Abstract

   Plastics in the environment can break down into nanoplastics (NPs),
   which pose a potential threat to public health. Studies have shown that
   the nervous system constitutes a significant target for nanoplastics.
   However, the potential mechanism behind nanoplastics’ neurotoxicity
   remains unknown. This study aimed to investigate the role of lncRNA in
   the depressive-like responses induced by exposure to 25 nm polystyrene
   nanoplastics (PS NPs). Forty mice were divided into four groups
   administered doses of 0, 10, 25, and 50 mg/kg via gavage for 6 months.
   After conducting behavioral tests, RNA sequencing was used to detect
   changes in mRNAs, miRNAs, and lncRNAs in the prefrontal cortex of the
   mice in the 0 and 50 mg/kg PS NPs groups. The results revealed that
   mice exposed to chronic PS NPs developed depressive-like responses in a
   dose-dependent manner. It was demonstrated that 987 mRNAs, 29 miRNAs,
   and 116 lncRNAs were significantly different between the two groups.
   Then, a competing endogenous RNA (ceRNA) network containing 6 lncRNAs,
   18 miRNAs, and 750 mRNAs was constructed. Enrichment results suggested
   that PS NPs may contribute to the onset of depression-like responses
   through the activation of axon guidance, neurotrophin-signaling
   pathways, and dopaminergic synapses. This study provided evidence of
   the molecular relationship between PS NPs and depression-like
   responses.

   Keywords: nano-polystyrene, depression-like responses, LncRNA,
   RNA-sequencing, ceRNA

1. Introduction

   In recent years, the threat of plastic pollution to human health has
   become a public health issue of global concern [[48]1,[49]2]. Global
   plastic production has soared over the past few decades due to the
   product’s widespread use in our lives and industries. It has been
   reported that the global production of plastics rose from 1.5 million
   tons in the 1950s to 359 million tons in 2018 [[50]3]. At the same
   time, a large amount of plastic waste has been sent to landfills,
   resulting in the pollution of the environment. In 2016, an estimated 19
   to 23 million tons of plastic waste generated globally reached water
   systems, and annual emissions could reach 53 million tons yearly by
   2030. The current amount of plastic production is about 450 million
   tons annually, which is projected to double by 2045 [[51]4,[52]5].
   However, most plastics are not easily degradable, and the continuous
   decomposition of plastic products as a result of thermal, chemical, and
   biological processes leads to the creation of microplastics (MPs)
   [[53]6]. MPs can be further broken down into smaller-particle-size
   nanoparticles (NPs < 0.1 μm) [[54]7]. Studies have shown that NPs may
   enter the body via multiple pathways and can be distributed to
   different tissues through the circulatory system [[55]8,[56]9,[57]10].
   At present, polystyrene (PS) is one of the most-produced plastic
   materials [[58]11]. Previous studies have evidenced that PS NPs can
   cross the blood–brain barrier into the brain [[59]12,[60]13], leading
   to various neurological disorders, such as anxiety [[61]14] and
   neurobehavioral impairments [[62]15,[63]16].

   Depression is one of the most common mental illnesses [[64]17]. It was
   reported that mental illness would be the leading cause of global
   health-related burdens by 2020, and depression is one of the major
   contributors to this burden [[65]18]. The World Health Organization
   (WHO) reported that depression ranked as the third leading contributor
   to the global burden of disease in 2004, and it is expected to rise to
   first place by 2030 [[66]19]. Previous studies have shown that
   environmental factors, including nanoparticle exposure, contribute to
   the onset of depressive disorders [[67]20,[68]21,[69]22]. It has been
   shown that the deposition of nanomaterials is associated with increased
   NO levels, increased thiobarbituric acid reactive species, and lower
   acetylcholinesterase activity in the brain (leading to cognitive
   impairment) [[70]23]. Our previous research has shown that chronic PS
   exposure can induce cognitive impairment in mice through damage to
   synaptic functions [[71]24]. However, the neurotoxic mechanisms of PS
   NPs still deserve to be fully investigated.

   Researchers have demonstrated that long noncoding RNAs (lncRNAs) are
   highly expressed in the brain and play a critical role in neural stem
   cell maintenance, brain patterns, synaptic and stress responses, and
   neuroplasticity [[72]25]. Evidence has suggested that lncRNA
   deregulation is one of the potential mechanisms behind many
   neurological disorders, including depression [[73]26,[74]27]. Previous
   study demonstrated that lncRNA has shown potential value as a
   diagnostic and therapeutic biomarker for depression [[75]27]. LncRNAs
   can regulate mRNA stability and translation in the cis or trans
   formation and interact with miRNAs to regulate mRNA transcription
   [[76]28]. Furthermore, a previous study demonstrated that PS NPs (1
   μg/L) increased the expression of linc-2, linc-9, linc-18, and linc-61
   and decreased the expression of linc-50 [[77]29]. RNA-sequencing
   (RNA-seq) results from another study showed differential expression of
   circRNA and lncRNA in the lung tissue of rats exposed to PS MPs
   [[78]30]. However, the mechanism of lncRNAs in the neurotoxicity
   induced by PS NPs exposure still requires exploration.

   In the present study, C57BL/6 mice were treated with PS NPs via gavage
   for 6 months. Our findings suggest that chronic exposure to PS NPs can
   lead to injury in the prefrontal cortex (PFC), leading to
   depressive-like responses in mice. RNA-seq was used to detect
   differentially expressed transcripts in the PFC to elucidate the
   mechanics behind the depression-like responses caused by PS NPs. The
   potential mechanism by which PS NPs cause depression-like responses in
   mice is as follows: the dysregulation of lncRNAs activates multiple
   important pathways, including axon guidance, dopaminergic synapses, and
   neurotrophin signaling pathways. The present study provides potential
   strategies for the early diagnosis of depression-like responses induced
   by PS NPs and deeply explains the potential pathological mechanisms of
   PS-NP-induced depressive behavior in mice.

2. Materials and Methods

2.1. Characterization of PS NPs

   We purchased 25 nm polystyrene samples from Bangs Laboratories, Inc.
   (Fishers, IN, USA). PS NPs were characterized via scanning electron
   microscopy (SEM) using an IT-500HR instrument (JEOL, Tokyo, Japan).
   Size distribution and zeta potential of PS NPs were examined using
   dynamic light scattering (DLS) at 25 °C (PALS, Brookhaven National
   Laboratory, New York, NY, USA).

2.2. Animals and Treatment

   Five-week-old male C57BL/6 mice were purchased from Vital River
   Laboratory (Beijing Vital River Laboratory Animal Technology Co., Ltd.,
   Beijing, China) and housed in individually ventilated cages. The mice
   were maintained in a temperature- (25 ± 3 °C) and humidity (50 ±
   5%)-controlled environment under a 12 h light/dark cycle. Water and
   standard laboratory feed were made available ad libitum.

   A total of 40 mice were divided into a control group, a low-dose group
   (10 mg/kg body weight (bw) PS NPs), a middle-dose group (25 mg/kg bw PS
   NPs), and a high-dose group (50 mg/kg bw PS NPs). PS NPs were dissolved
   in ultrapure water and sonicated for 5 min before each application.
   Mice in PS NPs groups were administered different concentrations of PS
   NPs via oral gavage daily for 6 months, while the control group was
   given equal amounts of distilled water. After the behavioral tests,
   mice were euthanized via cervical dislocation under anesthesia (1%
   sodium pentobarbital intraperitoneal injection), and the prefrontal
   cortex of each mouse was removed rapidly on ice and stored at −80 °C.
   The layout of our study is shown in [79]Figure 1. The Animal Care and
   Use Committee of Hebei Medical University (Hebei, China) approved the
   experimental procedures.

Figure 1.

   [80]Figure 1
   [81]Open in a new tab

   Flow chart illustrating the design of the experiment.

2.3. Behavioral Tests

2.3.1. Open Field Test (OFT)

   The states of nervousness and anxiety in the mice were assessed using
   OFT and with reference to a previous study [[82]31]. The experiments
   were conducted in a 30 cm × 30 cm × 25 cm square white square box. Mice
   were free to move around, and each mouse was observed for 5 min. The
   time and distance travelled in an open field were recorded and analyzed
   with a digital tracking system.

2.3.2. Novelty-Suppressed Feeding Test (NSF)

   The NSF assay is commonly used to evaluate depressive-like states in
   mice. Mice were subjected to a 24 h water-and-food fast before the
   novel inhibition of feeding experiment. On the following day, each
   mouse was subjected to a test individually in a corner of the field,
   and the latency period regarding the point at which each mouse started
   feeding was recorded. Observations were made for a maximum of 10 min.

2.3.3. Sucrose Preference Test (SPT)

   The SPT was used to detect anhedonia and evaluate depressive behavior
   in animals. Every mouse was housed in a cage with two bottles,
   containing a solution of 3% sucrose and water, respectively. Mice were
   allowed to freely choose to drink from either of the two water bottles.
   Liquid intake was measured daily for 3 days, and bottles were inverted
   daily to prevent position preference bias. The sucrose preferences of