Introduction

Nano- and microplastics (NMPs) are particles and fibers with diameters smaller than 5 µm. These tiny particles are generated from a variety of sources, such as the degradation of larger plastic items, microbeads in personal care products, and fibers from synthetic clothing. According to a study by Ahmed I Osman et al., NMPs can be classified based on their chemical composition, including polystyrene (PS), polypropylene, polyethylene, polyurethane, polyvinyl chloride and other materials1. Among these microplastics, PS is particularly significant and prevalent in the seafood, suggesting its widespread presence in the environment2. Recently, there has been a growing focus on studying the toxic effects of PS on human health2. Therefore, the objective of this study is to investigate the impact of PS microplastics on liver injury.

Long-term exposure to NMPs inevitably leads to the accumulation of NMPs in the human body through ingestion, inhalation, and dermal contact3,4,5. Accumulated studies on the harm of NMPs exposure to animals have demonstrated that NMPs may potentially harm humans by causing immune and stress responses, reproductive toxicity, etc.6. However, the mechanism by which NMPs exposure harms organisms is still unclear.

Liver fibrosis is an intermediate stage of chronic liver disease caused by various pathogenic factors. It can progress to liver cirrhosis and even to liver cancer, which is characterized by excessive accumulation of extracellular matrix and structural and functional changes in liver parenchymal and nonparenchymal cells under continuous liver injury and chronic inflammation7,8,9. The main pathogenesis involved in the development of liver fibrosis includes inflammation, apoptosis, lipid metabolism disorder and oxidative stress9,10,11. NMPs have been considered as hepatotoxic components. In a previous study, long-term exposure to NMPs promoted the accumulation of NMPs in the liver, digestive tract and kidney of mice and led to lipid metabolism disorder and oxidative stress in the liver3. Recent studies have found that long-term exposure to NMPs caused an imbalance in lipid metabolism, an inflammatory response and oxidative stress in the liver of mice and finally led to liver fibrosis12,13. Moreover, NMPs exposure exacerbated liver fibrosis in HFD-fed mice by promoting inflammation and oxidative stress14. Currently, the molecular mechanism of liver fibrosis induced by chronic exposure to NMPs remains unclear.

In the present study, we evaluated the impacts of long-term exposure to polystyrene nano- and microplastics (PS-NMPs) with different diameters (80 nm, 0.5 µm, and 5 µm) on liver fibrosis in vivo. Here, we reported that three hub genes, Acot3, Abcc3, and Nr1i3, play potential roles in the development of liver fibrosis induced by chronic exposure to PS-NMPs.

Materials and methods

Materials and reagents

PS-NMPs of three different diameters (80 nm, 0.5 µm, and 5 µm) were purchased from Baseline ChromTech Research Center (Tianjin, China) and utilized for animal toxicological experiments. Primary antibodies against α-SMA (Proteintech, #14395-1-AP; 1:1000) and CD68 (Boster, #BA3638, 1:1000) were used in this study.

Animal model

Five-week-old ICR male mice were used in the present experiments. All mice were housed under standard conditions (specific pathogen-free environment; 12 h light/dark cycle; room temperature at 21–23 °C) and had free access to their diets and water. All mouse experiments were approved by the Animal Ethics Committee of Wuhan Third Hospital (permission number: SY2021-021) and were carried out in keeping with the guidelines of the National Health and Medical Research Council of China. Animal studies are reported in accordance with ARRIVE guidelines. Forty mice were equally and randomly divided into four groups, including the control group (H2O) (n = 10), the 80 nm group (n = 10), the 0.5 µm group (n = 10), and the 5 µm group (n = 10). PS-NMPs were diluted in sterilized water to a final concentration of 1 mg/L. PS-NMPs-exposed mice directly drank the diluted PS-NMPs water for 12 weeks. The control mice (H2O) were exposed to normal water without PS-NMPs. After 12 weeks of administration, all mice were sacrificed for further experiments.

Evaluation of liver function indices

Body weight was measured before the mice were sacrificed. After sacrifice, the livers of the mice were weighed. Meanwhile, the serum of each mouse was obtained by centrifugation for 10 min at 3000 rpm and 4 °C and utilized to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) according to the manuscript’s protocol [Jiancheng Bioengineering, Nanjing, China].

Histology and immunohistochemistry

Paraffin-embedded liver sections (4 mm) were stained with hematoxylin and eosin (H&E), Masson’s trichrome staining (MTS), or Oil Red O staining following their respective standard procedures. Antibodies against α-SMA (Proteintech, #14,395–1-AP; 1:500) and CD68 (Boster, #BA3638, 1:100) were used for immunohistochemistry staining. For each mouse, ten random fields were selected at × 400 magnification and analyzed by ImageJ.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was obtained from the isolated livers. cDNA was acquired by mRNA transcription. qRT-PCR was performed using the Bio-Rad CFX Manager system. The primer sequences are shown in Supplementary Table 1.

Western blot analysis

Total protein lysates of mouse livers were obtained and used to perform western blotting as described15. The proteins were electrophoresed by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). After blocking with TBS containing 5% skim milk for 1 h at room temperature, the PVDF membrane was incubated with rabbit polyclonal antibody against α-SMA (Proteintech, #14395-1-AP, 1:1000) and mouse polyclonal antibody against IL-1β (Proteintech, #66737-1-lg, 1:1000). Rabbit polyclonal antibody against β-actin was utilized as a normalized control (ABclonal, #AC026, 1:1000). The indicated protein band densitometry was obtained using the gel imaging system (Bio-Rad Image Lab™ Software, USA) and quantified using ImageJ software.

Oxidative stress (GPX, MDA, SOD) analysis

The levels of the oxidative stress-related proteins superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in ovaries were measured by biochemical kits (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions.

Transmission electron microscopic analysis

The liver tissues were used to investigate the ultrastructure by transmission electron microscopy (TEM). Pieces of liver tissues were fixed in 2.5% glutaraldehyde (340855, Sigma-Aldrich), and postfixed in 1% osmium tetroxide. Subsequently, the fixed livers were dehydrated in graded alcohols and embedded in Epon. Ultrathin sections were obtained through cutting on copper grids, and stained with uranyl acetate and lead citrate. Finally, sections were examined and imaged using a digital electron microscope (HT7800, HITACHI, Japan).

RNA-seq and data analysis

Total RNA was extracted from the livers of mice after exposure to different diameters of PS-NMPs (80 nm, 0.5 µm, and 5 µm) according to the manufacturer’s instructions. Transcriptome sequencing of RNA was completed by the Beijing Genomics Institution (BGI). Four independent biological replicate samples were sequenced for each group. Differentially expressed genes (DEGs) between the H2O and NMPs groups were analyzed using linear models for the microarray data (limma) package in R. A |log2 (fold change)| > 1 and an adjusted P value < 0.05 were considered the threshold. The heatmap was drawn by the R package pheatmap. The shared DEGs between each NMPs group and the H2O group were identified by drawing a Venn diagram. Gene ontology (GO, http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.kegg.jp/) pathway enrichment analyses of DEGs were performed by using DAVID online tools (https://david.ncifcrf.gov/). The protein-protein interaction (PPI) network was predicted using the STRING database (http://string-db.org, version 11.5), which is an online database to investigate the functional interactions between proteins. The cutoff value for the minimum required interaction score is 0.4. The PPI network was further visualized and analyzed with the Cytoscape (version 3.4.0) plug-in app CytoHubba, which could analyze data using different algorithms. UpSet plots were drawn by the R package UpSetR.

Statistical analysis

The results are expressed as the mean ± S.E.M. Statistical analyses were implemented with SPSS version 23 (IBM-SPSS Inc., Chicago, IL, USA). All results in PS-NMPs-exposed mice were compared to the control group using one-way ANOVA with multiple comparisons. Post hoc multiple comparison LSD was applied.

Results

Effects of PS-NMPs of different diameters on the livers

In this study, we observed that long-term exposure to PS-NMPs led to remarkable decreases in liver weight and liver/body weight ratio as PS-NMPs particle size increased (Fig. 1A and C). The effect of PS-NMPs exposure at 0.5 and 5 µm on liver weight and liver/body weight ratio showed a similar trend (Fig. 1A and C). However, no effect of PS-NMPs on body weight or liver function indices (ALT and AST) was observed under the different diameters of PS-NMPs (Fig. 1B, D, E). Moreover, after PS-NMPs exposure, we observed liver injury in mice, including hepatocellular vacuolar degeneration and edema, irregularly arranged hepatic cords, cell dikaryon, and inflammation (Fig. 1F, G). These data indicate that PS-NMPs could induce liver injury.

Fig. 1
figure 1

Physiological characteristics and liver histology changes after PS-NMPs exposure (80 nm, 0.5 µm, and 5 µm). The levels of liver weight (A), body weight (B), liver/body weight index (liver weight/body weight (%) (C), ALT (D), and AST (E) at 12 weeks after different diameters of PS-NMPs exposure (n = 5–8). (F) Representative images of mice livers by HE staining (left panel) and quantification of histological changes in liver exposure to different diameters of PS-NMPs (right panel) (n = 8). Red arrow indicated binuclear cells, red triangle indicated hepatocellular vacuolar deformation and edema, black arrow indicated infiltration of inflammatory cells, and black triangle indicated irregularly arranged hepatic cords. Scale bar, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

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Liver fibrosis was promoted by long exposure to PS-NMPs

To investigate the impact of PS-NMPs exposure on liver fibrosis, we conducted MTS to assess the levels of fibrosis in the liver. We observed a significant increase in the percentage of tubulointerstitial fibrosis in mice exposed to PS-NMPs with diameters of 0.5 and 5 µm, compared to the control group (Fig. 2A, B). To further confirm the induction of liver fibrosis by PS-NMPs exposure, we examined the mRNA and protein expression levels of the fibrosis marker, alpha-smooth muscle actin (α-SMA), using qRT-PCR, western blotting, and immunohistochemical staining. While no significant differences were observed in mRNA expression of α-SMA among the groups (Fig. 2C), the protein expression level of α-SMA showed a significant increase with the increase in PS-NMPs particle size (Fig. 2D–F). These findings provide evidence that prolonged exposure to PS-NMPs with larger particle sizes (0.5 µm and 5 µm) can significantly induce liver fibrosis.

Fig. 2
figure 2

PS-NMPs exposure promotes liver fibrosis. (A) Representative images of MTS of liver tissue and (B) semi-quantitative results of liver fibrosis. Scale bar, 100 μm. (C) The mRNA level of α-sma was analyzed by qRT-PCR and normalized by β-actin (n=6). (D) α-SMA protein level was analyzed by western blotting and normalized by β-actin (n=5). (E) Representative images of IHC staining for α-SMA and (F) semiquantification of α-SMA based on IHC staining (n=8). Scale bar, 50 μm *p < 0.05, **p < 0.01, ***p < 0.001.

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Long-term exposure to PS-NMPs promotes liver inflammation

Subsequently, we investigated the role of PS-NMPs exposure in liver inflammation. We observed that CD68, a macrophage and monocyte marker, increased dramatically after long exposure to 80 nm- and 0.5 µm-diameter PS-NMPs via immunohistochemistry staining (Fig. 3A). We then analyzed the mRNA expression of the inflammatory markers Il-6, Il-1β, Tnf-α, Cxcl12, and Areg16, which is a kind of epidermal growth factor receptor (EGFR) ligand that responds to inflammatory conditions, to further confirm liver inflammatory status. The mRNA expression levels of Il-6 and Il-1β were markedly increased after long exposure to PS-NMPs that were 80 nm and 0.5 µm in diameter (Fig. 3B, C). The expression levels of Tnf-α and Cxcl2 were increased after exposure to 5 µm PS-NMPs (Fig. 3D–E). Meanwhile, Cxcl12 and Areg mRNA expressions were significantly increased after exposure to 0.5 µm- and 80 nm-diameter PS-NMPs, respectively (Fig. 3F, G). Subsequently, we observed the protein expression level of IL-1β and IL-6. The IL-1β and IL-6 protein expression levels were significantly increased in mice after chronic PS-MPs exposure (Fig. 3 H, I). These results indicated that long exposure to 80 nm and 0.5 µm PS-NMPs significantly induced liver inflammation.

Fig. 3
figure 3

Inflammation in the liver after long exposure to NMPs. (A) Representative images of IHC for CD68 (left panel) and semi-quantification of CD68 based on IHC staining (right panel) (n = 8). The mRNA levels of Il-1 (B), Il-6 (C), Tnf-a (D), Cxcl2 (E), Cxcl12 (F), and Areg (G) were analyzed by qPCR and normalized by β-actin (n = 6). (H) The protein level of IL-1β was performed by western blotting and normalized by β-actin (n = 5). (I) The protein level of IL-6 was performed by western blotting and normalized by β-actin (n = 5). Scale bar, 100 µm for × 200 and 50 µm for × 400. *p < 0.05, **p < 0.01, ***p < 0.001.

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Long-term exposure to PS-NMPs did not induce liver oxidative stress

We further investigated the expression levels of oxidative stress biomarkers, glutathione peroxidase activity (GPX), malondialdehyde (MDA), and superoxide dismutase (SOD) after long exposure to PS-NMPs. As shown in Supplementary Fig. 1, the levels of GPX, MDA, and SOD were not increased by exposure to PS-NMPs of any of the three diameters. In contrast to other data, our results showed that oxidative stress is not involved in the development of liver fibrosis under long-term exposure to PS-NMPs.

Liver lipid deposition was induced by chronic PS-NMPs exposure

To examine whether lipid metabolism disorder was induced in our mouse model, we investigated lipid deposition through TEM and Oil Red O staining. Lipid deposition was increased in mice exposed to PS-NMPs compared to that in the control group. Moreover, the highest increase in lipid deposition was observed by Oil Red O staining in mice exposed to 80 nm-, 0.5 µm-, and 5 µm-diameter PS-NMPs (Fig. 4A, B), which was further confirmed by TEM (Fig. 4C). Consistent with previous studies, our data suggested that PS-NMPs could induce lipid deposition. Interestingly, the PS-NMPs with the smallest diameter led to more lipid deposition.

Fig. 4
figure 4

Abnormal lipid deposition was increased in the liver with long exposure to NMPs. (A) Representative images of Oil Red O for livers with H2O treatment, 80 nm, 0.5 µm and 5 µm NMPs exposure. Scale bar, 50 µm for 200 magnification and 20 µm for 400 magnifications. (B) Quantification of Oil Red O positive area. (C) Representative images of TEM for livers with H2O treatment, 80 nm, 0.5 µm, and 5 µm NMPs exposure.

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Bioinformatics analyses of RNA-seq in livers exposed to PS-NMPs

To further investigate the mechanism of the progression of liver fibrosis induced by long-term exposure to PS-NMPs, we performed RNA-seq using mouse livers with or without PS-NMPs exposure. A total of 333, 602, and 758 DEGs were obtained by comparing the H2O with 80 nm, 0.5 µm, and 5 µm PS-NMPs (Supplementary Fig. 2). The up- and down-regulated DEGs between the control and different PS-NMPs exposures are indicated by volcano plots (Supplementary Fig. 2A). The mRNA expression levels of DEGs in these datasets were visualized by heatmap (Supplementary Fig. 2B). To further analyze the GO enrichment of DEGs and potential candidate pathways in long exposure to different diameters of PS-NMPs, GO term, KEGG pathway, and REACTOME pathway analyses for these DEGs were conducted by DAVID. The biological process (BP) related to lipid metabolism was significantly enriched in all three datasets (Fig. 5A). The DEGs in the H2O vs. 0.5 µm and H2O vs. 5 µm datasets were dramatically enriched in glucose homeostasis. The processes, such as response to unfolded protein, response to endoplasmic reticulum stress, and cellular response to unfolded protein, were markedly enriched in the control vs. 5 µm dataset (Fig. 5A). The DEGs of the cellular component (CC) were most associated with endoplasmic reticulum, endoplasmic reticulum membrane, and cytoplasm in the three datasets, etc. (Supplementary Fig. 3A). The DEGs of molecular function in the three datasets were mainly associated with small molecule binding, etc. (Supplementary Fig. 3B). For the KEGG pathway, the DEGs of all datasets were most associated with metabolic pathways, etc. (Fig. 5B). For the enriched REACTOME pathways, the DEGs of all datasets were mainly related to the metabolism of lipids and metabolism, etc. (Fig. 5C).

Fig. 5
figure 5

Function enrichment analyses of DEGs. The top ten significantly enriched (A) GO term BP, (B) KEGG, and (C) REACTOME pathways are identified.

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Lipid metabolism was induced by PS-NMPs

NMPs have been considered harmful substances that induce abnormal lipid metabolism in mouse livers13,17. To investigate the effects of PS-NMPs exposure on lipid metabolism, we examined the mRNA levels of several genes involved in the processes of fatty acid oxidation, fatty acid synthesis, and fatty acid transport. We found that chronic exposure to 80 nm and 0.5 µm diameter PS-NMPs significantly increased the mRNA expression levels of Acox1, Ppara, Cpt1a, and Mcad, which are involved in the fatty acid oxidation process. Treatment with 5 µm PS-NMPs markedly upregulated the mRNA expression of Ppara. The mRNA expression of Cpt1b was dramatically increased in mice liver after exposure to 80 nm PS-NMPs (Fig. 6A). For the fatty acid synthesis process, the mRNA expression levels of Apoc4 and Fas were induced by PS-NMPs exposure at 0.5 µm. Pparg mRNA expression was significantly augmented in both the 0.5 µm and 5 µm diameter PS-NMPs treatment groups. Scd1 mRNA expression was markedly induced by exposure to 80 nm diameter PS-NMPs (Fig. 6B). For the fatty acid transport process, the mRNA expression of Fatp1 was significantly increased in chronic exposure to 80 nm and 0.5 µm diameter PS-NMPs. Fatp2 mRNA expression was induced by 0.5 µm diameter PS-NMPs (Fig. 6C). These findings provide evidence that chronic exposure to PS-NMPs can lead to lipid metabolism disorders in mouse livers.

Fig. 6
figure 6

The lipid metabolism in the liver after chronic exposure to NMPs. (A) The mRNA levels of fatty acid oxidation-associated biomarkers, including Acox1, Ppara, Cpt1a, Cpt1b, Cpt1c, and Mcad, were analyzed by qRT-PCR and normalized by β-actin (n = 5–6). (B) The mRNA levels of fatty acid synthesis-related biomarkers, containing Acc, Apoc4, Pparg, Scd1, and Fas, were analyzed by qRT-PCR and normalized by β-actin (n = 5–6). (C) The mRNA levels of fatty acid transport-related biomarkers, including Fatp1 and Fatp2, were performed by qPCR and normalized by β-actin (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001.

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Shared DEGs confirmed the lipid metabolism disorder induced by PS-NMPs

We identified a total of 87 DEGs that were shared among all four RNA-seq datasets, with 46 upregulated and 41 downregulated DEGs in mice exposed to PS-NMPs compared to control mice (Fig. 7A). To gain insight into the potential functions and pathways affected by PS-NMPs exposure, we performed GO enrichment and KEGG pathway analysis on the shared DEGs using the DAVID database. The GO analysis revealed that the shared DEGs were predominantly enriched in processes related to positive regulation of lipid metabolism, negative regulation of lipid biosynthetic process, negative regulation of lipid storage, and cellular response to lipid in the biological process category (Fig. 7B). In terms of cellular components, the shared DEGs were significantly associated with the extracellular space and mitochondrial matrix (Supplementary Fig. 4). Regarding molecular functions, the shared DEGs were mainly enriched in small molecule binding (Supplementary Fig. 4). The KEGG pathway analysis showed that the shared DEGs were most significantly associated with fatty acid elongation, biosynthesis of unsaturated fatty acids, and metabolic pathways (Fig. 7C). Furthermore, the REACTOME pathway analysis revealed significant enrichment of shared DEGs in pathways related to lipid metabolism and fatty acid metabolism (Fig. 7D). These findings provide valuable insights into the potential molecular mechanisms underlying the effects of PS-NMPs exposure on lipid metabolism.

Fig. 7
figure 7

Screening of NMPs exposure-associated hub genes in the liver. (A) Venn diagram of 87 shared DEGs. The top ten significantly enriched BP (B), KEGG pathway (C), and REACTOME pathway (D) of shared DEGs are ranked. (E) The top four hub genes were identified by cytohubba based on 12 algorithms. (F) These four hub genes gene–gene interaction network and its neighboring genes showed different correlations.

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Four hub genes were identified

We constructed a protein–protein interaction (PPI) network for the 87 shared DEGs using the online database STRING, which comprised 85 nodes and 51 edges (Supplementary Fig. 4). Subsequently, we identified four hub genes, namely Acot3, Abcc3, Nr1i3, and Fmo2, using 12 algorithms in the Cytoscape App CytoHubba (Fig. 7E). To explore the interaction network of these hub genes with their neighboring genes, we utilized the online database GeneMANIA. The interaction network revealed enrichments in physical interactions (45%), predicted interactions (22.45%), co-expression (17.96%), other interactions (8.09%), pathway associations (2.07%), genetic interactions (1.81%), co-localization (1.57%), and shared protein domains (1.04%) (Fig. 7F).

To validate the mRNA levels of these four hub genes, we utilized RNA-seq data and performed qRT-PCR in vivo. The mRNA levels of Acot3, Abcc3, Nr1i3, and Fmo2 were significantly upregulated in the RNA-seq samples following exposure to PS-NMPs (Fig. 8A–D). To further confirm these results, we expanded the sample size and assessed the mRNA expression of the four selected genes using qRT-PCR. Consistent with the RNA-seq findings, chronic exposure to PS-NMPs significantly induced the mRNA levels of Acot3, Abcc3, and Nr1i3. However, the mRNA level of Fmo2 did not show a significant change (Fig. 8E–H). These results suggest that three hub genes, namely Acot3, Abcc3, and Nr1i3, are involved in PS-NMPs exposure-induced liver fibrosis in mice.

Fig. 8
figure 8

mRNA expression levels of hub genes. (A–D) The mRNA expression levels of four hub genes in RNA-sequencing datasets are presented. (E–H) The mRNA expression levels of four hub genes were confirmed by increasing the samples (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001.

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Discussion

In this study, we have provided evidence that long-term exposure to 0.5 µm- and 5 µm-diameter PS-NMPs can induce liver fibrosis, even in the absence of elevated levels of ALT and AST. Our findings suggest that inflammation and abnormal lipid deposition are the primary mechanisms underlying the development of liver fibrosis in response to chronic exposure to PS-NMPs, rather than oxidative stress. Through a combination of bioinformatics analysis and experimental validation using RNA-seq data from PS-NMPs-exposed liver tissue, we have identified dysfunction in lipid metabolism as a key mechanism in the development of liver fibrosis induced by chronic PS-NMPs exposure. Furthermore, we have identified three hub genes, namely Acot3, Abcc3, and Nr1i3, as potential molecular targets involved in the progression of liver fibrosis under PS-NMPs exposure. These findings contribute to our understanding of the pathogenesis of liver fibrosis associated with exposure to PS-NMPs and may have implications for the development of targeted therapeutic interventions.

Long-term exposure to NMPs with different particle diameters and concentrations leads to the aggregation of NMPs in the liver, which promotes liver injury and fibrosis3,12,13,17,18,19. While numerous studies have demonstrated the ability of NMPs to induce liver injury or fibrosis, it is important to note that these studies have typically used higher concentrations of NMPs than what animals are typically exposed to in their natural environment. In order to better mimic the level of environmental pollution, we selected a concentration of 1 mg/L for our experiments. This concentration is believed to be more representative of the actual exposure levels of NMPs in the environment12. In this study, we conducted an experiment in which mice were exposed to sterilized water containing three different particle diameters of PS-NMPs (80 nm, 0.5 µm, and 5 µm) at a concentration of 1 mg/L for a period of 12 weeks. Our findings revealed that long-term exposure to PS-NMPs with diameters of 0.5 µm and 5 µm resulted in a reduction in liver weight, liver weight/body weight ratio, and liver fibrosis. However, there were no significant changes observed in the levels of liver function markers such as ALT and AST. These results are in line with previous observations that ALT levels often remain within the normal range in patients with liver cirrhosis20,21. As a biomarker of fibrosis, α-SMA was dramatically increased in protein level but not in mRNA level, which may be associated with posttranscriptional regulation22. In our study, we also examined the effects of chronic exposure to 80 nm NMPs on liver fibrosis. While we did not observe liver fibrosis in mice exposed to 80 nm NMPs, we did find a significant induction of the fibrotic marker α-SMA. These findings suggest that even though visible fibrosis may not be present, there are still underlying molecular changes occurring in the liver due to microplastics (MPs) exposure.

Furthermore, our study identified oxidative stress, inflammation, and abnormal lipid metabolism as important mechanisms involved in the development of liver fibrosis induced by MPs exposure9,10,11. In previous studies, exposure to NMPs promoted liver oxidative stress3,13 Contrary to previous studies, our research findings suggest that oxidative stress may not be a necessary mechanism involved in the development of liver fibrosis in our model. Our analysis of GPX, MDA, and SOD protein levels did not indicate significant oxidative stress in the liver exposed to NMPs. However, our study did reveal that exposure to 0.5 µm PS-NMPs predominantly induced an inflammatory response. This was supported by the results of qRT-PCR, western blotting, and immunohistochemistry staining of various inflammatory biomarkers. Additionally, our investigation using Oil Red O staining and electron projection microscopy demonstrated that PS-NMPs promoted lipid accumulation in hepatocytes. Notably, both the 80 nm and 0.5 µm NMPs induced greater lipid droplet formation compared to other MPs-exposed groups. In recent studies, disordered lipid metabolism has been observed in livers exposed to NMPs13,17. Further analyses, including experimental verification and bioinformatics analyses of RNA sequencing datasets, revealed that lipid metabolism is a critical mechanism involved in liver fibrosis induced by chronic exposure to NMPs of different sizes. This finding was consistent across individual analyses for each dataset and was confirmed through integrative analysis of three datasets, which identified lipid metabolism, particularly fatty acid metabolism, as being induced by chronic exposure to PS-NMPs regardless of particle diameter. Deep screening and qRT-PCR verification of additional samples led us to identify three hub genes, Acot3, Abcc3, and Nr1i3, which play a crucial role in regulating abnormal lipid metabolism caused by chronic exposure to PS-NMPs. These findings contribute to our understanding of the molecular mechanisms underlying liver fibrosis induced by MP exposure and offer potential targets for therapeutic interventions.

Acyl-CoA thioesterase 3 (ACOT3) is a member of the acyl-CoA thioesterase family that hydrolyzes a variety of CoAs in peroxidases involved in lipid metabolism23. ACOT3 plays a regulatory role in the b-oxidation of mouse peroxidase but not in human peroxidase. Acot3 mRNA was abundantly expressed in the liver and kidney24. Although the role of ACOT3 in the development of liver fibrosis remains unclear, ACOT4 expressed in human peroxidase has been found to play a regulatory role in nonalcoholic fatty liver-related liver fibrosis23,25. Therefore, the role of ACOT3 in chronic exposure to NMPs-induced liver fibrosis still needs further study.

ATP-binding cassette subfamily C member 3 (ABCC3), also known as MRP3, is an important ATP-binding cassette transporter that is mainly expressed in the liver and mediates the efflux of multiple substrates containing lipids26,27. A recent study demonstrated that quercetin, a kind of antioxidant with anti-inflammatory and antifibrotic effects, reduced collagen accumulation and ABCC3 expression and ultimately inhibited the growth of tumors in early hepatocellular carcinoma28. In an idiopathic pulmonary fibrosis (IPF) mouse model, the significant increase in ABCC3 expression levels was accompanied by the development of fibrosis29. In a previous study, the ABCC3 expression level was dramatically upregulated in the liver at 14 days after bile duct ligation, although the researchers focused on BDL-induced liver necrosis30. Therefore, ABCC3 may have a potential effect on the development of liver fibrosis, and its mechanism in long-term exposure to PS-NMPs needs to be deeply researched.

Nuclear receptor subfamily 1 Group I member 3 (NR1I3), also named constitutive androstane receptor (CAR), is an important member of the nuclear receptor (NR) superfamily that regulates the gene expression of xenobiotics and endogenous metabolizing enzymes31,32. A previous study demonstrated that NR1I3 was involved in the induction of liver fibrotic markers, collagen Ia and TIMP-1, in methionine- and choline-deficient (MCD) diets for 16 weeks; however, these fibrotic markers were dramatically downregulated in MCD-dieted NR1I3-deficient mouse livers. Although there was no difference between WT and NR1I3-deficient mice fed an MCD diet, NR1I3-induced liver lipid peroxidation, which was associated with liver inflammation32. Meanwhile, NR1I3 also regulates lipid metabolism gene expression33. Whether NR1I3 promotes liver fibrosis induced by long-term exposure to PS-NMPs by regulating lipid metabolism remains to be further explored.

In the present study, we found that the impact of particle size on the hazard of microplastics is not singular and linear, which is different from previous studies on microplastics that have often argued that the smaller the particle size, the more dangerous it is34. For example, fat metabolism abnormalities stand out in the 80 nm NP exposure group while liver fibrosis stands out in the 5 μm MP exposure group. Due to their extremely small size, 80 nm NPs possess a high specific surface area and surface energy, making them more prone to interacting with molecules within organisms. This interaction may lead to changes in the activity of enzymes related to fat metabolism, thereby affecting the processes of fat synthesis and decomposition35. Additionally, 80 nm NPs have the ability to penetrate cell membranes and may directly enter cells, interfering with intracellular metabolic processes. When organelles related to fat metabolism, such as mitochondria and endoplasmic reticulum, are disturbed, it can more easily lead to abnormal fat metabolism36. On the other hand, when the particle size increases to a certain extent, such as 5 μm, which reaches the optimal diameter for entry into liver cells through the liver sinusoidal endothelial cells, it will be more likely to deposit in organs such as the liver. This deposition may compress and cause severe damage to liver cells, triggering inflammatory responses and fibrosis processes. Long-term deposition and repeated damage of MPs in the liver may lead to continuous repair and regeneration of liver cells. During this process, fibrous connective tissue may overproliferate, resulting in liver fibrosis.

Conclusion

The present study indicated that chronic exposure to all sizes of PS-NMPs at low concentrations resulted in upregulation of the expression of the fibrotic marker α-SMA, and exposure to 0.5 µm- and 5 µm-diameter PS-NMPs further led to liver fibrosis. The aberrant expression of three hub genes, Acot3, Abcc3, and Nr1i3, may be potential factors that regulate PS-MP exposure-induced lipid or fatty acid metabolism disorders. These findings confirm that chronic exposure to low concentrations of NMPs is sufficient to cause liver fibrosis and provide new evidence to prevent liver fibrosis induced by NMPs.