice per group. ∗P<0:05 for Con group vs PM
2.5
group and PM
2.5
group vs PM
2.5
+Mel group.
3Oxidative Medicine and Cellular Longevity

Con
DAPIFITCMERGE
Mel PM2.5 PM2.5+Mel
(a)
0.08
0.06
0.04
AOD (pixel)
0.02
0.00
Con
Mel
PM
2.5
PM
2.5
+Mel
⁎⁎
(b)
5
4
3
2
MDA (nmol/mgprrot)
1
0
Con
Mel
PM
2.5
PM
2.5
+Mel
⁎⁎
(c)
70
60
50
40
4-HNE (𝜇mol/L)
Con
Mel
PM2.5
PM2.5+Mel
⁎⁎
(d)
600
400
200
0
GSH-PX (𝜇mol/mgprot)
Con
Mel
PM2.5
PM2.5+Mel
⁎
⁎
(e)
Figure 2: Continued.
4 Oxidative Medicine and Cellular Longevity

100
80
60
SOD vatality (U/mgprot)
40
20
0
Con
Mel
PM2.5
PM2.5+Mel
⁎⁎
(f)
4
3
2
1
0
mRNA expression
Nrf2
Keap1
SOD
Con
Mel
PM2.5
PM2.5+Mel
⁎
#
##
⁎
⁎
(g)
Con Mel PM2.5 PM2.5+Mel
Nrf2
Keap1
SOD
GAPDH
(h)
1.5
1.0
0.5
0.0
Relative Nrf2 protein levels
Con
Mel
PM
2.5
PM
2.5
+Mel
⁎⁎
(i)
2.0
1.5
1.0
0.0
0.5
Relative Keap1 protein levels
Con
Mel
PM
2.5
PM
2.5
+Mel
⁎⁎
(j)
Figure 2: Continued.
5Oxidative Medicine and Cellular Longevity

(Beijing, China) for the entire year of 2017. The physico-
chemical characterization of PM
2.5
was described in detail
in our previous study. Tables S1 and S2 show the results of
element analysis. S, Ca, Na, Si, and Fe are the most
abundant elements. Toxic heavy metals (including Mn, Cd,
Cr, Ni, and Sb), toxic nonmetallic elements (As), and
water-soluble ions (NO
3-
, SO4
2-
, and NH4
+
) were detected
in PM
2.5
[33, 34]. Sampled filters were placed in ultrapure
water for 3 hours using an ice-water bath ultrasonic
instrument. Then, freeze-dried samples were irradiated
with ultraviolet light for 2 hours, diluted and mixed with
pure water, and suspended in PM
2.5
by ultrasonication for
30 minutes for later use.
2.2. Animals and Treatments. Seven-week-old male ApoE
-/-
mice (specific-pathogen free) were obtained from the Exper-
imental Laboratory Animal Technology Co., Ltd. (Vital
River, Beijing, China). Animal experimental procedures
were approved by the Experimental Animal Welfare Com-
mittee (Capital Medical University; AEEI-2016-076). All
mice were fed a high-fat diet (0.15% cholesterol and 21%
fat). After acclimatization for one week, a total of 60 mice
were randomly divided into four groups: (i) control group
(Con): animals were treated with saline; (ii) PM
2.5
group
(PM
2.5
): animals were treated with PM
2.5
; (iii) melatonin
group (Mel): animals were treated with melatonin; and (iv)
melatonin and PM
2.5
group (PM
2.5
+Mel): animals were
treated with PM
2.5
and melatonin. Mice were orally gavaged
with melatonin (20 mg/kg•bw, in 20~25 μL of 0.5% ethanol
solution) daily and PM
2.5
(5 mg/kg•bw, in 20~25 μLof
saline) via intratracheal instillation twice a week for 4 weeks.
The control mice received a corresponding volume of blank
filters eluted with saline by intratracheal instillation. The
vehicle mice were gavaged with the same amount of sterile
water (0.5% ethanol).
According to the concentration and intervention method
of melatonin in previous studies, melatonin (Sigma, USA)
was dissolved in absolute ethanol and diluted in sterile water
to a final concentration of 0.5% ethanol, with the oral gavage
at a dose of 20 mg/kg/day [35, 36]. The dose of PM
2.5
expo-
sure was based on the respiratory physiological parameters
of mice and the annual mean PM
2.5
concentration (35 μg/
m
3
), according to the WHO air quality guidelines [37].
The respiratory volume of an adult mouse (25 g) is 0.15 mL
at each breath, and the breath rate is 163 times per min,
and respirato ry volume for one day reaches 0.035208 m
3
.
For this reason, the daily exposure of mice was 0:035208 ∗
35 μg/m3=1:23228 μg. Based on the body weight of mice
25 g and the extrapolation coefficient of species 100, the vol-
ume of intratracheal instillation was 1:23228 μg/25 g ∗100
=4:93 μg/g. Previous studies have demonstrated that
PM
2.5
at 5 mg/kg can cause varying degrees of organ damage
[38, 39]. Therefore, a dose of 5 mg/kg was selected for animal
modeling.
2.3. Ultrasonic Examination of Liver. Before ultrasound
imaging, the mice were fasted for 12 h, the abdominal
regions were shaved, and then the mice were anaesthetized
with a saturated tribromoethanol solution via intraperito-
neal injection. We acquired transcutaneous ultrasound
images using a Vevo2100 Ultrasonic Doppler System (Fuji-
film Visual Sonics, US).
2.4. Histopathological Examination. Both haematoxylin-
eosin (H&E) and Oil Red O staining are effective and repro-
ducible methods for quantifying hepatic steatosis [40]. For
1.5
1.0
0.5
0.0
Relative SOD protein levels
Con
Mel
PM2.5
PM2.5+Mel
⁎⁎
(k)
Figure 2: Melatonin improved liver oxidative damage induced by PM
2.5
. (a) Production of ROS detected by the fluorescent probe DHE
(magnification, 200; scale bar, 20 μm). (b) Quantitative analysis of ROS production is reflected by the mean fluorescence intensity as
shown in different groups. (c) The level of MDA. (d) The level of 4-HNE. (e) The level of GSH-PX. (f) The vitality of SOD. (g) The
mRNA expression of Nrf2, Keap1, and SOD. (h) Western blotting of Nrf2, Keap-1, and SOD. (i) Protein quantification of Nrf2. (j)
Protein quantification of Keap1. (k) Protein quantification of SOD. All values are presented as the mean ± SD (n=6). ∗P<0:05 for Con
group vs PM
2.5
group and
#
P<0:05 for PM
2.5
group vs PM
2.5
+Mel group.
6 Oxidative Medicine and Cellular Longevity

10
8
Con
Mel
PM2.5
PM2.5+Mel
6
4
2
0
mRNA expression
PTP1B
PP2A
SREBP-1
SP1
⁎
⁎
#
#
#
#
⁎
⁎
(a)
Con
PTP1B
PP2A
SP1
GAPDH
P‑PP2A
P‑SP1
SREBP‑1
Mel PM2.5 PM2.5+Mel
(b)
0.5
0.4
0.3
0.2
0.1
0.0
Con
Mel
PM2.5
PM2.5+Mel
Relative PTP1B protein levels
⁎⁎
(c)
0.0
0.5
1.0
1.5
Con
Mel
PM2.5
PM2.5+Mel
Relative P‑PP2A/PP2A protein levels
⁎⁎
(d)
0.6
0.4
0.2
0.0
Con
Mel
PM2.5
PM2.5+Mel
Relative P‑SP1/SP1 protein levels
⁎⁎
(e)
0.25
0.20
0.15
0.10
0.05
0.00
Con
Mel
PM2.5
PM2.5+Mel
Relative SREBP‑1 protein levels
⁎⁎
(f)
Figure 3: Continued.
7Oxidative Medicine and Cellular Longevity

histological examination, liver specimens were fixed over-
night with 4% paraformaldehyde and then embedded in par-
affin sections (4–6μm). Tissue sections were counterstained
with H&E. To visualize lipid droplet accumulation, frozen
liver sections (10 μm) were taken, stained with Oil Red O
(0.5%) for 10 min, washed and rinsed with isopropanol,
and counterstained with haematoxylin for a few seconds.
Representative photographs were taken at 200x and 400x
magnification using an in-microscope system. There were
6 samples in each group, and twenty regions were randomly
selected from each separate section. The “color picker”in
Image-Pro-Plus was used to select the red fat droplets in
images until all the red fat droplets were marked. Then,
Oil Red O-stained area was measured, and its ratio to the
total tissue area was calculated.
2.5. Ultrastructural Observation by Transmission Electron
Microscopy (TEM). Lipid accumulation in the liver tissue
was observed by transmission electron microscopy. The liver
tissues were immediately placed into 2.5% glutaraldehyde
for 10 min at 4
°
C and then washed with PBS 3 times and
dehydrated. Sample sections (60 nm) were stained on copper
mesh and assessed using TEM (JEM-2100plus).
2.6. Detection of ROS Levels in Liver Tissue. Frozen sections
of the liver were washed, DHE solution (10 μM) was added,
and the sections were incubated at room temperature for
30 min. A confocal microscope (LSCM, TCS SP8 STED,
Germany) was used to capture fluorescence images. There
were 6 samples in each group, and 3 visual fields were ran-
domly selected for each sample. Then, the ratio of red area
to total area was statistically analyzed by Image-Pro-Plus.
2.7. Cell Culture and Treatment. The human normal liver
cell line L02 was obtained from Shanghai Institutes for Bio-
logical Sciences (SIBS, China). Cells were cultured in Dul-
becco’s modified Eagle’s medium (DMEM; Corning, USA)
containing 1% penicillin-streptomycin solution and 10%
foetal bovine serum (Corning, USA) at 37
°
C in a humidified
incubator with 5% CO
2
. Palmitic acid (PA) is an inducer for
cell steatosis. For treatment before each experiment, cells
were treated with PA solution dissolved in DMEM for
24 h. When the cell density reached 70%-80%, DMEM
(without serum) containing PM
2.5
or melatonin was added
and then cultured for 24 h. The control group was cultured
in a constant volume of pure medium.
2.8. Assessment of Cytotoxicity. A total of 1×10
4L02 cells
per well were seeded in 96-well culture plates. When the cell
density reached 50%, the L02 cells were exposed to gradient
concentrations of PM
2.5
(0, 12.5, 25, and 50 100 μg/mL), PA
(0, 50, 100, 200, 400, 800, and 1600 μmol/L), and melatonin
(0, 12.5, 25, 50, 100, and 200 μmol/L). According to the pro-
tocols, cell viabilities were measured by Cell Counting Kit-8
(CCK-8, Tongren, Japan), and the absorbance was measured
at 450 nm using a microplate reader (Thermo, USA).
2.9. Biochemical Parameter Analysis. Triacylglycerols
(TAGs), total cholesterol (TC), low-density cholesterol
(LDL-C), high-density cholesterol (HDL-C), glutathione
peroxidase (GSH-Px), superoxide dismutase (SOD), and
malonaldehyde (MDA) levels were measured spectropho-
tometrically according to the instructions of the kit (Nanjing
Jiancheng Institute of Biotechnology, Nanjing, China). Pro-
tein concentration was determined using a BCA protein
assay kit (Dingguo Changsheng Biotech, China). The 4-
hydroxynonenal (4-HNE) activity was determined using a
Hailian Biotechnology Co. Ltd. ELISA (enzyme-linked
immunosorbent assay) kit (Jiangxi, China).
2.10. Cellular BODIPY Staining. BODIPY™493/503
(Thermo, USA) is a lipophilic fluorescent probe targeting
polar lipids that can be used to label cell neutral lipid con-
tent, especially lipid content localized to lipid droplets. It
was dissolved in anhydrous ethanol to generate a 10 mM
stock solution, which was frozen, dried, and stored away
Con
Mel
PM2.5
PM2.5+Mel
PP2A activity (U/L)
3
2
1
0
(g)
Con
Mel
PM2.5
PM2.5+Mel
SP1 activity (𝜇g/ml)
0.4
0.3
0.2
0.1
0.0
(h)
Figure 3: Melatonin ameliorated abnormal liver lipid metabolism caused by elevated PTP1B expression induced by PM
2.5
. (a) The mRNA
expression of PTP1B, PP2A, SP1, and SREBP-1. (b) Western blotting of PTP1B, PP2A, P-PP2A, SP1, P-SP1, and SREBP-1. (c) Protein
quantification of PTP1B. (d) Protein quantification of P-PP2A/PP2A. (e) Protein quantification of P-SP1/SP1. (f) Protein quantification
of SREBP-1. (g) The activity of PP2A. (h) The activity of SP1. All values are presented as the mean ± SD (n=6). ∗P<0:05 for Con
group vs PM
2.5
group and
#
P<0:05 for PM
2.5
group vs PM
2.5
+Mel group.
8 Oxidative Medicine and Cellular Longevity

PM2.5 concentration (𝜇g/ml)
0 6.25 12.5 25 50 100
0.5
0.6
0.7
0.8
0.9
Cell viability (% of control)
1.0
1.1
⁎
⁎
⁎
(a)
PM2.5 concentration (𝜇g/ml)
0 12.5 25 50 100
0.00
0.01
0.02
0.03
T-CHO (mmol/g protein)
0.04
0.05
⁎⁎
⁎
(b)
PM2.5 concentration (𝜇g/ml)
0 12.5 25 50 100
0.00
0.05
0.10
0.15
TAGs (mmol/g protein)
0.20
⁎
⁎
(c)
PM2.5 concentration (𝜇g/ml)
0 12.5 25 50 100
0
2
4
6
8
ROS uorescence intensity
(fold control)
10 ⁎
⁎
⁎
(d)
101
0
20
40
60
80
100
Count (%)
0 102103104
FITC-A
0 𝜇g/ml
105106107.2
12.5 𝜇g/ml
25 𝜇g/ml
50 𝜇g/ml
100 𝜇g/ml
(e)
Figure 4: Continued.
9Oxidative Medicine and Cellular Longevity

MERGE FITC DAPI
0 𝜇g/ml 12.5 𝜇g/ml 25 𝜇g/ml 50 𝜇g/ml 100 𝜇g/ml
(f)
PM2.5 concentration (𝜇g/ml)
0 12.5 25 50 100
PTP1B
PP2A
P-PP2A
SP1
P-SP1
SREBP-1
GAPDH
(g)
0
12.5
25
50
100
0.00
PM2.5 concentration
(𝜇g/ml)
0.05
0.10
0.15
0.20
Relative PTP1B protein levels
⁎
⁎
(h)
0
12.5
25
50
100
0.0
0.5
1.0
1.5
Relative P-PP2A/PP2A
protein levels
⁎
⁎
PM2.5 concentration
(𝜇g/ml)
(i)
0
12.5
25
50
100
0.0
0.2
0.4
0.6
0.8
1.0
Relative P-SP1/SP1 protein levels
⁎
⁎
PM2.5 concentration
(𝜇g/ml)
(j)
Figure 4: Continued.
10 Oxidative Medicine and Cellular Longevity

from light. PBS was used to dilute the solution to 10 μM,
which was used for incubation with the cells at room tem-
perature in the dark for 20 min; finally, the cells were
observed via a confocal microscope (LSCM, TCS SP8 STED,
Germany).
2.11. Detection of ROS Levels in L02 Cells. The level of ROS
in L02 cells was analyzed by flow cytometry. After the cells
were infected for 24 h, a 2′,7′-dichlorofluorescein diacetate
(DCFH-DA, Sigma, USA) working solution (10 μM) was
added followed by incubation at 37
°
C for 30 min. The cells
were washed twice with PBS, and ROS levels were deter-
mined by flow cytometry. The single-parameter histograms
were obtained by taking the logarithm of fluorescence signal
as abscissa and the number of cells as ordinate, which could
intuitively reflect the relative intensity of ROS in living cells.
The average fluorescence intensity was the number of cells
divided by the area under each peak. ROS fluorescence was
measured with a confocal scanning laser microscope. To
quantify the ROS production in L02 cells treated with
0
12.5
25
50
100
0.00
PM2.5 concentration
(𝜇g/ml)
0.02
0.04
0.06
0.08
0.10
Relative SREBP-1 protein levels
⁎
⁎
(k)
0
12.5
25
50
100
0.0
0.2
0.4
0.6
0.8
Relative Nrf2 protein levels
⁎
⁎
⁎
PM2.5 concentration
(𝜇g/ml)
(l)
0
12.5
25
50
100
0.0
0.1
0.2
0.3
0.4
Relative Keap1 protein levels
⁎
PM2.5 concentration
(𝜇g/ml)
(m)
0
12.5
25
50
100
0.0
0.2
0.4
0.6
0.8
Relative SOD protein levels
⁎
PM2.5 concentration
(𝜇g/ml)
(n)
0 12.5 25 50 100
GAPDH
SOD
Keap1
Nrf2
PM2.5 concentration (𝜇g/ml)
(o)
Figure 4: PM
2.5
induced lipid accumulation in hepatocytes. (a) Cell viability. (b) Total cholesterol lipid levels (mmol/g). (c) Triacylglycerol
lipid levels (mmol/g). (d) Representative fluorescence intensity images obtained from flow cytometry in L02 cells. (e) Analysis of
fluorescence intensity obtained from flow cytometry. (f) Representative confocal images of ROS. (g) Western blotting of PTP1B, PP2A,
P-PP2A, SP1, P-SP1, and SREBP-1. (h) Protein quantification of PTP1B. (i) Protein quantification of P-PP2A/PP2A. (j) Protein
quantification of P-SP1/SP1. (k) Protein quantification of SREBP-1. (l) Protein quantification of Nrf2. (m) Protein quantification of
Keap1. (n) Protein quantification of SOD. (o) Western blotting of Nrf2, Keap1, and SOD. All values are presented as the mean ± SD.∗P
<0:05.
11Oxidative Medicine and Cellular Longevity

MERGE FITC DAPI
Con NAC PM2.5 PM2.5+NAC
(a)
0
0
20
40
60
80
100
101102103
Count (%)
104105
FITC-A
106107.2
Con
NAC
PM2.5
PM2.5+NAC
(b)
Con
NAC
PM2.5
PM2.5+NAC
ROS uorescence intensity
(fold of control)
0
1
2
3
4
5⁎
⁎⁎
(c)
MERGE FITC DAPI
Con NAC PM2.5 PM2.5+NAC
(d)
GAPDH
SREBP-1
P-SP 1
SP1
P-PP2A
PP2A
PTP1B
Con NAC PM2.5 PM2.5+NAC
(e)
Con
NAC
PM2.5
PM2.5+NAC
0.0
0.2
0.4
0.6
0.8
Relative PTP1B protein levels
⁎⁎
(f)
Figure 5: Continued.
12 Oxidative Medicine and Cellular Longevity

PM
2.5
and/or melatonin, cells were pretreated with the ROS
inhibitor N-acetylcysteine (NAC; Sigma, USA) (1 mM) for
1 h before PM
2.5
and/or melatonin exposure.
DCFH-DA, intracellular reactive oxygen species detec-
tion probe, is a universal indicator of oxidative stress. After
it enters the cell, it is hydrolyzed to produce DCFH. Intracel-
lular reactive oxygen species can oxidize nonfluorescent
DCFH to produce fluorescent DCF. Intracellular reactive
oxygen species (ROS) levels were obtained by measuring
the fluorescence intensity of DCF.
2.12. Real-Time Polymerase Chain Reaction (qPCR). Total
RNA from L02 cells and liver tissue was extracted using TRI-
zol™Reagent (Thermo, USA). According to the protocol,
the RNA was reverse transcribed into cDNA with Prime-
Script RT Master Mix (Takara, China). SYBR® Premix Ex
Taq™II (Takara, China) was used for quantitative PCR on
a Realplex2 (Eppendorf, Germany). The mRNA primers
are listed in Table 1.
2.13. Western Blot Analysis. Protein extracts of mouse liver
tissue and L02 cells were prepared by a Whole Cell Lysis
Assay Kit (Keygen Biotech, China), and the concentrations
were determined by a BCA protein quantitative assay kit
(Dingguo Changsheng Biotech, China). The same amounts
of protein were separated by electrophoresis using 8%–12%
SDS-PAGE gels and transferred to suitably sized nitrocellu-
lose membranes (Pall Corp., USA). After blocking with 5%
BSA or 5% skim milk in Tris-buffered saline (TBS), the
membranes were incubated with primary antibodies at 4
°
C
overnight, including PTP1B (Abcam, UK), SP1 (Abcam,
UK), P-SP1 (Abcam, UK), SOD (Abcam, UK), Nrf2
(Abcam, UK), Keap1 (Abcam, UK), PP2A (Santa, USA), P-
PP2A (Santa, USA), SREBP-1 (Santa, USA), and GAPDH
(CST, USA). The next day, the membranes were washed
three times with TBST and incubated with an anti-rabbit/
mouse IgG secondary antibody (CST, USA). A LI-COR
Odyssey system (LI-COR Biosciences, USA) was used for
detection of the protein bands, which were quantified using
Image Studio software (NIH, Bethesda, MD).
2.14. The Addition of PTP1B Inhibitor. We dissolve PTP1B
inhibitor PTP1B-IN-1 (MedChemExpress, USA) in DMSO
to prepare a stock solution at a concentration of 20 mM.
Then, L02 cells were treated with PTP1B-IN-1 (5 μg/mL,
10 μg/mL, 20 μg/mL, and 40 μg/mL) diluted with DMEM
for 12 h. After qPCR anal ysis, 10 μg/mL was selected as the
dose of PTP1B-IN-1.
2.15. Statistical Analysis. SPSS 24.0 software was used to ana-
lyze all experimental data. Data are presented as the mean
±SD. Data consistent with a normal distribution and an
Con
NAC
PM2.5
PM2.5+NAC
0
1
2
3
Relative P-PP2A/PP2A
protein levels
⁎⁎
(g)
Con
NAC
PM2.5
PM2.5+NAC
0.0
0.5
1.0
1.5
Relative P-SP1/SP1
protein levels
⁎⁎
(h)
Con
NAC
PM2.5
PM2.5+NAC
0.0
0.1
0.2
0.3
Relative SREBP-1 protein levels
⁎⁎
(i)
Figure 5: PM
2.5
induced lipid accumulation in hepatocytes by increasing ROS levels and PTP1B expression. (a) BODIPY staining of L02
cells (scale bar, 50 μm). (b) Representative fluorescence intensity images obtained from flow cytometry in L02 cells. (c) Analysis of
fluorescence intensity obtained from flow cytometry. (d) Representative confocal images of ROS. (e) Western blotting of PTP1B, PP2A,
P-PP2A, SP1, P-SP1, and SREBP-1. (f) Protein quantification of PTP1B. (g) Protein quantification of P-PP2A/PP2A. (h) Protein
quantification of P-SP1/SP1. (i) Protein quantification of SREBP-1. All values are presented as the mean ± SD.∗P<0:05.
13Oxidative Medicine and Cellular Longevity

Melatonin concentration (𝜇mol/L)
0
12.5
25
50
100
200
0.6
0.8
1.0
1.2
1.4
Cell viability (% of control)
⁎
⁎
⁎
(a)
0.7
0.8
1.0
0.9
1.1
1.2
Cell viability (% of control)
Con
Mel
PM2.5
PM2.5+Mel
⁎
(b)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.5
1.0
1.5
2.0
2.5
T-CHO (mmol/g protein)
⁎⁎
(c)
Con
Mel
PM2.5
PM2.5+Mel
0.1
0.3
0.2
0.5
0.4
TAGs (mmol/g protein)
⁎⁎
(d)
Con
MERGE FITC DAPI
Mel PM2.5 PM2.5+Mel
(e)
–102.9 0
0
20
40
60
80
100
Count (%)
103104105
FITC-A
106106.8
Con
Mel
PM2.5
PM2.5+Mel
(f)
Figure 6: Continued.
14 Oxidative Medicine and Cellular Longevity

ROS uorescence intensity
(fold of control)
Con
0
1
2
3
4
Mel PM2.5 PM2.5+Mel
⁎⁎
(g)
0
1
2
###
#
3
mRNA expression
4
PTP1B SP1 PP2A SREBP-1
Con
Mel
PM2.5
PM2.5+Mel
⁎
⁎
⁎
⁎
(h)
Con
GAPDH
SREBP-1
P-SP1
SP1
P-PP2A
PP2A
PTP1B
Mel PM2.5 PM2.5+Mel
(i)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.1
0.2
0.3
Relative PTP1B protein levels
⁎⁎
(j)
Con
Mel
PM2.5
PM2.5+Mel
0
2
1
4
3
5
Relative P-PP2A/PP2A
protein levels
⁎⁎
(k)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.2
0.1
0.3
0.4
0.5
Relative P-SP1/SP1 protein levels
⁎⁎
(l)
Con
Mel
PM2.5
PM2.5+Mel
0.00
0.10
0.05
0.15
0.20
Relative SREBP-1 protein levels
⁎⁎
(m)
Con
Mel
PM2.5
PM2.5+Mel
80
100
120
140
160
PP2A activity (mU/ml)
⁎⁎
(n)
Figure 6: Continued.
15Oxidative Medicine and Cellular Longevity

⁎⁎
Con
Mel
PM2.5
PM2.5+Mel
6
8
10
12
14
SP1 activity (ng/ml)
(o)
0
1
2
mRNA expression
3
Nrf2 Keap1
##
#
SOD
Con
Mel
PM2.5
PM2.5+Mel
⁎⁎
⁎
(p)
Con
Nrf2
Keap1
SOD
GAPDH
Mel PM2.5 PM2.5+Mel
(q)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.4
0.2
0.6
0.8
Relative Nrf2 protein levels
⁎⁎
(r)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.4
0.2
0.6
Relative Keap1 protein levels
⁎⁎
(s)
Con
Mel
PM2.5
PM2.5+Mel
0.0
0.4
0.2
1.0
0.6
0.8
Relative SOD protein levels
⁎⁎
(t)
Figure 6: Melatonin alleviated PM
2.5
-induced oxidative damage and upregulated PTP1B expression in vitro. (a) and (b) Cell ability. (c)
Total cholesterol lipid levels (mmol/g). (d) Triacylglycerol lipid levels (mmol/g). (e) Representative confocal images of ROS. (f)
Representative fluorescence intensity images obtained from flow cytometry in L02 cells. (g) Analysis of fluorescence intensity obtained
from flow cytometry. (h) The mRNA expression of PTP1B, PP2A, SP1, and SREBP-1. (i) Western blotting of PTP1B, PP2A, P-PP2A,
SP1, P-SP1, and SREBP-1. (j) Protein quantification of PTP1B. (k) Protein quantification of P-PP2A/PP2A. (l) Protein quantification of
P-SP1/SP1. (m) Protein quantification of SREBP-1. (n) The activity of PP2A. (o) The activity of SP1. (p) The mRNA expression of Nrf2,
Keap-1, and SOD. (q) Western blotting of Nrf2, Keap-1, and SOD. (r) Protein quantification of Nrf2. (s) Protein quantification of Keap-
1. (t) Protein quantification of SOD. All values are presented as the mean ± SD.∗P<0:05 for Con group vs PM
2.5
group and
#
P<0:05
for PM
2.5
group vs PM
2.5
+Mel group.
16 Oxidative Medicine and Cellular Longevity

0
0
PM2.5
Mel
PTP1B-inhibitor
101102103104105106107.2
20 40 60
Count (%)
80 100
−−−−
−−
−+
++
++++
++
+
+
+
−
−−
−−
FITC-A
(a)
0
1
2
ROS uorescence intensity
(fold of control)
3
4
PM2.5
Mel
PTP1B-inhibitor
−−
−−
−−
−+
++
++++
++
+
+
+
−
−−
−−
⁎
⁎
(b)
PM2.5
Mel
PTP1B-inhibitor
PTP1B
PP2A
P-PP2A
SREBP-1
GAPDH
SP1
P-SP 1
−−−−
−−
−+
++
++++
++
+
+
+
−
−−
−−
(c)
0.0
0.2
0.4
0.6
0.8
Relative PTP1B protein levels
1.0
PM2.5
Mel
PTP1B-inhibitor
−−−−
−−
−+
++
++++
++
+++−
−−
−−
⁎
⁎
⁎
(d)
Figure 7: Continued.
17Oxidative Medicine and Cellular Longevity

equal variance were tested by one-way ANOVA or two-way
ANOVA. Among them, the data with only one variable of
PM
2.5
adopted one-way ANOVA, and the data with two var-
iables of melatonin and PM
2.5
adopted two-way ANOVA.
The Kruskal-Wallis test was used for nonparametric data.
A value of p<0:05 indicates statistical significance. Each
experiment was repeated at least three times.
3. Results
3.1. Melatonin Alleviated the PM
2.5
-Induced Fatty Increase
and Steatosis in ApoE
-/-
Mice. To evaluate the effects of
PM
2.5
on liver lipid accumulation in mice, we first confirmed
that PM
2.5
induced liver changes by ultrasound examination.
The contrast of liver-kidney echo is one of the obvious man-
ifestations of a fatty liver. Compared to the control group,
ultrasonography showed that the echo of the liver paren-
chyma was high and dense, and the contrast sign of the liver
and kidney was positive in the PM
2.5
group, but this expres-
sion was relieved in the melatonin group (Figure 1(a)). The
anterior-posterior diameter and left-right diameter of the
liver can reflect changes in liver size. Although these two
indicators did not change significantly between the PM
2.5
and control groups, there was a significant difference
between the PM
2.5
and melatonin groups (Figure 1(e)). Con-
cordant with this, analysis of the body weights, liver weights,
and liver coefficient of the mice showed that the liver size of
the PM2.5 group was significantly higher than that of the
control group, and that melatonin had a slight alleviating
effect on liver weight gain (Supplementary Figure S1E-1G).
Histological examinations of the liver are presented in
Figures 1(b)–1(d). Electron microscopy images showed
large lipid droplets, and HE and Oil Red O staining
revealed notably enlarged adipocytes, fatty degeneration,
and specific lipid accumulation in PM
2.5
-treated mice
compared to the control group. However, treatment with
melatonin visibly alleviated these alterations. Next, changes
in lipid content in the liver were examined. The levels of
total cholesterol (TC) and triacylglycerols (TAGs) in the
livers increased in response to PM
2.5
, while melatonin
treatment significantly decreased lipid levels (Figures 1
(g)and 1(h)). In addition, the Masson staining and qPCR
analysis results of inflammatory factors (IL-1, IL-6, and
TNF-α) in liver tissue showed that PM
2.5
could cause liver
injury, and melatonin had a mitigating effect
(Supplementary Figure S1A-1D). Taken together, these
results suggested that PM
2.5
exposure could induce hepatic
lipid metabolism disorders and that melatonin treatment
had a beneficial effect on the liver.
3.2. Protective Effects of Melatonin on PM
2.5
-Induced
Oxidative Damage in Liver. Multiple studies have shown
PM2.5
Mel
PTP1B-inhibitor
−−−−
−−
−+
++
++++
++
+++−
−−
−−
0.0
0.5
1.0
1.5
2.0
Relative P-PP2A/PP2A
protein levels
⁎
⁎
⁎
(e)
PM2.5
Mel
PTP1B-inhibitor
−−−−
−−
−+
++
++++
++
+++−
−−
−−
0.0
0.5
1.0
1.5
Relative P-SP1/SP1
protein levels
⁎⁎
⁎
(f)
PM2.5
Mel
PTP1B-inhibitor
−−−−
−−
−+
++
++++
++
+++−
−−
−−
0.0
Relative SREBP-1
protein levels
0.1
0.2
0.3
0.4 ⁎
(g)
Figure 7: PTP1B inhibitor preconditioning eliminated lipid dysregulation in hepatocytes caused by PM
2.5
and melatonin intervention. (a)
Representative fluorescence intensity images obtained from flow cytometry in L02 cells. (b) Analysis of fluorescence intensity obtained from
flow cytometry. (c) Western blotting of PTP1B, PP2A, P-PP2A, SP1, P-SP1, and SREBP-1. (d) Protein quantification of PTP1B. (e) Protein
quantification of P-PP2A/PP2A. (f) Protein quantification of P-SP1/SP1. (g) Protein quantification of SREBP-1. All values are presented as
the mean ± SD.∗P<0:05.
18 Oxidative Medicine and Cellular Longevity

that PM
2.5
aggravates lipid metabolism disorder by inducing
oxidative stress. To determine the effects of PM
2.5
/melatonin
on ROS production, liver sections were stained with the fluo-
rescent probe DHE to evaluate ROS levels. As shown in the
representative fluorescence micrographs (red fluorescence)
and histogram of ROS relative fluorescence density
(Figures 2(a) and 2(b)), PM
2.5
treatment increased ROS gen-
eration, while melatonin supplementation alleviated ROS
generation. MDA and 4-HNE are important indexes of lipid
peroxidation. According to the quantitative analysis, expo-
sure to PM
2.5
resulted in significantly increased levels of
MDA and 4-HNE, whereas melatonin treatment reversed
these effects (Figures 2(c) and 2(d)). Moreover, the degree
of oxidative stress was detected by examination of GSH-Px
and SOD. As anticipated, PM
2.5
treatment reduced the activ-
ity of GSH-Px and SOD in the liver compared to the control.
However, these two indicators were reversed by melatonin
(Figures 2(e) and 2(f)). Subsequent analysis of the protein
and mRNA expression levels of known indicators of oxida-
tive stress, including Keap1, Nrf2, and SOD, was performed.
In the present study, PM
2.5
exposure decreased Nrf2 and
SOD mRNA expression and increased Keap1 mRNA expres-
sion, and these negative effects were mitigated by melatonin
(Figure 2(g)). Consistently, compared with the control
group, the expression of Nrf2/Keap1 and the SOD protein
in the PM
2.5
group was not significantly different, but there
were significant changes after melatonin treatment
(Figures 2(h)–2(k)). However, this result did not rule out
an impact on their gene expression. Overall, these data sug-
gested that the antioxidative stress effects of melatonin
might protect the liver from PM
2.5
exposure.
3.3. Melatonin Ameliorated Abnormal Liver Lipid
Metabolism and Caused Elevated PTP1B Expression
Induced by PM
2.5
.To identify the potential mechanisms by
which PM
2.5
or melatonin induced gene expression in lipid
accumulation, qPCR analysis of genes related to lipid metab-
olism was examined in liver samples. Analysis of the mRNA
expression levels showed that PM
2.5
exposure was associated
with lipid metabolism disorder, and PTP1B, which is closely
related to MAFLD, was an important upregulated transcrip-
tion factor (Supplementary Figure S2). PTP1B is a key
regulator of the antioxidant system and an activator of
liver adipogenesis. Next, the regulation of downstream
PTP1B on genes related to liver lipid metabolism was
verified by qPCR analysis. As expected, PM
2.5
exposure
significantly increased the expression of lipid accumulation
markers (PP2A, SP1, and SREBP-1), whereas exogenous
melatonin treatment decreased their levels (Figure 3(a)).
Lipid accumulation plays an important role in the
progression of MAFLD. Additionally, for further
verification, Western blotting was carried out. PM
2.5
administration led to an increase in the accumulation of
PTP1B, P-PP2A/PP2A, P-SP1/SP1, and SREBP-1, and
subsequent analysis showed that melatonin inhibited their
expression (Figures 3(b)–3(f)). However, the enzymatic
activities of PP2A and SP1 did not change significantly in
PM
2.5
-exposed mice (Figures 3(g) and 3(h)). These results
PM2.5
ROS
Melatonin
PP2A PP2A
PTP1B
SP1 SP1
SREBP-1
Normal liver cells
Metabolic-associated fatty liver disease
pY
pY
Nrf2/Keap1
SOD
Lipogenesis / Steatosis
Lipid drops
Figure 8: Schematic of melatonin ameliorating PM
2.5
-induced hepatic lipid accumulation.
19Oxidative Medicine and Cellular Longevity

could be greatly downregulated by melatonin treatment. All
of the above data indicated that melatonin supplementation
reduced adiposity accumulation triggered by PM
2.5
.
3.4. PM
2.5
Exposure Caused Lipid Accumulation in L02 Cells
by Inducing ROS Production. The cytotoxic effects of PM
2.5
on L02 cell viability was assessed by CCK-8 assay. As evi-
denced in Figure 4(a), the viability of L02 cells decreased
with increasing doses of PM
2.5
. Treatment with between 25
and 100 μg/mL PM
2.5
for 24 h showed significant differences
compared with untreated cells. To detect the effects of PM
2.5
on lipid synthesis, L02 cells were exposed to various doses of
PM
2.5
(0–100 μg/mL) for 24 h. As expected, the contents of
T-CHO and TAGs in L02 cells gradually increased as the
concentration of PM
2.5
increased (Figures 4(b) and 4(c)).
Next, ROS generated by PM
2.5
treatment were detected by
flow cytometry analysis quantification and confocal micros-
copy (Figures 4(d)–4(f)). Compared to the control group,
the ROS fluorescence intensity was observably increased in
PM
2.5
-treated L02 cells, which occurred in a manner depen-
dent on the PM
2.5
concentration. Then, activation of the
PTP1B pathway was determined after PM
2.5
exposure.
Quantitative measurements of protein expression showed
that P-PP2A/PP2A, P-SP1/SP1, and SREBP-1 expression
was dependent on the PM
2.5
concentration (Figures 4(g)–
4(k)). In addition, activation of the Nrf2/Keap1 pathway
was detected by Western blot. Low-dose PM
2.5
induced the
upregulation of Nrf2 and SOD protein expression, while
high-dose PM
2.5
inhibited their expression. The opposite
trend was observed for Keap1 (Figures 4(l)–4(o)). Given
the above data, although the effect indexes in the 25 μg/mL
dose group were significantly different, the expression of
the proteins in the PTP1B pathway was significantly differ-
ent in the 50 μg/mL dose group, and the Nrf2/Keap1 path-
way was inhibited in the 50 μg/mL dose group. For this
reason, the concentration of PM
2.5
(50 μg/mL) was selected
for subsequent experiments.
To obtain more details on the role of oxidative stress in
PM
2.5
-induced liver lipid accumulation, NAC (N-acetylcys-
teine) was added to L02 cells exposed to PM
2.5
(Figure 5).
Representative images of BODIPY staining are shown in
Figure 5(a). NAC treatment significantly abolished the
PM
2.5
-induced increase in lipid content in L02 cells. Flow
cytometry and confocal microscopy data analysis indicated
that the fluorescence intensity of the ROS generated in the
NAC-treated cultures exposed to PM
2.5
(50 μg/mL) was sig-
nificantly less than that in PM
2.5
-exposed cells
(Figures 5(b)–5(d)). Additionally, NAC treatment restored
the expression of the proteins PTP1B, P-PP2A/PP2A, P-
SP1/SP1, and SREBP-1 in PM
2.5
-treated L02 cells to levels
comparable with the control (Figures 5(e)–5(i)). PM
2.5
expo-
sure initially triggered oxidative stress, which further led to
lipid peroxidation. In short, the effects of PM
2.5
exposure
on lipid accumulation in L02 cells were ROS dependent
and involved PTP1B signalling.
3.5. Melatonin Alleviated PM
2.5
-Induced Oxidative Damage
and Lipid Accumulation In Vitro. To determine the dosage
of melatonin to be used, CCK-8 assays were applied to
examine cell viability. With increasing concentrations of
melatonin, the viability of L02 cells first increased and then
decreased, and 200 μmol/L melatonin showed a significant
decrease compared to the control group (Figure 6(a)). More-
over, the effects of PM
2.5
and melatonin on cell viability were
investigated (Figure 6(b)). Finally, 100 μmol/L melatonin
was selected for further experiments. First, the protective
effects of melatonin on lipid accumulation were detected
by measuring the levels of TC and TAGs (Figures 6(c) and
6(d)). The results indicated that melatonin decreased the
lipid levels in PM
2.5
-induced L02 cells compared with
PM
2.5
treatment alone. Flow cytometry and confocal micros-
copy data analysis indicated that treatment with melatonin
could restore ROS to control levels (Figures 6(e)–6(g)). Fur-
thermore, melatonin altered the mRNA levels of PTP1B,
PP2A, SP1, and SREBP-1 in the presence of PM
2.5
(Figure 6(h)). Similarly, the levels of the proteins in the
PTP1B pathway showed that melatonin reduced PM
2.5
-
induced lipid accumulation (Figures 6(i)–6(m)). Intrigu-
ingly, melatonin restored the PM
2.5
-mediated increase in
PP2A and SP1 activity to a normal level (Figures 6(n) and
6(o)). Moreover, PM
2.5
exposure decreased the Nrf2 and
SOD mRNA expression and increased the Keap1 mRNA
expression, and these negative effects were mitigated by mel-
atonin (Figure 6(p)). Melatonin activation of the Nrf2/Keap1
pathway represented relief of oxidative stress after PM
2.5
treatment. Compared to the control group, Nrf2/Keap1
and SOD protein expression was significantly affected in
the melatonin group but only slightly changed after PM
2.5
exposure (Figures 6(q)–6(t)). It could therefore be concluded
that the relief of oxidative stress after melatonin treatment
could alleviate fat accumulation in L02 liver cells.
3.6. Melatonin Regulated Hepatic Lipid Metabolism through
the PTP1B and Nrf2 Signalling Pathways in PM
2.5
-Treated
L02 Cells. To confirm that PM
2.5
-induced hepatocyte steato-
sis occurred through the upregulation of PTP1B, a specific
inhibitor of PTP1B (10 μg/mL) was used in this study.
Firstly, the dose was determined by PCR to detect the inhib-
itory effect on PTP1B mRNA expression (Supplementary
Figure S3). As shown in Figures 7(a) and 7(b), both
melatonin and the PTP1B inhibitor had significant ROS
scavenging ability. Compared with PM
2.5
treatment alone,
melatonin treatment decreased the ROS levels by
approximately 40%, and the PTP1B inhibitor decreased the
ROS levels by approximately 80%. The levels of melatonin
and PTP1B inhibitor in PM
2.5
-exposed cells were also
significantly lower than those in the group treated with
both melatonin and PM
2.5
. Similar to melatonin, the
inhibitor downregulated the expression of PTP1B and its
downstream molecular proteins (PTP1B, P-PP2A/PP2A, P-
SP1/SP1, and SREBP-1) that regulate lipid production. The
synergistic effects of melatonin and the inhibitor were
greater than the effects of melatonin alone (Figures 7(c)–
7(g)). Consistent with the results above, PTP1B was the
direct target gene of PM
2.5
-induced oxidative stress, and
inhibition of PTP1B expression downregulated the
expression of its downstream gene SREBP-1 and reduced
ROS production, thus reducing lipid production in L02 cells.
20 Oxidative Medicine and Cellular Longevity

4. Discussion
Environmental PM
2.5
has been recognized as the largest
global threat affecting human health, including the develop-
ment of MAFLD [4]. The pathogenesis and molecular mech-
anisms of PM
2.5
-induced MAFLD have not yet been well
elucidated. In this study, we found that PM
2.5
induced oxi-
dative stress, which activated PTP1B and in turn regulated
ROS release with positive feedback. Moreover, melatonin
alleviated the interference of liver fat metabolism disorder
caused by PM
2.5
through regulation of the ROS-mediated
PTP1B and Nrf2 signalling pathways.
MAFLD is a redefinition of NAFLD (nonalcoholic fatty
liver disease). MAFLD represents a general overview of com-
mon liver metabolic disorders (not just nonalcoholics) and
has multiple dominant drivers of subphenotypic response
diseases. It covers more than NAFLD and has more specific
diagnostic criteria [41]. Therefore, the more accurate and
clear terminology of MAFLD was adopted in this work,
which may provide a much wider applicability for our subse-
quent study to explore the toxic mechanism of PM
2.5
. In our
study, ultrasonography showed that in the PM
2.5
exposure
group, the liver had a smooth contour, sharp edges, and
increased parenchymal echo density, and positive contrast
signs were also observed in the liver and kidney (Figure 1).
The hepatorenal index is an important indicator of a fatty
liver [42]. Similarly, the histopathological observation results
(Oil Red O and HE staining) showed obvious fat vacuoles of
varying sizes in the livers of PM
2.5
-treated mice, which pre-
sented with severe steatosis (Figure 1). Exposure to PM
2.5
has been reported to cause systemic IR and increase the
accumulation of hepatic lipids in the liver, which is consis-
tent with our findings [43]. In addition, we observed
increases in T-CHO and TAGs in mouse livers and human
hepatocytes exposed to PM
2.5
, indicating that the induced
lipid metabolism disorders were severe, as evidenced by the
lipid index (Figures 1 and 4). The liver is a central organ
for lipid homeostasis and energy metabolism [44]. Liver ste-
atosis is caused by an imbalance in lipid homeostasis, where
lipid absorption or de novo fat production exceeds lipid oxi-
dation or output [45]. Here, our results revealed that PM
2.5
exposure induced significant lipid accumulation in the liver
accompanied by an increase in liver volume, suggesting that
PM
2.5
exposure triggered pathological changes in liver mor-
phology and function.
To date, many studies have demonstrated that MAFLD
is closely related to oxidative stress induced by PM
2.5
expo-
sure [5]. Oxidative stress is caused by the imbalance between
the production of ROS and the ROS scavenging activity [46].
Excessive ROS results in increased adipogenesis and
decreased β-oxidation of fatty acids, which leads to the accu-
mulation of triacylglycerols in hepatocytes [47]. We studied
the induction of ROS generation by PM
2.5
in ApoE
-/-
mice,
and the data revealed that PM
2.5
promoted ROS production,
decreased SOD activity, and induced lipid peroxidation (as
evidenced by the levels of 4-HNE and MDA) (Figure 2).
Consistent with the results of the animal experiments,
PM
2.5
increased intracellular ROS in a dose-dependent man-
ner in L02 cells (Figure 4). Compared with reliable evidence
that PM
2.5
exposure can upregulate ROS pathways, the reg-
ulation and defence of antioxidant mechanisms are relatively
scarce. We further examined the mRNA and protein levels
of antioxidant stress markers, namely, Nrf2/Keap1 and
SOD. The qPCR results showed that PM
2.5
acted by upregu-
lating the Nrf2 inhibitor Keap1. Compared with the control
group, Nrf2 protein expression was slightly downregulated
in the PM
2.5
group, but there was no significant difference
(Figure 2). The reason for this result may be that at the tran-
scriptional and translational levels, the course of mRNA
translation into proteins is adjusted by a variety of factors,
which may lead to an inconsistency between mRNA and
protein expression [48]. Interestingly, the expression of
Nrf2 was upregulated in the low-dose PM
2.5
group and
downregulated in the high-dose PM
2.5
group, while the
expression of Keap1 showed the opposite trend (Figure 4).
It may be that the low dose of PM
2.5
induces the body’s
stress response and activates Nrf2. However, with increasing
exposure dose, PM
2.5
inhibits the Nrf2 pathway. Similarly, it
has been found that low concentrations of PM
2.5
slightly
upregulate Nrf2 expression, and subsequently, PM
2.5
treat-
ment dose-dependently decreases Nrf2 expression [10].
These studies have shown that PM
2.5
induces ROS produc-
tion and changes in antioxidant genes, which play vital reg-
ulatory roles in the progression of MAFLD.
ROS overproduction can modulate many cellular events
involved in hepatic lipid metabolism diseases by regulating a
variety of disease-related targets, such as PTP1B [49]. In
addition, PTP1B levels were significantly elevated in the
hepatocytes of fructose-fed hamsters, HFD-fed mice, and
fatty liver and insulin-resistant animal models [50]. The
overexpression of PTP1B in liver cells increased the expres-
sion level and transcriptional activity of SREBP1, which
resulted in the increased synthesis of liver triacylglycerols
and fatty acids [51]. This was due to the enhanced transcrip-
tional activity of the recombinant SP1 site in the SREBP1
promoter by increasing the activity of PP2A when PTP1B
was overexpressed [52]. This study found that PM
2.5
could
upregulate PTP1B expression by inducing ROS generation,
and the expression level of PTP1B and ROS production
was dose-dependent (Figures 3, 4, and 6). Especially, PM
2.5
and melatonin did not affect the activity of PP2A and SP1
(Figure 3). This may be related to the posttranslational mod-
ification of proteins [53, 54]. NAC preconditioning inhibited
PM
2.5
-induced PTP1B overexpression, suggesting that ROS
plays an important role in PTP1B activation after PM
2.5
exposure (Figure 5). Surprisingly, we found that PTP1B reg-
ulated by PM
2.5
had positive feedback regulation on ROS
release. As shown in Figure 7, pretreatment of L02 cells with
PTP1B inhibitors significantly reduced ROS production and
subsequently downregulated the PTP1B downstream pro-
teins PP2A, SP1, and SREBP1. Based on previous evidence,
PTP1B KO mice showed decreased ROS production and
lipid peroxidation in the liver [55]. To our knowledge, our
results provide new evidence of the mechanism by which
PM
2.5
exposure promotes the occurrence and development
of MAFLD, demonstrating that PM
2.5
exposure activates
the ROS/PTP1B pathway and that PTP1B regulates ROS
by positive feedback.
21Oxidative Medicine and Cellular Longevity

Many studies have shown that the nocturnal indole
melatonin produced by the pineal gland is effective against
metabolic syndrome [56]. More recently, melatonin has
been shown to reverse the harmful effects of fructose in
the diet, and this animal model modulates metabolic path-
ways such as lipid production, β-oxidation, lipolysis, and
gluconeogenesis [57]. Melatonin may also be ingested in
the liver in a dose-dependent manner through specific cel-
lular and nuclear receptors [58]. The pathogenesis of
MAFLD is complex, but melatonin may be the key to
the treatment of MAFLD. Furthermore, melatonin allevi-
ated hepatic steatosis and lipid accumulation in ApoE
-/-
mice under different experimental conditions [59]. A pre-
vious animal study showed that ROS mediated
lipopolysaccharide-induced SREBP-1c activation and lipid
accumulation in the liver. Melatonin might be used as a
pharmacological agent to prevent endotoxin-induced
MAFLD [60]. Liver lipotoxicity is closely related to hepatic
metabolic disorders caused by impaired fatty acid oxida-
tion and increased ROS production [61]. It may be helpful
to study the protective effects of melatonin on PM
2.5
-
induced hepatic fatty degeneration. Therefore, more
research is encouraged to explore this issue. In our study,
as expected, melatonin mitigated steatosis and decreased
the lipid content of the liver during PM
2.5
damage. Both
animal and cell experiments showed that melatonin effec-
tively reduced ROS levels and helped to downregulate
PTP1B and increase Nrf2 expression in PM
2.5
-treated
groups, thereby changing the effects of PM
2.5
exposure
on liver lipid accumulation.
The schematic diagram summarizing these results and
mechanisms shows that PM
2.5
-induced ROS accumulation
simultaneously promotes fat generation signal transduction
by activating the PTP1B-PP2A-SP1-SREBP1 axis and inhi-
biting the Nrf2/Keap1-SOD axis, resulting in lipid accumu-
lation and promotion of the occurrence and development
of MAFLD. Moreover, melatonin plays an antioxidative
stress role and regulates the ROS-mediated PTP1B and
Nrf2 signalling pathways by inhibiting ROS production to
alleviate the harmful effects induced by PM
2.5
(Figure 8). A
comprehensive study of ROS targets could not only provide
insight into the mechanism of PM
2.5
-induced MAFLD but
also give more evidence for the clinical applications of
melatonin.
5. Conclusions
In summary, this study shows that PM
2.5
promoted the
occurrence and development of MAFLD in ApoE
-/-
mice.
Excess accumulation of PM
2.5
-induced ROS could activate
PTP1B, which in turn had a positive feedback regulation
effect on ROS release. Our study is the first to show that mel-
atonin alleviated the disturbance of PM
2.5
-triggered hepatic
steatosis and liver damage by regulating the ROS-mediated
PTP1B and Nrf2 signalling pathways. These results suggest
that melatonin administration may be a prospective therapy
for the prevention and treatment of MAFLD associated with
air pollution.
Data Availability
Most of data and materials generated or analyzed during this
study are included in this manuscript. Other data are avail-
able from the corresponding authors on reasonable request.
Ethical Approval
The animal experiment in this study was approved by the
Animal Ethics Committee of Capital Medical University
(Ethics No. AEEI-2016-076).
Consent
Not applicable.
Conflicts of Interest
The authors declare they have no conflict of interest.
Authors’Contributions
Zhiwei Sun and Junchao Duan conceived and designed the
experiments. Zhou Du, Shuang Liang, Jingyi Zhang, and
Qing Xu performed the experiments. Zhou Du, Yang Li,
Yang Yu, and Qing Xu analyzed the data. Zhiwei Sun and
Junchao Duan contributed reagents/materials/analysis tools.
Zhou Du and Junchao Duan wrote the paper.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (91943301, 92043301, 81930091, and
81973077), Beijing Natural Science Foundation Program
and Scientific Research Key Program of Beijing Municipal
Commission of Education (KZ202110025040).
Supplementary Materials
Figure S1: liver damage effects of PM
2.5
. Figure S2: mRNA
expression levels associated with PM
2.5
exposure to liver
injury. Figure S3: mRNA expression of PTP1B after treat-
ment with PTP1B inhibitor. Table S1: concentrations of
inorganic elements in PM
2.5
. Table S2: content of water sol-
uble ions in PM
2.5
. Table S3: introduction of genes related to
liver injury caused by PM
2.5
.(Supplementary Materials)
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24 Oxidative Medicine and Cellular Longevity