Received: 20 June 2022 Revised: 2 September 2022 Accepted: 5 September 2022

DOI: 10.1002/tox.23663

RESEARCH ARTICLE

Polystyrene microplastics induce autophagy and apoptosis in

birds lungs via PTEN/PI3K/AKT/mTOR

Hongmin Lu 1 | Kai Yin 1 | Heng Su 2 | Dongxu Wang 1 | Yue Zhang 1 |

Lulu Hou 1 | Jun bo Li 1 | Yu Wang 1 | Mingwei Xing 1

1
College of Wildlife and Protected Area,
Northeast Forestry University, Harbin, Abstract
People's Republic of China
Microplastics (MPs) seriously pollute and potentially threaten human health. Birds
2
College of Resources and Environment,
Northeast Agricultural University, Harbin,
are sentinels of environmental pollutants, which respond quickly to contamination
People's Republic of China events and reveal current environmental exposure. Therefore, birds are good bioindi-

Correspondence
cators for monitoring environmental pollutants. However, the mechanism of lung
Mingwei Xing and Yu Wang, College of injury in birds and the role of the PTEN/PI3K/AKT axis are unknown. In this study,
Wildlife and Protected Area, Northeast
Forestry University, Harbin 150040, broilers treated with different polystyrene microplastics (PS-MPs) (0, 1, 10, and
Heilongjiang, People's Republic of China. 100 mg/L) were exposed to drinking water for 6 weeks to analyze the effect of PS-
Email: xingmingwei@nefu.edu.cn and
wangyu2013@nefu.edu.cn MPs on lung injury of broilers. The results showed that with the increase of PS-MPs
concentration, malonaldehyde (MDA) content increased, and catalase (CAT) and glu-
Funding information
The Key Projects of Natural Science
tathione (GSH) activity decreased, further leading to oxidative stress. PS-MPs caused
Foundation of Heilongjiang Province of China, the PI3K/Akt/mTOR pathway to be inhibited by phosphorylation, and autophagy
Grant/Award Number: ZD2020C005
accelerated formation (LC3) and degradation (p62), causing autophagy. In PS-MPs
exposed lung tissues, the expression of Bax/Bcl-2 and Caspase family increased, and
MAPK signaling pathways (p38, ERK, and JNK) showed an increase in phosphoryla-
tion level, thus leading to cell apoptosis. Our research showed that PS-MPs could
activate the antioxidant system. The antioxidant system unbalance-regulated Cas-
pase family, and PTEN/PI3K/AKT pathways initiated apoptosis and autophagy, which
in turn led to lung tissue damage in chickens. These results are of great significance
to the toxicological study of PS-MPs and the protection of the ecosystem.

KEYWORDS
apoptosis, autophagy, birds lung, polystyrene microplastics, PTEN/PI3K/AKT/mTOR

1 | I N T RO DU CT I O N plastic production has been growing steadily.1,2 Yet, despite the gradual
improvement of people's environmental protection awareness, many
Microplastic (MPs) pollution is one of humanity's most urgent problems plastic materials were discarded, along with the garbage accumulating in
in the 21st century. Since the fifties of the last century, global annual the natural environment.3,4 In recent years discharge of plastic waste

Abbreviations: AKT, protein kinase B; ATG12, antithymocyte globulin 12; ATG5, antithymocyte globulin 5; Bax, Bcl-2 associated X protein; Bcl-2, B cell lymphoma/leukemia 2; Caspase3,
cysteinyl aspartate-specific proteinase 3; Caspase8, cysteinyl aspartate-specific proteinase 8; Caspase9, cysteinyl aspartate-specific proteinase 9; CAT, catalase; COPD, chronic obstructive
pulmonary disease; Cytc, cytochromec; DRP1, dynamin-related protein 1; ECL, chemiluminescence substrate kit; ERK, extracellular regulated protein kinases; FADD, Fas-associated death domain
protein; Fas, TNF receptor superfamily, member 6; GSH, glutathione; HE, hematoxylin–eosin; JNK, Jun N-terminal kinase; LC3, microtubule-associated protein 1A/1B-light chain; MARK,
mitogen-activated protein kinase; MDA, malonaldehyde; MFN, mitochondrial fission facto; MPs, microplastics; mTOR, mammalian target of rapamycin; OPA1, OpticAtrophy 1; p62, nuclear pore
glycoprotein P62; PBST, phosphate buffer containing Tween 20; PI3K, phosphatidylinositiol3-ki- nase; PS-MPs, polystyrene microplastics; PTEN, phosphatase and tensin homolog; PVDF,
polyvinylidene difluoride; QRT-PCR, quantitative real-time polymerase chain reaction; RIPA, radio-Immunoprecipitation assay; ROS, reactive oxygen species; UNEP, United Nations Environment
Program.

78 © 2022 Wiley Periodicals LLC. wileyonlinelibrary.com/journal/tox Environmental Toxicology. 2023;38:78–89.

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LU ET AL. 79

into the sea by coastal countries is about 9.6 million tons to 2540 million model; broilers treated with different concentrations of PS-MPs (0, 1,
tons.5 In the face of such a severe plastic pollution problem, the United 10, and 100 mg/L) were exposed to drinking water for 6 weeks to
Nations Environment Program (UNEP) has identified the pollution analyze the effect of PS-MPs on lung injury of broilers. PS-MPs could
caused by MPs as one of the top 10 global environmental problems cause oxidative stress, activate the MAPK pathway, reduce the
(UNEP, 2014). Plastic is everywhere: the sea, wastewater treatment expression level of PI3K/AKT in the lung, and finally induce lung
plants in oceans, lakes, rivers, estuaries, air, sediment, landfills, and injury. Our research aims to warn people about the harm of MPs to
6,7
water. The existence of polystyrene microplastics (PS-MPs), which is the environment and human health and provide new insights. PTEN/
as a new environmental pollutant because of their potential toxic effects PI3K/AKT and MAPK axis further clarified the toxicological mecha-
on humans and ecosystems, is causing concern.8 The invertebrates of nism of MPs. The specific relationship between MPs concentration
aquatic organisms, poultry, birds, vertebrates, and other invertebrates and tissue damage was explained. This work will provide a theoretical
often mistake PS-MPs for food, thus, causing them to accumulate along basis for the toxicological study of MPs, bird preservation, and ecolog-
the food chain to threaten human health.9,10 ical environment security.
Compelling evidence shows that ingested PS-MPs lead to various
aspects of tissue or organ dysfunction in vitro and vivo.11,12 PS-MPs
first enter the gastrointestinal tract, and these particles could subse- 2 | M A T E R I A L S A N D M ET H O D S
quently be transferred to tissue areas by cell internalization or blood
circulation.13 The mice were taken PS-MPs, resulting in intestinal dys- 2.1 | Treatment with test chemicals
regulation, inflammation, and disturbed liver metabolism.14 Related
studies have shown that short-term or long-term indraft PS-MPs can PS-MPs (5 μm) were purchased from Tesulang Chemical Company
significantly increase the morbidity and mortality of lung diseases, (China) and stored at room temperature. This study used a model ani-
including pulmonary fibrosis, pneumonia, asthma, chronic obstructive mal chicken belonging to birds. Birds belong to terrestrial vertebrate
pulmonary disease (COPD), and even lung cancer.15,16 However, and aquatic vertebrate species, and they mainly feed in aquatic envi-
whether ingestion of PS-MPs can cause lung damage is not completely ronments. Thus this study used concentrations associated with con-
clear. Studies have found that once PS-MPs accumulated in the intesti- tamination of aquatic MPs.
nal tract, they can be transferred from the intestinal cavity to the blood
and lymphoid organs and transferred to other tissues within the blood
circulatory system, such as the lungs and kidneys.13 PS-MPs can dam- 2.2 | Laboratory animals are raised
age the structure and function of birds' kidneys, cause increased
mucus secretion, and alter kidneys and other physiological responses, The Experimental Animal Committee of Northeastern Forestry Uni-
such as altered metabolic and reproductive activities.17,18 Moreover, versity approved the study (Approval No: UT-31; June 20, 2014). The
PS-MPs caused intestinal tract damage by producing reactive oxygen experimental process accorded with the regulations of the state and
species (ROS), inflammation, single-cell fibrosis, and DNA damage.19 Northeast Forestry University on the management and use of experi-
These studies suggest that exposure to PS-MPs causes damage to the mental animals.
digestive system and urinary systems. Nevertheless, the bird respira- In this study, 120 1-day-old high land broilers (purchased from
tory system is still unclear, and more data is urgently needed. Weiwei Co. Ltd, China) were adaptively reared for 1 week. One week
The imbalance of oxidation and antioxidation is necessary to later, they were randomly divided into four groups: standard diet
induce oxidative stress injury in cells.20 The signal pathway of phos- group (Con), low concentration group, PS-MPs 1 mg/L (L-MPs),
phatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinases, medium-concentration group PS-MPs 10 mg/L (M-MPs), and high
(PI3K)/protein kinase (AKT)/mammalian rapamycin target protein concentration group PS-MPs 100 mg/L (H-MPs). It is generally
(mTOR) regulates multiple cell behavior.21,22 Many studies have assumed that less than or equal to 1 mg/L is considered environmen-
shown that when cells undergo oxidative stress, many ROS can acti- tally relevant. Study data suggest that the toxicological effects of plas-
vate PTEN and inhibit the expression of the PI3K/AKT pathway, tic intake on health may not be as severe as commonly assumed, so
23
which is the main factor leading to cell death. In addition, ROS plays this study set a 10-fold gradient to study toxicity endpoints. Medium-
a crucial role in inducing apoptosis and autophagy by activating concentration (10 mg/L) and high concentrations (100 mg/L) were
mitogen-activated protein kinase (MAPK), including p38MAPK, extra- used as toxicological concentrations.27 There were 30 chickens in
cellular regulated protein kinases (ERK), and Jun N-terminal Kinase each group, and all four groups were fed with a regular basal diet. The
24
(JNK). The above studies suggest that oxidative stress regulates feed nutrient components are shown in Table S1. The drinking water
PTEN/PI3K/AKT and MAPK signaling pathways and plays a vital role was fed for 6 weeks with 0, 1, 10, and 100 mg/L PS-MPs. This paper's
in participating in cytopathological or toxicological changes. drinking water exposure treatment method is as follows: each time, a
Birds are sentinels of environmental pollutants, which respond new PS-MPs suspension is prepared and shaken with an ultrasonic
quickly to contamination events and reveal current environmental oscillator for 20 min. In addition, we remix the drinking water every
exposure. Therefore, birds are good bioindicators for monitoring envi- 2–3 h to ensure as little microplastic settling as possible. In this work,
ronmental pollutants.25,26 Here, We used broiler chickens as the study the experimental animal chicken's temperature, humidity, and light
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80 LU ET AL.

adjustment methods during the adaptation to the environment and (CAT), and malondialdehyde (MDA) (Nanjing Jiancheng Bioengineering
the experiment are as follows. One-day-old chickens were reared for Institute, Nanjing, China) kits. The activity of antioxidant CAT, the


a week at 33 C, moderate humidity (65%), and 16-h light length to level of antioxidant GSH, and the lipid peroxidation metabolite MDA
acclimate to the environment. According to the growing needs of the were measured.
chickens, gradually adjust the temperature and humidity, reduce the
temperature by 1
C every 3 days, and reduce the humidity by 5%
every week. Finally, the temperature was controlled at 22
C, the 2.7 | Western blotting
humidity was kept at 55%, and the sunshine time was kept at
about 16 h. Detection of the expression of autophagy and apoptosis-related pro-
teins. Lung tissues were collected after 6 weeks of MPs exposure in
all groups of broilers. The total protein was extracted by the Radio-
2.3 | Determination of organ index Immunoprecipitation Assay (RIPA, Beyotime, China) buffer (100 mg
tissue/0.5 ml RIPA added protease and 1 pl/ml phosphatase inhibitor)
Determination of growth performance: at the exposure test's begin- and quantified by the BCA method. The sample amount was calcu-
ning, broilers' body and lung weight were weighed in the 2nd, 4th, lated according to the determined protein concentration.28 The pro-
and 6th weeks. According to the weight record obtained, the organ tein was separated on SDS-PAGE gel and then transferred to the
index of the number of lungs was calculated: organ index (%) = (organ polyvinylidene difluoride (PVDF) membrane (200 mA, 220 V, 2 h).
weight/weight)  100. When 5% skim milk is added to the shaker and sealed for 15%, wash
the membrane with PBST buffer (5 min, 3 times). Rabbit-derived pri-
mary antibody (PTEN, PI3K, AKT, mTOR, anti-thymocyte globulin
2.4 | Hematoxylin–eosin (HE) staining observation (ATG12, ATG5), microtubule-associated protein 1A/1B-light chain
of chicken lungs (LC3), Nuclear Pore Glycoprotein P62 (p62), ERK, p-ERK, JNK, p-JNK,
p38, p-P38, B cell lymphoma/leukemia 2 (Bcl-2), Bcl-2 associated X
After 6 weeks of PS-MPs exposure, the pathological lung tissue protein (Bax), Cytochrome c (Cytc), dynamin-related protein 1 (DRP1),
changes were observed under a light microscope. The broilers were mitochondrial fission factor (MFN1 and MFN2), cysteinyl aspartate
dissected according to the routine method. According to the standard specific proteinase 3, 8, 9 (Caspase-3, 8, 9), Wanleibio, China). TNF
procedure of HE staining, embedding, slicing, dewaxing, hematoxylin receptor superfamily, member6 (Fas), optic atrophy 1 (OPA1, Abclo-
staining, hydrochloric acid alcohol differentiation, tap water returning nal, USA). Incubate overnight at 4
C. On the second day, after wash-
to blue, staining in eosin dye solution were carried out, and then dehy- ing the membrane with phosphate buffer containing Tween 20 (PBST)
dration, transparency, and sealing were imposed. Finally, the dyeing buffer (15 min, 3 times), the secondary antibody anti-diluent
effect was observed under a light microscope (Olympus, Japan). (1:10000, Biosharp, China) was added. When incubating at room tem-
perature for 2 h, wash the membrane (15 min, 3 times). Then, the spe-
cific bands were detected by a chemiluminescence substrate kit (ECL)
2.5 | Chicken lung tissue observation by electron and detection kit (Biosharp, China), and the image was quantified. The
microscopy details of the primary antibody used in this study are shown in
Table S3.
After 6 weeks, lung tissue samples were quickly extracted. Then the
following steps were carried out: fixed with 2.5% glutaraldehyde for
2 h. Fixed with 2% monosodium acid for 2 h. Dehydration: gradient 2.8 | Quantitative real-time polymerase chain
dehydration of each 15 min with 50%, 70%, 80%, 90% ethanol (over- reaction (qRT-PCR)
night in 70% ethanol), and then dehydration with 100% ethanol
3 times, each 20 min. Replacement: Replacement with acetone twice, The lungs of chickens in each group were taken. According to the
each time 15 min. The epoxy resin was embedded and cut into manufacturer's instructions, used kit TriZol (Invitrogen, USA) to
1  1  1 mm3 ultrathin slices. Uranyl acetate and lead citrate were extract total RNA. Synthesize cDNA according to HiScript II Q RT
stained and finally observed by transmission electron microscope SuperMix for qPCR reverse transcription kit. According to the
(GEM-1200ES day, Japan). ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, China). Find
the gene sequences in National Coalition Building Institute (NCBI)
and design primers with Primer Premier 5.0 software (Premier,
2.6 | Detection of oxidative stress index in lung Canada). Reaction conditions: 95
C, 30s (1 cycle), 95
C, 10s, 60
C,
serum 30s (40 cycles), 95
C, 15 s, 60
C, 60s, 95
C, 15 s. The melting curve
was prepared, and the relative quantitative analysis was carried out
The serum of broilers in each group was taken and operated according by the 244Ct method.29 The primer sequences are shown in
to the instructions of Glutathione (GSH) (Wanleibio, China), catalase Table S4.
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LU ET AL. 81

2.9 | Functional analysis of the target genes different letters are statistically significantly different simultaneously
(p < .05). * indicates significant differences between the Con group
After our target mixed gene list was completed, the term enrichment and experimental group (*p < .05 and **p < .01), and # indicates signif-
analysis was carried out using the online software Metascape (http:// icant differences between the M-MPs, H-MPs group and L-MPs
metascape.org), and the interaction network between genes was gen- group (#p < .05 and ##p < .01).
erated. Put our target differential genes into the functional annotation
of the target genes. Got all the functions that genes were involved
in. Post-benefit Fisher exact and multiple comparison tests calculated 3 | RE SU LT
each function's significance balance (p-value). The significant function
of genes was screened out from goats, and the standard of significant 3.1 | PS-MPs characterization and organ index
screening was: p-value < .05.
There were morphology and properties of PS-MPs. PS-MPs' circu-
larity and roundness were 15.66 ± 0.05 and 15.63 ± 0.06 mm,
2.10 | Statistical analysis respectively (mean ± SD). PS-MPs were spherical, and their surface
was well-round (Figure 1A, B). It can be seen from Figure 1C that at
The data are expressed as mean ± standard deviation (average ± SD), the 2nd week of PS-MPs exposure, the organ index of the L-MPs,
and were analyzed statistically by SPSS11.0 software. Bars with M-MPs, and H-MPs group was significantly higher than that of the

F I G U R E 1 Characterization of the physiochemical properties of the PS-MPs, antioxidase activity and the organ index. (A) Low-magnitude
SEM images, (B) high-magnitude SEM images, (C) pulmonary organ index, (D) CAT enzyme activity, (E) GSH enzyme activity, (F) MDA content.
Bars with different letters are statistically significantly different at the same time point (p < .05).* indicates significant differences between the
control group and experimental group (*p < .05 and **p < .01), and # indicates significant differences between the M-MPs, H-MPs group and L-
MPs group (#p < .05 and ##p < .01). Data are shown as three independent samples (n ≥ 3)
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82 LU ET AL.

Con group at the 2nd week of PS-MPs exposure (p < .05). It may be 3.3 | Transmission electron microscope
due to broilers' vigorous growth, and PS-MPs accumulate less and
cannot produce toxicological effects during the 2 weeks. In the 4th As shown in Figure 3, the Con group cell structure and lung gland cells
and 6th weeks, the organ index in the L-MPs, M-MPs, and H-MPs were normal (pink arrow). Compared with the Con group, the lung gland
group was significantly lower than that in the Con group (p < .05, cells in the L-MPs model group were atrophied, irregularly shaped, and
p < .01), which may be due to the effect of PS-MPs exposure on ruptured (pink arrow), and signs of apoptosis were observed in lung cells
the growth and development of the lungs (p < .05, p < .01). These (blue arrow). By contrast, in the Con group and L-MPs group, glandular
results confirmed that exposure to PS-MPs would cause tissue cells in the M-MPs group ruptured (pink arrow), apoptotic bodies
damage to broilers (p < .05, p < .01). increased (blue arrow), autophagosomes, and autophagy lysosomes
appeared (red arrow). In the H-MPs group showed injury, membrane
deformation, nuclear-condensed chromatin accumulation, apoptotic
3.2 | Pathological changes of lung tissue caused by bodies appeared and wrapped in the cell membrane (blue arrow), autop-
exposure to PS-MPs hagosomes and autophagy lysosomes increased (red arrow). Noticeable
swelling of mitochondria, the disappearance of a crest (yellow arrow),
The results of HE staining showed that in the Con group, the lung tis- vacuolization of mitochondria (green arrow), fragmentation, and severe
sue structure was intact, the size and structure of alveoli were stan- pathological changes in cells were observed.
dard, the alveolar wall was thin, and there was no hyperemia and
inflammation (Figure 2A). In the L-MPs group, the alveolar septum
was significantly thickened (black arrow), with a large number of 3.4 | PS-MPs induced oxidative stress in the lungs
inflammatory cell infiltration (blue arrow) and slight alveolar hemor-
rhage (green arrow) (Figure 2B). M-MPs group showed a large number In order to evaluate the effect of PS-MPs exposure on the activities
of inflammatory cell infiltration, a small number of neutrophils (blue of antioxidant enzymes in the lung, we measured CAT activity, GSH
arrow), alveolar wall thickening, edema (black arrow), alveolar hemor- levels, and MDA content. Compared with the Con group, the results
rhage (green arrow), bronchial obstruction (yellow arrow), and vascular showed that the activity of CAT and the level of GSH decreased
rupture (Figure 2C). In the H-MPs group, pathological lesions were (p < .05, p < .01), and the content of MDA increased (p < .01) signifi-
more serious (Figure 2D). Exposure to PS-MPs leads to lung tissue cantly in the lung tissue of chickens induced by PS-MPs
damage. The degree of injury increases with the increase of PS-MPs (Figure 1D–F). With the increase of PS-MPs concentration, the anti-
concentration. oxidant enzymes were rendered dose-dependent.

F I G U R E 2 (A–D) Effect of PS-MPs on the lung histopathological structure in broiler chickens. Histopathological sections of the lung
(200). (A) Control group, (B) L-MPs group, (C) M-MPs group, and (D) H-MPs group. Black arrow: The alveolar wall is thickened, blue
arrow: inflammatory fine cell infiltration, red arrow: alveolar hemorrhage. Yellow arrow: bronchial obstruction green arrow: vascular
rupture, thrombus
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LU ET AL. 83

3.5 | Effect of PS-MPs exposure on the expression Akt, and mTOR expression descended (p < .05, p < .01). It was sug-
of genes associated with the PTEN/PI3K/AKT gested that inhibition of PI3K/Akt/mTOR induces the formation of
pathway in the lung autophagosomes.

First, transmission electron microscope data showed many autop-

hagosomes in the PS-MPs group in Figure 3 (red arrow), which 3.6 | Exposure of PS-MPs induced apoptosis
enhanced the formation of autophagosomes (Figure 3). LC3, as a through both the mitochondrial pathway and the
critical autophagy regulator, promotes the formation of autophago- death receptor pathway
somes, while p62 acts as a substrate for autophagy-lysosome deg-
radation. Beclin1 is one of the essential regulatory proteins of First, transmission electron microscope data showed that PS-MPs
autophagy and participates in forming the autophagy membrane. exposure led to apoptosis. As shown in Figures 4 and 6, quantitative
Figures 4 and 5 showed that PS-MPs upregulated LC3II and Beclin1 detection of the Caspase gene and protein showed that after 6 weeks
(Figure 5A, F, and J) while down-regulating the level of p62 of treatment with PS-MPs, the expression levels of Cytc, Bax, Bcl-2,
(p < .05, p < .01) (Figure 5A and K), indicating that autophagy flux is DRP1, and Caspase-3, Caspase-8 and Caspase-9 in mitochondria of
induced. Some studies have shown that PTEN could promote broilers were significantly increased (p < .05, p < .01) (Figure 6A–C,
autophagy by blocking the activation of the PI3K/Akt/mTOR path- G–J). The expression levels of OPA1, MFN1, and MFN2 were signifi-
way. In order to study the mechanism of PS-MPs on the PTEN/ cantly decreased (p < .01) (Figure 6D–F, H), indicating that PS-MPs
PI3K/Akt signal pathway. We examined the expression of genes exposure led to mitochondrial dysfunction (Figure 6L). In addition, we
related to the PTEN/PI3K/AKT (p < .05, p < .01) signaling pathway also detected the exogenous apoptosis pathway and found that the
in the lung cells of broilers after 6 weeks of PS-MPs exposure, as expression of Fas, protein and Fas, FADD, Fasl gene increased in the
shown in Figures 4 and 5. We found that after PS-MPs exposure, PS-MPs exposure group (p < .05, p < .01) (Figures 4A and 6). These
compared with the Con group, the expression level of the PTEN results suggested that PS-MPs exposure induced apoptosis through
gene was significantly increased (p < .05, p < .01), but PI3K, Akt, p- the mitochondrial and death receptor pathways.

F I G U R E 3 Effect of PS-MPs on the ultrastructure of lung cells in broiler chickens. Pink arrow:lung gland cells. Blue arrow:apoptosis. Red
arrows:autophagosomes and autophagy lysosomes. Green arrow:cell vacuoles
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84 LU ET AL.

F I G U R E 4 Analysis of gene transcript levels and action of PS-MPs. (A) Heatmap, transcriptional water of related genes, (B) and
(C) Enrichment bars, colored with p-values. Bars with different letters are statistically significantly different at the same time point (p < .05).*
indicates significant differences between the control group and experimental group (*p < .05 and **p < .01), and # indicates significant differences
between the M-MPs, H-MPs group and L-MPs group (#p < .05 and ##p < .01). Data are shown as three independent samples (n ≥ 3)

3.7 | PS-MPs through MAPK pathway to mediates poultry into the digestive tract, resulting in a false sense of satiety
lung injury or damage to the intestinal structure.31,32 In addition, plastic frag-
ments are affected by physical, chemical, biological, and other fac-
To detect whether the MAPK signal pathway regulated PS-MPs on tors to break up into smaller particles, reaching different
lung cell apoptosis and autophagy in broilers. We detected the ecosystems with ocean currents. PS-MPs have been found in sea-
expression level of genes related to the MAPK signal pathway in food such as fish and mussels, tap water, honey and beer, and
broilers exposed to different concentrations of PS-MPs for 6 weeks. human feces.33 PS-MPs are ubiquitous in the natural environ-
Western blot results showed that the ratio of p-JNK/JNK (p < .01), p- ment.34 Animal ingestion of PS-MPs will cause digestive tract
ERK/ERK (p < .05), and p-p38/p38 (p < .05) increased, and PS-MPs blockage and false satiety, leading to tissue damage and even life-
were exposed for 6 weeks (Figure 7A–D). Gene expression level threatening.35 Our study showed that in 2 weeks, PS-MPs had no
results showed that the expression level of JNK (P < 0.05), ERK significant effect on the lung of broilers. However, at the 4th and
(p < .01), p38 (p < .05, p < .01) increased significantly after PS-MPs 6th weeks, the organ index of broiler lungs decreased (Figure 1).
stimulation of broiler lung cells for 6 weeks, as shown in Figure 7E. Similar to the results of this study, polyethylene beads (10–106 μm,
These results suggested that PS-MPs exposure mediates apoptosis 1.25  10(2)–1.25  10(7) particles/liter) were tested for 72 h, and
and autophagy through the MAPK pathway. it was found that PS-MPs caused tissue damage of phaeodactylum
tricornutum.36,37 Studies have shown that ingestion of PS-MPs in
broilers causes tissue damage.
4 | DISCUSSION Several studies have shown that birds, poultry, and fish can
ingest nanoparticles, then be absorbed by the digestive system and
Global MPs production increases yearly, and the low recycling rate transferred from the circulatory system to different organs.38,39 The
30
has caused increasingly severe environmental pollution. Plastic accumulation of nano-to-micron-sized was detected in the lung.
fragments entering the natural environment may be mistakenly Nano-to-micron-sized damaged lung tissue structure and led to
eaten by creatures such as humans, fish, seabirds, sea turtles, and pathological injury.40 In our study, when the concentration of
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LU ET AL. 85

F I G U R E 5 Changes in the autophagy pathway (panel A), and protein interaction (panel H). Bars with different letters are statistically
significantly different at the same time point (p < .05).* indicates significant differences between the control group and experimental group
(*p < .05 and **p < .01), and # indicates significant differences between the M-MPs, H-MPs group and L-MPs group (#p < .05 and ##P < .01).
Data are shown as three independent samples (n ≥ 3)

PS-MPs exceeded the environment-related concentration, there Recently, much evidence shows that oxidative stress initiates
was significant tissue damage in the lung tissue (Figures 2 and 3). apoptosis,43 and two Caspase activation pathways (endogenous and
These results suggested that PS-MPs could aggravate the damage exogenous) are involved in apoptosis.44 The endogenous pathway
to lung tissue structure and cell structure. Tissue structure damage releases cytochrome c first to activate caspase-9 and then to activate
in the body should be further analyzed from the enzyme activity caspase-3. The second pathway is exogenous, activating caspase-8
and molecular level. and caspase-3 to participate in apoptosis cascade by recognizing
Antioxidant enzymes are widely used as sensitive biomarkers in extracellular ligands such as Fas and FasL across cell membrane recep-
ecotoxicological assessment to understand the potential threat of tors.45,46 PS-MPs can induce apoptosis47 of human neural progenitor
environmental pollutants. Many studies on the toxicity of PS-MPs cells by inhibiting Bcl-2 and increasing the expression of Bax, Cyt-c,
have shown that they cause oxidative stress, damage the cell mem- and Caspase-3. Our study found that Caspase-3 expression was upre-
brane, and induce biochemical changes, resulting in inflammation and gulated in a dose-dependent manner after 6 weeks of exposure to
apoptosis.41,42 In our study, oxidative stress was considered the pri- PS-MPs in broiler chickens (Figures 4 and 6). PS-MPs can activate
mary mechanism of the targeted effect of PS-MPs. The PS-MPs death receptors (Fas, FADD, and Caspase-8), mitochondrial apoptosis
reduced the activities of GSH and CAT, increasing the accumulation pathways (Bcl-2, Bax, Cyt-c, Caspase-9, and Caspase-3) as shown in
of MDA, and cells were induced to undergo oxidative stress, which Figures 4 and 6, and finally induce cell apoptosis.
promoted pulmonary toxicity (Figure 1D–F). The results showed that It is also noteworthy that our study also found that PS-MPs can
oxidative stress promoted the pulmonary toxicity induced by PS-MPs. lead to different forms of cell damage. The researchers also found
It eventually leads to apoptosis.43 that oxidative stress can regulate the PTEN/PI3K/AKT signal
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86 LU ET AL.

F I G U R E 6 Protein expression of apoptotic pathway (panel A) and coexpression matrix (panel L). Bars with different letters are statistically
significantly different at the same time point (p < .05).* indicates significant differences between the control group and experimental group
(*p < .05 and **p < .01), and # indicates significant differences between the M-MPs, H-MPs group and L-MPs group (#p < .05 and ##p < .01).
Data are shown as three independent samples (n ≥ 3)

F I G U R E 7 Protein expression and mRNA transcription levels of the MAPK pathway. Bars with different letters are statistically significantly
different at the same time point (p < .05).* indicates significant differences between the control group and experimental group (*p < .05 and
**p < .01), and # indicates significant differences between the M-MPs, H-MPs group and L-MPs group (#p < .05 and ##p < .01). Data are shown
as three independent samples (n ≥ 3)

pathway, thus participating in cellular toxicological processes.48,49 study found that PS-MPs can trigger oxidative stress in the lungs.
After oxidative stress induced by PS-MPs, the PTEN/PI3K/Akt/ With the activation of the MAPK signal pathway (Figure 7), the
mTOR pathway was down-regulated (Figures 4, and 5). Our current expression of PTEN was increased, and the expression of
 15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23663 by Bahauddin Zakariya University, Wiley Online Library on [20/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
LU ET AL. 87

PI3K/AKT/mTOR was inhibited, thus promoting lung autophagy basis for studying the environmental toxicity and damage caused by
(Figures 4, and 5). In recent years, some studies have pointed out PS-MPs pollution. This paper explores the toxicity and molecular
that PTEN/PI3K/Akt/mTOR signaling pathway mainly regulates mechanism of PS-MPs to broiler lungs from the aspects of tissue,
autophagy and participates in cell proliferation, migration, invasion, enzyme activity, protein, and gene. Thus, it provides a necessary refer-
and angiogenesis. In addition, PI3K/AKT pathway is the upstream ence to evaluate the mechanism of toxicological damage of PS-MPs
pathway of LC3. Inhibition of PI3K/AKT expression can achieve on birds and poultry and the impact on human health.
50
autophagy by increasing LC3 expression (Figure 6). This study
found that inhibition of PTEN/PI3K/Akt/mTOR through Beclin1 AUTHOR CONTRIBU TIONS
protein expression and LC3-II/I ratio significantly increased, while Hongmin Lu: Conceptualization, data curation, software, formal analy-
p62 level decreased, which promoted autophagosome formation. At sis, writing-original draft, writing-review & editing. conceptualization,
the same time, establishing a connection between ATG12 and data curation, software, formal analysis, writing-original draft. Kai Yin:
ATG5 molecules helped LC3 attach lipids to form autophagosomes Conceptualization, software. Heng Su: Formal analysis, investigation.
(Figures 4 and 5). We found that PS-MPs induced the conversion of Dongxu Wang: Formal analysis. Yue Zhang: Inon, data curation. Lulu
LC3-I into LC3-II, which is an indicator of the separation of goods Hou: Review & editing, software. Junbo Li: Visualization. Yu Wang:
into vesicles. P62 directly binds to LC3, and its expression is nega- Validation, visualization, writing-review & editing. Mingwei Xing: Data
tively correlated with autophagy.51 Our current study showed that curation, validation, resources, funding acquisition, project
the level of p62 was down-regulated after exposure to PS-MPs, administration.
excessive autophagy flux (autophagy death) due to the continuous
clearance of necessary cellular components, suggesting autophagy FUNDING INF ORMATI ON
dysfunction, and similar results have been reported before.52 Simi- This work was supported by the Key Projects of Natural Science
larly, our results showed that although PS-MPs in the current envi- Foundation of Heilongjiang Province of China (Grant ZD2020C005).
ronmental concentrations do not cause autophagy, the exposure
concentration increases to damage autophagy flux. CONFLIC T OF INT ER E ST
Our study confirmed that the PTEN/PI3K/Akt/mTOR pathway is The authors declare that they have no known competing financial
involved in PS-MPs-induced pulmonary toxicity. However, its specific interests or personal relationships that could have appeared to influ-
mechanism needs to be further studied. In addition, some studies ence the work reported in this paper.
have shown that the down-regulation of PI3K and activation of the
MAPK pathway may promote apoptosis and autophagy and inhibit OR CID
advanced gastric cancer. Under excessive oxidative stress, the MAPK Mingwei Xing https://orcid.org/0000-0003-1636-897X
pathway regulates various physiological processes such as apoptosis,
cell development, and death by activating intracellular phosphorylated RE FE RE NCE S
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