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. 2026 Feb 23;4(6):1195–1209. doi: 10.1021/envhealth.5c00580

PM2.5-Induced Multinucleation of Bronchial Epithelial Cells due to Microtubule Overacetylation and Disruption of Microtubule-Associated Proteins

Liyan Xiao †,‡, Jinyan Pang †, Zhiling Guo §, Kexin Zhang †, Zhiqin Xiong †, Man Yang †, Ji Wang †, Yang Li †,§,*, Iseult Lynch §,*
PMCID: PMC13288244  PMID: 42344592

Abstract

The formation of multinucleated cells is associated with abnormal mitosis, which can lead to uncontrolled cell proliferation and even malignant transformation due to the continuous increase in chromosomal instability. Our previous study was the first to discover that PM2.5 induces a decrease in microtubule dynamics and promotes the formation of multinucleated cells in the human bronchial epithelial cell line (BEAS-2B). To elucidate the intrinsic mechanism by which PM2.5 causes multinucleation in bronchial epithelial cells, we conducted in vivo experiments using C57BL/6J mice exposed to PM2.5 via intratracheal instillation, as well as in vitro mechanistic studies using BEAS-2B cells. To verify the involvement of the PI3K/Akt/GSK-3β signaling pathway, we employed PI3K inhibitor LY294002 and GSK-3β-overexpressing BEAS-2B cells. In both in vivo and in vitro experiments, an increase in multinucleated cells was observed following PM2.5 treatment. Additionally, PM2.5 directly led to a higher proportion of polymerized microtubules and abnormal microtubule aggregation. In the mechanistic studies, we mainly investigated the impact of PM2.5 on the microtubule dynamics. The results demonstrated that PM2.5 activated the PI3K/Akt/GSK-3β signaling pathway, which inhibited the deacetylase enzyme HDAC6, resulting in excessive microtubule acetylation. Simultaneously, PM2.5 induced the dissociation of microtubule-associated protein MAP1B from the microtubules. Both processes contributed to the overstabilization of microtubule proteins. However, mitosis requires highly dynamic microtubules, characterized by rapid switching between growth and shrinkage. Therefore, the overstabilization of microtubule proteins induced by PM2.5 reduced the microtubule dynamics and disrupted spindle function during mitosis, ultimately leading to mitotic failure and the formation of multinucleated cells.

Keywords: PM2.5 , multinucleation, microtubule, acetylation, MAP1B

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Introduction

Air pollution has become one of the most important risk factors for many respiratory diseases. Fine particulate matter (PM2.5), particulate matter with an aerodynamic diameter ≤2.5 μm, has been recognized as a key harmful component of air pollution. It can be directly inhaled and penetrates deeply into the lung alveoli, causing severe lung dysfunction, including asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), and even lung cancer. − Li et al. demonstrated that PM2.5 may directly impair the function of airway Club cells, suggesting its direct cytotoxic effect on bronchial epithelial cells. And studies have shown that PM2.5 not only impairs lung function but also promotes pulmonary fibrosis and lung cancer development by inducing senescence in alveolar type II cells and damaging the regenerative capacity of lung stem cells. , A nationwide study across 295 Chinese counties reported that a 10 μg/m3 increase in PM2.5 exposure (based on the average PM2.5 concentration from 2007 to 2014) contributed to a 4.20% higher incidence of lung cancer in males and a 2.48% increase in females. A systematic review also indicated that short-term exposure to dust storms containing PM2.5 was significantly associated with all-cause and cardiovascular mortality, further corroborating the systemic health risks of PM2.5. In fact, PM2.5 was classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC) in 2013. Unfortunately, the molecular mechanisms underlying PM2.5-induced lung cancer are still not well understood.

Chromosomal instability (CIN) is one of the most common characteristics of cancer genomes, contributing to the broad genomic complexity, including loss or amplification of driver genes, extrachromosomal DNA, focal rearrangements, and micronuclei formation. CIN is considered a key factor in the intertumoral heterogeneity observed in cancers and plays a crucial role in tumor progression. , Multinucleated cells, multipolar mitoses, chromatin strings, chromatin bridges, and nuclear heterogeneity have been suggested as morphological indicators of CIN in the diagnosis and prognosis of tumors. , Our previous study found that PM2.5 affected the chromosome stability in human bronchial epithelial cells (BEAS-2B) by increasing the proportion of multinucleated cells. And increasing evidence indicated that CIN may initiate tumorigenesis. Drews et al. reported that CIN-related consequences, such as the deletion or amplification of driver genes, were associated with cancer stage, metastasis, and poor prognosis. Therefore, the increase in the number of multinucleated cells caused by PM2.5 could be a crucial factor in the malignant transformation of cells and even in the development of lung cancer. The aims of this study were thus to investigate the causes of multinucleated cell formation induced by PM2.5 and to provide experimental insights into the mechanism of PM2.5-induced lung carcinogenesis.

Multinucleated cells arise mainly from mitotic errors. Abnormalities in the structure or function of the spindle could lead to mitotic failure. The mitotic spindle is a microtubule-based assembly responsible for separating chromosomes during cell division. As microtubules (MTs) are the key components of the spindle, adverse effects on their structure might be the main cause of spindle abnormalities. As is well known, MTs are highly dynamic polymers composed of tubulin dimers, following an assembly/disassembly cycle known as dynamic instability. Moreover, the turnover of MT polymers in the spindle was more rapid during mitotic division. Consequently, imbalances in the dynamic instability of MT are more likely to result in abnormal spindle assembly or function. Maiato et al. showed that the correct assembly of the spindle acts as a safeguard, preventing chromosome mis-segregation during mitosis. Abnormal spindle assembly due to imbalance in MT dynamics was responsible for most chromosome segregation defects. Additionally, Blackwell et al. confirmed that MT dynamic instability facilitates the search and capture of kinetochores during spindle formation, which is a crucial process for accurate chromosome segregation. MT-stabilizing drugs might induce CIN by affecting spindle MT assembly. Our study found that PM2.5 led to abnormal spindle function by disrupting the dynamic instability of MT. Abnormal spindle function during cell division might be the main reason for the formation of multinucleated cells after PM2.5 treatment, making it worth further investigation of how PM2.5 disrupts the dynamic instability of MT.

MT were assembled from heterodimers of α-tubulin and β-tubulin into long polymers, with their main structure being a hollow tube formed by 13 linear protofilaments that were laterally connected and closed. The polymer was polar, with a fast-growing plus end and a slow-growing minus end. MTs displayed a behavior known as dynamic instability, where the ends of individual polymers transition between periods of growth and shortening. Goodson et al. demonstrated that the dynamic instability of MTs was driven by nucleotide hydrolysis and affected by a variety of specialized regulatory proteins, including MT-associated proteins (MAPs). MAPs played a crucial role in regulating MT dynamics by binding to and detaching from them. Lawrence et al. discovered that MAPs form extensions at the ends of growing MT and bind cooperatively to the MT lattice, protecting the MT from the severing activity of MT-cutting enzymes. Research indicated that the dynamic instability of MT was not only regulated by MAPs but was also linked to the post-translational modification (PTM) of tubulins. , Some of these modifications, such as phosphorylation, acetylation, and detyrosination, were found on a broad range of MT proteins, which influence MT properties directly, via altering the MT lattice structurally, or indirectly by changing MT interaction partners. Our previous study found that PM2.5 had a significant effect on MT acetylation. Acetylation of lysine 40 (K40) of α-tubulin was the only tubulin PTM found in the lumen of MT, and has been identified as being closely related to MT instability. Therefore, it was possible that the overstability of MT induced by PM2.5 might have been related to the changes in MAPs binding to MT and the acetylation state of MT.

In this work, an mRNA microarray was used to identify the effects of low-dose PM2.5 on the gene expression profiles of BEAS-2B cells. Bioinformatics techniques were then employed to analyze the functions and signaling pathways involved in the differentially expressed genes. The PI3K/Akt/GSK-3β pathway was found to be closely related to the regulation of cell proliferation and cytoskeleton. Yang J et al. demonstrated that the PI3K/AKT/GSK-3β pathway was one of the major pathways regulating MT dynamics. Therefore, the imbalance in the MT dynamic instability caused by PM2.5 might be closely related to the activation of the PI3K/AKT/GSK-3β pathway. Research has shown that glycogen synthase kinase-3β (GSK-3β) is an evolutionarily conserved serine/threonine kinase that affected not only the binding of MT-associated proteins to MT but also the expression of enzymes that regulated MT acetylation. , Thus, it was hypothesized that PM2.5 could activate the PI3K/AKT/GSK-3β pathway, which, on the one hand, affects the binding of MAPs to MT, and on the other hand, induces alterations in the acetylation state of MT. These changes led to an imbalance in MT dynamic instability and abnormal spindle MT assembly, ultimately resulting in failed mitosis and multinucleated cells.

Experimental Section

Chemicals and Antibodies

Rabbit anti-PI3K 110α (#4249), rabbit anti-PI3K 110β (#3011), rabbit anti-AKT (#4691), rabbit antiphospho-Akt (ser473) (#88599), rabbit anti-GSK-3β (#12456), rabbit antiphospho-GSK-3β (ser9) (#5558), anti-rabbit IgG (H+L) (DyLight 680 Conjugate) (#5366), antimouse IgG (H+L) (DyLight 680 Conjugate) (#5470) antibodies were purchased from Cell Signaling Technology. Mouse antiphospho-GSK-3β (Tyr216) (AB2533691) antibody was obtained from Thermo Fisher. Mouse anti-HDAC6 (12834-1-AP), rabbit antiacetylated Tubulin (Lys40) (66200-1-Ig), and mouse anti-GAPDH (60004-1-Ig) antibodies were bought from Proteintech. Mouse anti-MAP1B (ab11266), chicken antialpha tubulin (ab89984), donkey antimouse IgG H&L (Alexa Flour 594) preadsorbed (ab150112), donkey antigoat IgG H&L (Alexa Flour 594) preadsorbed (ab150136), and donkey anti-rabbit IgG H&L (Alexa Flour 647) preadsorbed (ab150063) antibodies were obtained from Abcam.

PM2.5 Collection and Stock Solution Preparation

The PM2.5 used in this study was collected at the building roof, which was about 6 m above the groundin urban area of Beiing, China, from January to December in 2017, using two air particle samplers (TH-1000cl, Wuhan Tianhong, China: PMS-104, APMEngineering Co. Ltd., Korea). Following collection, the PM2.5 was subjected to further extraction for use in subsequent experiments. The extraction procedure of PM2.5 and the analysis of its chemical composition have been described in our group’s previously published article. In summary, the chemical composition of PM2.5 was comprehensively analyzed by using spectroscopic and chromatographic methods. Analysis of 24 inorganic elements revealed that S, Ca, Na, Si, and Fe were the most abundant. Several toxic components were also detected, including the heavy metals Mn, Cd, Cr, Ni, Sb, and the metalloid As. Among water-soluble ions, NO3 –, SO4 2–, and NH4 + were present at notably higher concentrations compared to those of other ions. To prepare the PM2.5 stock solution, the freeze-dried PM2.5 samples were weighed, resuspended in 0.9% saline, and stored in a refrigerator at 4 °C. The stock solution was prepared at a concentration of 10 mg/mL.

In Vivo Experimental Design

The experiment was conducted using male C67BL/6J mice, aged 7 weeks, purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). After purchase, the mice were acclimated to an environment with a temperature of 20–24 °C, humidity of 40–60%, and a 12 h light/dark cycle for 7 days prior to the start of the experiments. The experiment was divided into two groups: the control group and the PM2.5-treated group, each group comprised 10 mice (a total of 20 mice). The mice in the PM2.5-treated group received intratracheal instillation of PM2.5. The mice in the experimental group were administered a suspension (10 mg of PM2.5/kg of bw in 20–50 μL of saline) every 3 days for 30 consecutive days, resulting in a total of 10 administrations. The exposure dose was derived by translating the World Health Organization’s Air Quality Guideline (WHO, 2005) interim target-1 (IT-1) for the 24 h mean PM2.5 concentration (75 μg/m3) to a relevant rodent dose. This translation considered standard physiological respiratory parameters for mice (minute volume and exposure duration) and incorporated a 100-fold uncertainty factor, as established in toxicological experimental design. This factor accounts for interspecies variability (mouse-to-human) and intraspecies variability (within humans). The mice in the control group received an intratracheal instillation of saline. At the conclusion of the experiment, all mice were euthanized and lung tissues were collected for subsequent analysis. The animal husbandry and experimental procedures were approved by the Animal Ethics Committee of Capital Medical University (Ethics No. AEEI-2016-076).

Histopathological Analysis

Lung tissues were processed for histopathological evaluation by following standard laboratory procedures. Briefly, lung tissues were excised, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological examination. The sections were then examined under a light microscope (Olympus X71-F22PH, Japan).

Cell Culture and Exposure to PM2.5

Bronchial epithelial cells (BEAS-2B) were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences (SIBS, China). These cells were cultured in DMEM (Corning, Corning, USA) supplemented with 10% fetal bovine serum (PAN, GRE), penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were cultured in a humidified environment with 5% CO2 at 37 °C to 80% confluence and then subcultured. For exposures, cells were seeded at 1500–2000 cells/cm2 in 10 cm2 plates and allowed to adhere for 24 h.

The PM2.5 suspension was initially dispersed by a sonicator (160 W, 20 kHz, 15 min; Bioruptor UDC-200, Belgium) and then immediately added to BEAS-2B cells after being diluted to different concentrations with DMEM medium. The control group was cultured with DMEM without PM2.5. Both the treated and control groups were cultured for 24 h postexposure, after which the cells were collected for subsequent experiments. Each group had three replicate wells.

Multinucleation Analysis

The staining procedure was performed according to the instructions in the Giemsa Staining Kit (Maxim). After staining, the cells were gently rinsed with deionized water to remove any excess dye. The morphological alterations in the cell nuclei were observed under a light microscope (Olympus IX81, Japan). The number of cells exhibiting multiple nuclei was manually counted by two independent observers in randomly selected fields of view (with 500 cells per group, totaling three groups). The total ratio of multinucleated cells was then calculated.

Immunofluorescence Analysis

BEAS-2B cells were exposed to 25 μg/mL of PM2.5 for 24 h. The concentration of 25 μg/mL for BEAS-2B cells was selected based on preliminary cytotoxicity assessments. This dose reduced cell viability to approximately 70–75%, clearly demonstrating toxicity while retaining sufficient cells for subsequent research. Following the conclusion of the treatment period, the cells were fixed with 4% formaldehyde, and the cell membranes were permeabilized with 0.2% Triton X-100. The cells were then incubated for 1 h with a solution comprising 1% Triton X-100, 5% FBS, and 85% PBS. Subsequently, the cells were incubated with the primary antibody (overnight at 4 °C), the fluorescent secondary antibody (1 h at room temperature), and with 4′,6-diamidino-2-phenylindole (DAPI, 15 min at room temperature) to stain the nuclei. The cells were observed using a confocal laser scanning microscope (LSCM, Leica TCS SP8, Germany). MT were stained using a chicken-derived primary antibody against α-tubulin and a donkey-derived antichicken fluorescent secondary antibody (Alexa Fluor 488). Acetylated MT were stained using a rabbit-derived primary antibody against the acetylated MT protein (Lys40) and a donkey-derived anti-rabbit fluorescent secondary antibody (Alexa Fluor 647). Similarly, MAP1B was stained with a rabbit-derived primary antibody and a donkey-derived anti-rabbit fluorescent secondary antibody (Alexa Fluor 647). The chromosomes or nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Western Blot Analysis

Cellular protein samples were prepared by disrupting cells using RIPA lysis buffer (Solario, China), which contained protease and phosphatase inhibitors. The concentration of total cellular proteins was determined using a bicinchoninic acid (BCA) protein quantification kit from Dingguo Biotechnology Co. Ltd. (China). Aliquots of protein samples were subjected to SDS-PAGE electrophoresis (using a 10% separator gel) and then transferred to PVDF membranes (Millipore). The membranes were closed at room temperature for 15 min using a rapid closure solution from NCM Biotechnology (China). They were then incubated overnight at 4 °C with different primary antibodies. On the subsequent day, the membranes were washed three times with Tris buffered saline with a Tween-20 solution and incubated with the corresponding IRDye-labeled secondary antibodies for 1 h at room temperature. Protein bands were detected using the Li-COR Odyssey imaging system (LI-COR Biosciences). Western blot image analysis was conducted in gray scale mode using ImageJ software.

Microtubule Dynamics Assay

The altered dynamics of MT was reflected by the change in the ratio of MT/tubulin proteins in the cells, and the extraction of MT/tubulin was carried out according to the kit instructions (Microtubules/Tubulin In Vivo Assay Kit, Cytoskeleton). The detailed experimental methodology was as follows: (1) the centrifuge was preheated for at least 2 h prior to the experiment; (2) 2 mL of prewarmed Lysis and Microtubule Stabilization Buffer was incubated in a 6 cm Petri dish at 37 °C for 3 min (1 μL of 100 mmol/L GTP, 10 μL of 100 mmol/L ATP, and 10 μL of protease inhibitor was added to each mL of LMS2); (3) Centrifuge tubes, including sample tubes, positive control, and negative control were labeled. The positive control used was paclitaxel diluted 1:100 added to the lysate, while the negative control was CaCl2 1:100 dilution added to the lysate; (4) Using a cell scraper, the cells and lysate were collected from the Petri dish into a centrifuge tube, and were centrifuged rapidly at 2000g 37 °C for 5 min. The supernatant was transferred to a special high-speed centrifuge tube and centrifuged at 1,00,000g 37 °C for 30 min. The labeled centrifuge tubes were placed on ice and 10 mL ultrapure water added to each. After centrifugation, the supernatant was quickly transferred to a new tube and placed on ice; The MT depolymerization control solution was diluted 1:100 and placed on ice, and the same volume of this liquid was added to the supernatant and resuspended, depolymerized for 15 min at room temperature and mixed every 2 min with a 200 μL spiker; (9) 10 μL of the supernatant and precipitated solutes from steps 7 and 8 and add 90 μL of pure water and 25 μL of 5× SDS sample buffer were mixed and placed in a metal bath at 95 °C for 2 min; and (10) 20 μL from each group of samples was used for Western blot.

LY294002 Pretreatment

LY294002 (CST #9901) was dissolved in DMSO. For a 10 mmol/L stock solution, 1.5 mg of LY294002 was reconstituted in 488 μL of DMSO. The doses (10 μmol/L) were based on preliminary experiments and were chosen to specifically inhibit PI3-kinase activity. LY294002-pretreated BEAS-2B cells were treated with LY294002 for 24 h. In the PM2.5 plus LY294002-pretreated group, the cells were treated with LY294002 and PM2.5 (25 μg/mL) for 24 h. The cells were incubated with DMEM as a control.

Construction of GSK-3β Overexpression Cells by Lentiviral Transfection

Lentiviral transfection was performed when the cell density in the culture dishes reached 30%. First, 980 μL of DMEM, 20 μL of virus suspension, and 0.5 μL of Polybrene were added to the cells, and the cells were incubated for 24 h. The virus-containing medium was then removed, and fresh complete medium was added to the cells, which were incubated for an additional 24 h. After transfection, 2 μg/mL puromycin was used to screen the effectively transfected cells. A negative control group was set up to determine the end time of the resistance screening. The drug screening was terminated when all negative control cells had died. At the end of the drug selection, the surviving cells in the transfected group were further cultured, and when the cell density reached 80% or higher, the cells were harvested and proteins were extracted for Western blot. If the expression of the GSK-3β in the lentivirus-treated cells increased significantly compared to the normal cell line, the GSK-3β overexpression cells were considered to have been successfully established.

Statistical Analysis

Data on the occurrence of multinucleated cells were expressed as incidence rates and analyzed using the chi-square test. Other data sets were presented as the mean plus or minus the standard deviation (SD). Statistical significance was determined by one-way ANOVA, followed by the least significant difference (LSD) post hoc test for comparing differences among groups. A p-value of less than 0.05 was deemed to indicate statistically significant differences.

Results

Multinucleation of Bronchial Epithelial Cells Induced by PM2.5 In Vivo and In Vitro

After the mice were treated with PM2.5 (10 mg/kg·bw) via intratracheal instillation, histopathological analyses of the lung tissues were performed following H&E staining. As shown in Figure A, PM2.5 exposure resulted in lung injury, including alveolar lumen dilatation, alveolar formation, capillary stasis, inflammatory cell infiltration, and widening of the pulmonary septa, compared to the control group. Additionally, the ratio of multinucleated bronchial epithelial cells in the PM2.5-treated group increased to 2.06%, which was significantly higher than that of the control group (1.29%, p < 0.05) (Figure B).

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Histopathological changes of lung tissue and multinucleation of bronchial epithelial cells after exposed to PM2.5. (A) Representative HE staining results of lung tissue of C57BL/6 mice. n = 3 per group. (a) Control group, (b–d) PM2.5-treated group: alveolar dilatation, capillary stasis, infiltration of inflammatory cells in alveolar septa, and thickening of alveolar septa were observed. Scale bar: 200 μm. (B) Multinucleated cells formation in bronchial epithelium induced by PM2.5. (a) Control group and (b) multinucleated cells (yellow arrows) were observed in the bronchial epithelium of mice in the PM2.5-treated group (10 mg/kg bw), scale bar: 50 μm. (c) The ratio of multinucleated cells after exposed to PM2.5 in lung tissue. *p < 0.05, compared with the control group. (C) Binucleated and multinucleated cells formation in BEAS-2B cells after 24 h exposed to different concentrations of PM2.5. (a) Control group, (b–d) PM2.5-treated group (6.25, 12.5, 25 μg/mL), abnormal nuclei including binuclei and multinuclei were shown by the yellow arrows, scale bar: 20 μm. (e) The ratio of multinucleated cells after exposed to different concentrations of PM2.5 in BEAS-2B cells. *p < 0.05, compared with the control group.

In vitro, BEAS-2B cells were exposed to different concentrations of PM2.5 (6.25, 12.5, and 25 μg/mL) for 24 h. Abnormal cell morphology, including binucleate, multinucleate, and irregular nuclei, was observed in the PM2.5-treated group (Figure C, yellow arrows). When the PM2.5 concentration reached 25 μg/mL, the multinucleation ratio of BEAS-2B cells increased to 6.0%, approximately 4.3 times higher than that of the control group (p < 0.05). These results suggested that PM2.5 exposure might cause lung damage and induce the formation of multinucleated cells in the bronchial epithelial cells.

Abnormal MT Dynamics and Spindle Morphology Induced by PM2.5 in BEAS-2B Cells

The dynamic balance between polymerization and depolymerization of MT proteins is crucial for maintaining spindle function. Abnormal MT dynamics can lead to abnormal mitosis, resulting in the formation of binucleation and multinucleation. Free and polymerized MT were extracted using the Microtubule/Tubulin In Vivo Assay Kit, and protein levels were determined by Western blot. The results showed that the level of polymerized MT significantly increased after BEAS-2B cells were exposed to PM2.5 (25 μg/mL) for 24 h, compared to the control group (Figure A). Next, morphological changes in MT during mitosis were observed by immunofluorescence. Under normal conditions, intracellular MT were evenly distributed in the cytoplasm (Figure B­(a)). However, after PM2.5 treatment, an abnormal aggregation of MT was observed (Figure B­(b)). Figure C shows the spindle morphology at the metaphase of mitosis. In the control group (Figure C­(a)), the spindles showed normal morphology with narrow ends and a wide middle and well-localized bipolarity, whereas multipolar spindles, bipolar asymmetric spindles, and abnormal aggregation of spindle filaments were observed in the PM2.5-treated group (Figure C­(b–d)). These findings indicated that the dynamic balance of MT might be altered after PM2.5 treatment, with increased levels of polymerized MT and decreased MT dypolymerization. In addition, PM2.5 affected the normal arrangement of MT in the cells and the formation of spindle filaments during mitosis.

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Effect of PM2.5 on microtubule dynamics and spindle morphology. (A) Westen blot results of polymerized/depolymerized microtubules in PM2.5-treated group (25 μg/mL) and control group. (B) Immunofluorescence results of microtubule morphology in the interphase of mitosis. (a) Control group and (b) PM2.5-treated group (25 μg/mL). (C) Immunofluorescence results of spindle morphology at the telophase of mitosis, (a) normal bipolar spindle in control group, (b–d) multipolar spindle, asymmetric spindle, and spindle filament abnormally aggregated spindle in PM2.5-treated group. Green: microtubules, scale bar: 2 μm.

The Upregulation of MT Acetylation Level Induced by PM2.5 in BEAS-2B Cells

Post-translational modification of MT can affect MT dynamics by altering the conformational state, with an increase in MT acetylation levels being a sign of a decrease in MT dynamics. Western blot results showed that the level of MT acetylation gradually increased with higher PM2.5 concentrations. Statistically significant differences (p < 0.05) were observed in the 12.5 and 25 μg/mL PM2.5-treated groups compared to the control group (Figure A). To further explore the PM2.5-induced changes in MT dynamics, the distribution and localization of acetylated MT during mitosis were observed by immunofluorescence. The results showed that the fluorescence intensity of acetylated MT was upregulated with increasing PM2.5 concentration in the prophase, metaphase, anaphase, and telophase of mitosis. An increase in the aggregation of MT labeled with green fluorescence was found (Figure B). These results suggested that exposure to PM2.5 led to increased levels of MT acetylation, indicating the reduction of MT dynamic instability.

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Effect of PM2.5 on microtubule acetylation level. (A) Western blot results of acetylated microtubule proteins after 24 h exposed to PM2.5 (6.25, 12.5, 25 μg/mL). n = 3 per group, data were expressed as mean ± SD *p < 0.05 and **p < 0.01, compared with the control group. (B) Immunofluorescence results of spindle morphology and microtubule protein acetylation level in each mitotic period. Green: tubulin, blue: DAPI, red: acetylated microtubules, scale bar: 2 μm.

The Downregulation of Deacetylase HDAC6 Induced by PM2.5 in BEAS-2B Cells

Cellular MT acetylation levels are regulated by two main types of enzymes, acetylase α-TAT1 and deacetylases (HDAC6 and SIRT2). To further investigate the changes in MT acetylation levels induced by PM2.5, the above enzymes were examined by Western blot. The expression level of HDAC6 decreased in a dose-dependent manner with increasing PM2.5 concentrations (p < 0.01), while the expression levels of α-TAT1 and SIRT2 did not show significant differences compared to the control group (Figure A). Next, the cellular level and localization of HDAC6 were detected by immunofluorescence, and the fluorescence intensity of HDAC6 (red) in the PM2.5-treated group was obviously reduced and was mainly distributed in the cytoplasm compared to the control group (Figure B). These results indicated that PM2.5 inhibited the expression of deacetylase HDAC6, which further contributed to the increase in MT acetylation.

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Effect of PM2.5 on enzymes responsible for microtubule acetylation/deacetylation. (A) Western blot results of microtubule regulatory α-tubulin deacetylases, histone deacetylase-6 (HDAC6) and SIRT2, and microtubule regulatory α-tubulin acetylases, α-tubulin acetyltransferase (αTAT1) in BEAS-2B cells after 24 h exposed to PM2.5. n = 3 per group, data were expressed as mean ± SD *p < 0.05 and **p < 0.01, compared with the control group. (B) Immunofluorescence results of PM2.5 effected on the fluorescence intensity of HDAC6 in BEAS-2B cells, blue: DAPI, red: HDAC6, scale bar: 10 μm.

Abnormal Localization of MT-Binding Protein MAP1B Induced by PM2.5 in BEAS-2B Cells

MAP1B is a major MT-binding protein involved in MT assembly and the regulation of MT dynamics. The binding of MAP1B to MT promotes MT dynamics, while the dissociation of MAP1B from MTs results in overstability of the MTs. In this study, multiple immunofluorescence staining was used to observe the binding status of MAP1B to MTs in BEAS-2B cells treated with different concentrations of PM2.5 (6.25, 12.5, and 25 μg/mL) for 24 h. The results are shown in Figure . In the control group, the red fluorescence of the labeled MAP1B protein and the green fluorescence of the labeled MTs overlapped, which indicated that MAP1B colocalized well with MTs at all stages of mitosis. However, in the PM2.5-treated groups, MAP1B (red), which was originally bound to MTs (green), gradually dissociated from the MTs as the PM2.5 concentration increased during mitosis. These results indicate that PM2.5 affected the binding of MAP1B to MTs, with MAP1B progressively dissociating from the MTs as the level of PM2.5 exposure increases. This dissociation contributed to a decrease in MT dynamics.

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Effect of PM2.5 on the binding status of microtubule-binding proteins (MAP1B) to microtubules. Immunofluorescence results of the altered microtubule-binding status of MAP1B to microtubules in BEAS-2B cells treated with different concentrations of PM2.5 (6.25, 12.5, and 25 μg/mL) during different stages of mitosis. Blue: DAPI; green: microtubule; red: MAP1B, scale bar: 2 μm.

Activation of PI3K/AKT/GSK-3β Signaling Pathway Induced by PM2.5 in BEAS-2B Cells

In our previous study, transcriptomic analyses of BEAS-2B cells exposed to PM2.5 revealed that the PI3K/AKT/GSK-3β signaling pathway is closely related to cell division and cytoskeletal regulation. To better understand the mechanism underlying multinucleation caused by PM2.5, the expression of PI3K/AKT/GSK-3β pathway-related proteins was analyzed by Western blot after 24 h of PM2.5 treatment. The protein expression levels of the PI3K regulatory subunits p110α and p110β were measured. The results showed that PI3K p110β expression was significantly upregulated at 25 μg/mL PM2.5 (p < 0.05), while PI3K p110α was not affected by PM2.5. Next, the phosphorylation level of AKT, a serine/threonine kinase activated downstream of PI3K, was determined. Phosphorylated AKT (Ser473) expression increased after PM2.5 treatment, especially in the 12.5 and 25 μg/mL PM2.5 treatment groups. These results indicated that PM2.5 activated AKT in mitosis through the p110β subunit of PI3K. GSK-3β, which is a negative feedback loop of AKT, plays a key role in MT dynamics and cellular division. The activity of GSK-3β is mainly determined by the phosphorylation of the different sites. Phosphorylation at Ser9 inhibits GSK-3β activity, while phosphorylation at Tyr216 enhances GSK-3β activity. Thus, protein levels of the GSK-3β Ser9 and Tyr216 sites were determined. The results are shown in Figure . Compared to the control group, PM2.5 significantly increased the phosphorylation of GSK-3β Ser9 at 25 μg/mL (p < 0.05), while it significantly decreased Tyr216 phosphorylation at 12.5 and 25 μg/mL (p < 0.05). These results indicated that PM2.5 inhibits GSK-3β activity by activating PI3K 110β and increasing the phosphorylation level of AKT (Ser473), which might lead to the increase of MT acetylation and the decrease of MT dynamics.

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Effects of PM2.5 on PI3K/AKT/GSK-3β signaling pathway in BEAS-2B cells. (A) Western blot results of PM2.5 effect on the PI3K/AKT/GSK-3β signaling pathway-related proteins in BEAS-2B cells. (B) Gray scale analysis of PI3K/AKT/GSK-3β pathway-related proteins using ImageJ. (a–g) PI3K p110α, PI3K p110β, AKT, p-AKT (ser473), GSK-3β, p-GSK-3β (ser9), p-GSK-3β (Tyr216). n = 3 per group, and the data were expressed as mean ± SD *p < 0.05 and **p < 0.01, compared with the control group.

Upregulation of MT Acetylation via the AKT/GSK-3β Signaling Pathway Induced by PM2.5 In Vivo

To further explore the relationship between the AKT/GSK-3β signaling pathway with HDAC6 activity and MT acetylation, the expression levels of AKT/GSK-3β pathway-related proteins in mouse airway epithelial cells after PM2.5 treatment (10 mg/kg·bw) were analyzed by immunohistochemistry. As shown in Figure , exposure to PM2.5 was found to significantly increase the expression of p-AKT (ser473) (p < 0.05). The decreasing expression of p-GSK-3β (Tyr216) indicated the inhibition of GSK-3β activity compared to that of the control group (Figure B). Next, the levels of deacetylase HDAC6 and MT acetylation were also analyzed in mouse airway epithelial cells. The results, shown in Figure C,D, indicated a decrease in the expression of the deacetylase HDAC6 as well as a significant increase in the expression level of MT acetylation ACE40 (p < 0.05). These findings suggest that PM2.5 activated AKT, negatively regulated GSK-3β activity, inhibited the expression of the deacetylase HDAC6, and increased MT acetylation levels in vivo.

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Effects of PM2.5 on AKT/GSK-3β signaling pathway in mice lung tissues. (A–D) Immunohistochemical and quantitative analysis results of p-AKT (ser473), p-GSK-3β (Tyr216), HDAC6, and acetylated microtubules in mice lung tissues, respectively. n = 5 per group. (a) Control group and (b) PM2.5-treated group (10 mg/kg bw). (c) The corresponding gray value analysis results. The IOD values of the above protein expressions were analyzed using ImageJ, and the data were expressed as mean ± SD *p < 0.05, compared with the control group, scale bar: 200 μm.

LY294002 Decreased the Formation of Multinucleation in BEAS-2B Cells by Affecting MT Acetylation and Localization of MAP1B

To further explore the mechanism by which PM2.5 induced multinucleation and MT acetylation via the PI3K/AKT signaling pathway, the inhibitor LY294002 (10 μmol/L) was used to suppress the PI3K activity before exposing BEAS-2B cells to PM2.5. The level of PI3K downstream protein expression as well as MT acetylation and multinucleation formation were determined. The experimental groups were set as control group, 10 μmol/L LY294002-pretreated group, 25 μg/mL PM2.5-treated group, and 25 μg/mL PM2.5 plus 10 μmol/L LY294002-pretreated group. The results, shown in Figure A,B, demonstrate that compared to the PM2.5-treated group, the PM2.5 plus LY294002-pretreated group restored the protein expression level of deacetylase HDAC6 (p < 0.05). The expression of the MT acetylating protein ACE40 was decreased after PM2.5 treatment. However, there was a rising trend in the PM2.5 plus LY294002 group, compared to the PM2.5 group, indicating that LY294002 could effectively increase the level of HDAC6 and reduce the level of MT acetylation by inhibiting PI3K/AKT signaling. Additionally, the results of multiple immunofluorescence staining showed the status of the MAP1B protein in the cytoplasm. MAP1B protein gradually dissociated from MTs in the PM2.5-treated group, but the colocalization levels of MAP1B and MTs were restored to the normal state in the PM2.5 plus LY294002 group (Figure C), which indicated that LY294002 could also effectively reverse the colocalization of MAP1B and MTs to the normal level by inhibiting PI3K activity. The multinucleation ratio of the cells was detected by Giemsa staining, and the results are shown in Figure D, while the multinucleation ratio of the PM2.5 plus LY294002-treated group was significantly decreased compared with the PM2.5-treated group (p < 0.05). These findings suggest that LY294002 could further suppress PM2.5-induced multinucleation in BEAS-2B cells by increasing the expression of HDAC6 and reducing the level of MT acetylation as well as by restoring the status of the MT-binding protein MAP1B.

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Pretreatment of LY294002 reversed the effect of PM2.5 on PI3K/Akt/GSK-3β signaling, microtubule-associated proteins and multinucleated cells formation. (A) Western blot results of PM2.5 effect on p-AKT (ser473), p-GSK-3β (ser9), HDAC6, and acetylated microtubules protein levels with or without the pretreatment of LY294002. (B) Quantitative analysis of the gray scale values of the above proteins using ImageJ, n = 3 per group, and the data were expressed as mean ± SD *p < 0.05 and **p < 0.01, compared with the control group. (C) Immunofluorescence results of PM2.5 effect on the colocalization of MAP1B with microtubules with or without the pretreatment of LY294002. Blue: DAPI; green: microtubules; red: MAP1B, scale bar: 2 μm. (D) Multinucleation analysis results of PM2.5 effect on the multinucleated cells formation with or without the pretreatment of LY294002. (a) Control group, (b) LY294002 pretreatment group, (c) PM2.5 (25 μg/mL)-treated group, (d) PM2.5 plus LY294002-pretreated group; yellow arrows indicated were abnormal nuclei, including binucleated, multinucleated, and irregular nuclei cells. (e) Ratio of multinucleated cells in each group. *p < 0.05, compared with the control group, scale bar: 20 μm.

GSK-3β Overexpression Reduced the Effect of PM2.5 on MT Acetylation and Multinucleation

To investigate the relationship between PM2.5-induced multinucleation and GSK-3β/HDAC pathway-related protein levels, lentiviral transfection technology was used to construct BEAS-2B cells overexpressing GSK-3β. The transfected cells were selected with puromycin, and the overexpression efficiency was greater than 80%. The changes in the expression levels of GSK-3β and MT acylation-related proteins were detected by Western blot after exposure to PM2.5. Compared to the control group, the protein level of GSK-3β increased significantly in the GSK-3β overexpressing cells, confirming the successful construction of the overexpression cell line. The experimental groups were set as control group, 12.5 μg/mL PM2.5-treated group, GSK-3β overexpression group, and 12.5 μg/mL PM2.5 plus GSK-3β overexpression group. As shown in Figure A, compared to the PM2.5-treated group, the protein level of deacetylase HDAC6 was upregulated in the PM2.5 plus GSK-3β overexpression group. Similarly, MT acetylation protein ACE40 was downregulated after GSK-3β overexpression (p < 0.05). Additionally, after BEAS-2B cells were stained with Giemsa staining, the number of multinucleated cells was observed and counted under a microscope, and then the results were statistically analyzed. The experimental results are shown in Figure B. The multinucleation ratio in the PM2.5-treated group was 4.68%, significantly higher than the multinucleation ratio of the control group (approximately 2.07%). However, compared to the PM2.5-treated group, the multinucleation ratio decreased to 2.19% in the PM2.5 plus GSK-3β overexpression group. The results showed that overexpression of GSK-3β led to an increase in the level of deacetylase HDAC6 and a decrease in the level of MT acetylation, which further affected the formation of multinucleation induced by PM2.5.

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GSK-3β overexpression inhibited the effect of PM2.5 on the GSK-3β/HDAC6 pathway. (A) Western blot results of PM2.5 effect on GSK-3β, HDAC6, and acetylated microtubules protein in GSK-3β overexpressing cell lines and the wild type cell lines, n = 3 per group, and data were expressed as mean ± S.D., *p < 0.05, compared with the control group. (B) Comparison of multinucleation formation ratio in GSK-3β overexpressing cell lines and the wild type cell lines after PM2.5 exposure, *p < 0.05, compared with the control group.

Discussion

In recent years, numerous studies have shown that PM2.5 is one of the main risk factors for many respiratory diseases, especially lung cancer. Wang et al. found that long-term exposure of lung cancer cells to PM2.5 promoted cell proliferation and tumor growth. A previous study by our group found that short-term exposure to PM2.5 significantly increased the proportion of multinucleated bronchial epithelial cells. As is well known, most bronchial epithelial cells have only one nucleus, and multinucleated cells, as a morphological indicator of CIN, have been suggested for use in tumor diagnosis and prognosis. Therefore, investigating the molecular mechanisms underlying the formation of multinucleated cells induced by PM2.5 might provide a new theoretical basis for the emergence of lung tumors caused by PM2.5.

At the in vivo level, this study employed intratracheal instillation to administer short-term PM2.5 exposure to mice. Its primary objective was to elucidate the early critical events and associated signaling pathways involved in PM2.5-induced malignant transformation of lung cells rather than to directly simulate the complete process of lung cancer development under long-term environmental exposure. The intratracheal instillation method was chosen due to its precise dose control and high reproducibility, making it suitable for effectively inducing and observing early toxic phenotypes, such as the formation of multinucleated cells, within a relatively short time frame. At the same time, it was important to fully recognize the limitations of this method in simulating natural inhalation deposition patterns. Therefore, future research will focus on developing chronic, low-dose inhalation models that simulate real-world conditions to validate the translational relevance of these mechanisms throughout carcinogenesis. The current study, to investigate whether short-term PM2.5 exposure induces multinucleated cell formation, includes parallel in vivo and in vitro studies. Histopathological analysis of mouse lung tissue revealed that PM2.5 caused lung damage, mainly characterized by alveolar lumen dilatation, capillary congestion, inflammatory cell infiltration, and widening of the pulmonary septa (Figure A). Furthermore, the PM2.5-treated group exhibited a higher proportion of multinucleated cells compared to the control group (Figure B). Multinucleated cells are mainly observed within pseudostratified ciliated columnar cells. However, they are very rarely found in the normal airways of the control group. The occasional presence of these multinucleated cells in control animals may be attributed to low-frequency cytokinesis failure or tissue-sectioning artifacts. However, PM2.5 exposure induced a significant (1.6-fold) increase in the number of multinucleated cells, indicating its role in promoting mitotic disruption and genomic instability in bronchial epithelium. This change underscores a PM2.5-driven early cellular abnormality that is relevant to carcinogenic progression. Consistent with these in vivo findings, the proportion of multinucleated cells also increased in a dose-dependent manner with higher PM2.5 exposure in vitro (Figure C). These results demonstrate that PM2.5 could induce the formation of multinucleated bronchial epithelial cells both in vivo and in vitro. It is noteworthy that recent studies have also shown that PM2.5 can induce senescence in alveolar type II cells and impair their proliferative and self-renewal capacities. This suggests that the toxic effects of PM2.5 on pulmonary epithelial cells are specific to the cell types and mechanisms. However, the underlying mechanisms remained unclear, prompting further investigation.

Some studies have shown that multinucleated cells mainly arose from mitotic arrest. − Post-arrest, cells either initiated apoptosis or exit without dividing into daughter cells resulting in multiple small nuclei. , Once multinucleated, these cells generally could not recover normal nuclear morphology and either die, remain arrested, or attempt another mitosis that was typically multipolar (e.g., tripolar and not bipolar). Over time, the CIN index increased and the cells became more susceptible to malignant transformation. Chenet et al. also suggested that aneuploidy due to mitotic errors contributed to both tumorigenesis and the progression of cancer toward more aggressive genotypes. Mitotic arrest might occur due to perturbations in MT dynamics. MT stabilizers have been shown to affect mitotic morphology, causing errors in chromosome alignment and segregation, spindle multipolarity, and multipolar division. In other words, reduced MT dynamics played a crucial role in mitotic arrest and formation of multinucleated cells. The results of the MT protein dynamics assay showed that the MT/tubulin ratio in the PM2.5-treated group was higher than that in the control group (Figure A). Morphological observations suggested that MTs were uniformly distributed in the cytoplasm of the control group, whereas PM2.5 treatment caused local aggregation and structural disorganization of MTs (Figure B). Additionally, the PM2.5-treated group showed several structural abnormalities in the spindle, including spindle multipolarity, asymmetry of the two poles of spindles, and abnormal aggregation of the spindle filaments (Figure C). Therefore, it could be speculated that PM2.5 caused an increased proportion of multinucleated cells, which was associated with impaired MT dynamics. Exposure to PM2.5 resulted in an increase in polymerized MTs and a decrease in MT dynamics as well as an abnormal aggregation of MTs in the cytoplasm. Thus, PM2.5 decreased the dynamics during mitosis, which prevented the successful assembly of spindles, leading to mitotic arrest and ultimately the formation of multinucleated cells.

MTs exhibited MT dynamics, which was mainly related to their PTMs and the binding state of MT proteins to them. PTMs could affect MT properties directly by structurally altering the MT lattice, or indirectly by altering MT interaction partners. Existing research showed that tubulins could undergo many chemically distinct PTMs. MT acetylation was the only post-translational modification that occurred within the MT lumen and was closely associated with MT instability. Our results also showed that MT acetylation levels increased in a dose-dependent manner with increasing PM2.5 concentrations (Figure A). This suggested that PM2.5-promoted MT acetylation may contribute to the reduced intracellular MT dynamics after PM2.5 treatment. The relationship between MT hyperacetylation and mitotic abnormalities was observed by confocal microscopy. The results showed that the acetylated spindle MT protein increased with increasing PM2.5 concentrations in the process of mitosis. Additionally, the PM2.5-treated group exhibited a slight aggregation of spindle filaments, resulting in an abnormal spindle morphology (Figure B). As a result, PM2.5 caused persistent MT hyperacetylation throughout the cell cycle, leading to decreased MT dynamics and impaired spindle assembly, which, in turn, resulted in mitotic arrest and multinucleated cell formation.

MT acetylation is regulated by multiple enzymes. α-Tubulin-N-acetyltransferase 1 (aTAT1) mediates MT acetylation on the lysine 40 residue located inside the MT. Deacetylation also occurred on tubulin dimers and was mediated by several enzymes, such as HDAC6 and sirtuin 2 (SIRT2). , In the current research, PM2.5 inhibited HDAC6 expression in a dose-dependent manner, but did not significantly affect α-TAT1 and SIRT2 (Figure A). Similarly, HDAC6 was uniformly distributed in the cytoplasm and decreased with an increasing PM2.5 dose (Figure B). Osseni et al. reported that HDAC6 regulated MT acetylation independently of other enzymes and found that pharmacological inhibition of HDAC6 expression alone prevented MT disorganization. Therefore, the inhibition of HDAC6 expression by PM2.5 might be the primary reason for MT hyperacetylation.

The binding state of MT-associated proteins to MTs represents another factor that affects MT dynamics. MT-associated protein 1B (MAP1B), as a lattice protein, could be involved in regulating MT dynamics by reducing MT stability or by regulating the recruitment plus-end tracking proteins to the plus-end of the MTs. Tymanskyj et al. also showed that MAP1B enhanced MT assembly rates by binding to dynamic MTs. Our study found that PM2.5 affected MAP1B in MTs. In the control group, MAP1B was bound to MTs. However, when the cells were treated with PM2.5, MAP1B gradually dissociated from MTs during mitosis as the concentration of PM2.5 increased (Figure ). This phenomenon suggested that PM2.5 had a negative effect on the binding of MAP1B to MTs. Thus, the altered binding state of MAP1B to MTs after PM2.5 exposure might be one of the reasons for the imbalance in the MT dynamic instability and abnormal mitotic spindle MT assembly caused by PM2.5. In conclusion, PM2.5 caused MT overacetylation by inhibiting the expression of the deacetylase HDAC6. Additionally, PM2.5 inhibited the binding of MT-binding protein MAP1B to dynamic MTs. In both cases, PM2.5 caused a reduction in MT dynamics, leading to an imbalance in spindle MT dynamics and abnormal spindle assembly, which ultimately led to mitotic failure and the formation of multinucleated cells.

To investigate the molecular mechanism of PM2.5 in regulating the actin cytoskeleton to promote multinucleated cells formation, we used mRNA microarrays to detect the effects of PM2.5 on the gene expression profiles of BEAS-2B cells and bioinformatics techniques to analyze the functions and pathways in which the differentially expressed genes are involved. The results showed that PM2.5 caused upregulation of the PI3K/AKT/GSK-3β pathway, which was closely related to cytoskeletal regulation. Yang et al. showed that the PI3K/AKT/GSK-3β signaling pathway regulates MT dynamics. To understand how PM2.5 disrupted MT dynamics, the expression of the PI3K/AKT/GSK-3β signaling pathway was detected in vitro (in BEAS-2B cells) and in vivo (male C67BL/6J mice). At the in vitro level, Figure shows that PM2.5 did not affect PI3K p110α subunit protein levels, but PI3K p110β, p-AKT (ser473), and p-GSK-3β (s9) protein levels tended to increase with increasing PM2.5 concentration, and p-GSK-3β (Tyr216) protein expression showed a decreasing trend. The activity of GSK-3β was determined by its phosphorylation status. Phosphorylation of the N-terminal serine-9 residue (p-GSK-3β (S9)) had a negative correlation with GSK-3β activity, while hyperphosphorylation at Tyr216 (p-GSK-3β (Tyr216)) had a positive correlation with it. Therefore, PM2.5 could activate the PI3K/AKT pathway and inhibit the GSK-3β activity. GSK-3β is a kinase that regulates the activity of HDAC6, and it could interact directly with HDAC6, thereby increasing its activity and inducing an increased level of MT acetylation. At the same time, the study suggested that MAP1B was one of the substrates for GSK-3β. The binding of MAP1B to MTs was also affected by GSK-3β activity. At the in vivo level, PM2.5 entering the lungs via the respiratory tract increased the expression of p-AKT and p-GSK-3β (S9) and decreased the expression of HDAC6 in lung tissue cells, which led to an increase in the level of MT acetylation (Figure ). Taken together, the decrease in HDAC6 expression and dissociation of MAP1B from dynamic MTs caused by PM2.5 might be linked to the activation of the PI3K/AKT pathway followed by GSK-3β inhibition. To confirm this hypothesis, we selected the PI3K inhibitor LY294002 and the GSK-3β overexpressing cell lines for the next experiments.

LY294002 is an inhibitor that effectively blocks AKT phosphorylation downstream of PI3K. It is commonly used to inhibit the PI3K/AKT/GSK-3β signaling pathways. The results showed that compared to the control group, PM2.5 caused an increase in the expression levels of p-AKT and p-GSK-3β (S9), resulting in a decrease in GSK-3β activity, which in turn inhibited HDAC6 and led to MT hyperacetylation. However, following treatment with PM2.5 plus LY294002, the expression levels of p-AKT and p-GSK-3β (S9) decreased, while HDAC6 expression increased and the level of MT acetylation decreased compared to the PM2.5-treated group (Figure A,B). Furthermore, Figure C indicates that PM2.5 led to the dissociation of MAP1B from MTs. However, this dissociation was reduced when cells were treated with PM2.5 plus LY294002 (Figure C). This phenomenon suggested that the reduction in GSK-3β activity caused by PM2.5 activated the PI3K/AKT pathway, which inhibited the phosphorylation of MAP1B and caused it to dissociate from dynamic MTs. Figure D confirms that exposure to PM2.5 increased the proportion of multinucleated cells. The number of multinucleated cells was significantly lower in the PM2.5 plus LY294002 group compared to the PM2.5-treated group. Meanwhile, in order to investigate the regulatory relationship between GSK-3β activity and HDAC expression, we developed a cell model with a high level of GSK-3β expression. As shown in Figure A, the protein level of HDAC6 was upregulated in the PM2.5-treated group in the GSK-3β overexpressing cell line, and MT acetylation was downregulated in the PM2.5-treated group compared with that in the normal cell line (Figure A). Thus, it was demonstrated that PM2.5 inhibited GSK-3β activity by activating the PI3K/AKT pathway, which in turn inhibited HDAC6 protein expression, leading to increased levels of MT acetylation. Figure B also found that in wild-type cell lines, the PM2.5-treated group exhibited a higher number of multinucleated cells compared to the control group. However, the proportion of multinucleation in GSK-3β overexpressing cell lines was observed to decrease compared with wild-type cell lines following simultaneous exposure to PM2.5. This suggested that PM2.5 activated the PI3K/AKT/GSK-3β pathway, which was linked to multinuclear cell formation.

Conclusions

PM2.5 activated the PI3K/AKT/GSK-3β pathway and inhibited HDAC6 in the BEAS-2B cells. This led to the overacetylation of MTs, on the one hand, and the inhibition of MAP1B binding to MTs on the other hand. Both of the processes resulted in the overstabilization of the MTs, which in turn caused a disturbance in the dynamic balance of the spindle filaments, and finally contributed to spindle MT dysfunction, mitotic arrest, and the formation of multinucleated cells.

Acknowledgments

Y.L. would like to acknowledge the support of China Scholarship Council and also wishes to express gratitude to Dr. Emilie Brun from School of Geography, Earth & Environmental Sciences, University of Birmingham, for her invaluable guidance and assistance during the experimental process. Z.G. and I.L. acknowledge funding from the Horizon Europe Guarantee Fund via Innovate UK (Grant No. 10066165) to enable participation in the MACRAME project (Grant Agreement No. 101092686).

The experimental design was devised by Y.L. and L.X., with L.X. responsible for the experimental manipulation. L.X., J.P., and Z.G. were involved in the initial drafting of the manuscript, while K.Z., Z.X., M.Y., and J.W. were responsible for data organization. Y.L., Z.G., and I.L. were responsible for the review and editing. All authors have read and approved the final manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

All animal care and experimental procedures were approved by the Animal Ethics Committee at Capital Medical University (approval number AEEI-2019–003).

The authors declare no competing financial interest.

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