Protective Effect of Biobran/MGN-3, an Arabinoxylan from Rice Bran, Against the Cytotoxic Effects of Polyethylene Nanoplastics in Normal Mouse Hepatocytes: An In Vitro and In Silico Study

Abstract

Background: Plastic is one of the most versatile and widely used materials, but the environmental accumulation of nanoplastics (NPs) poses a risk to human health. Preclinical studies have verified that the liver is one of the main organs susceptible to NPs. Biobran/MGN-3, an arabinoxylan from rice bran, has been shown to have hepatoprotective effects; here, we show Biobran's ability to alleviate polyethylene nanoplastics (PE-NPs)-induced liver cell toxicity by reversing apoptosis and restoring G2/M cell arrest in mouse liver cells (BNL CL.2).

Methods: Toxicological effects were measured using the sulforhodamine B (SRB) assay for cell viability and flow cytometry for cell cycle analysis and apoptosis. An in silico study was also used to demonstrate the docking of PE-NPs to pro-inflammatory mediator proteins (IL-6R, IL-17R, CD41/CD61, CD47/SIRP), cell cycle regulators (BCL-2, c-Myc), as well as serine carboxypeptidase, which is an active ingredient of Biobran.

Results: Exposing liver cells to PE-NPs caused a significant decrease in cell viability, with an IC50 value of 334.9 ± 2.7 µg/mL. Co-treatment with Biobran restored cell viability to normal levels, preserving 85% viability at the highest concentration of PE-NPs. Additionally, total cell death observed after exposure to PE-NPs was reduced by 2.4-fold with Biobran co-treatment. The G2/M arrest and subsequent cell death (pre-G0 phase) induced by PE-NPs were normalized after combined treatment. The in silico study revealed that Biobran blocks the nucleophilic centers of PE-NPs, preventing their interaction with pro-inflammatory mediators and cell cycle regulators.

Conclusions: These findings highlight the potential use of Biobran as a hepatoprotector against NP toxicity.

Keywords: Biobran; G2/M cell arrest; MGN-3; apoptosis; hepatocytes; polyethylene nanoplastics.

Figure 1

Flow diagram illustrating the synthesis process of PE-NPs via the sonochemical method. The process is divided into four main stages: (1) initial materials preparation, showing raw PE powder and stock solution preparation; (2) sonochemical synthesis, detailing ultrasonic processing conditions; (3) post-processing, including membrane filtration; and (4) final stages comprising characterization and storage conditions.

Figure 2

Multi-technique characterization of PE-NPs: (A) TEM image showing spheroidal morphology with some aggregation (scale bar 10 nm); (B) XRD pattern displaying characteristic orthorhombic polyethylene peaks at 19.4°, 21.6°, and 23.9°; (C) 3D AFM topography revealing quasi-cubic surface structures with height variations up to 72.4 nm; (D) Raman spectrum with distinctive polyethylene vibrational modes at 752, 1129, 1298, 1444, 2847, and 2887 cm⁻¹; (E) DLS analysis showing narrow size distribution centered at ~70 nm; and (F) Zeta potential distribution with peak at approximately −25 mV.

Figure 3

Inverted light microscopic (ILM) photographs displaying changes in cell count throughout the cytotoxicity experiment in the BNL CL.2 cells after 72 h of different treatments. The photos show the normal architecture of the untreated control (A), treatment with Biobran (B), PE-NPs (C), or their combination (D). After removing the media, the cells were fixed with TCA, stained with SRB dye, and visualized under an ILM at 100×.

Figure 4

SRB assay evaluating cytotoxic effects of PE-NPs, Biobran, and their combination in normal liver cells (BNL CL.2) after treatment for 72 h. Data are presented as mean ± SD.

Figure 5

(A) Cell cycle distribution in BNL CL.2 cells after treatment with PE-NPs, Biobran, and their combination for 48 h was determined using DNA cytometry analysis compared with control (untreated cells). (B) The percentage of cells in all cell cycle phases, including the pre-G1 phase, was plotted as a bar graph of mean ± SD; n = 3. * Statistically significant from control at p < 0.05.

Figure 6

(A) Apoptosis/necrosis assessment by flow cytometry in BNL CL.2 cells with PE-NPs, Biobran, and their combination for 48 h and stained with PI/Annexin V-FITC. (B) Different cell populations at apoptosis and necrosis were plotted as percentages of total events. Data are presented as mean ± SD; n = 3. * Statistically significant from control at p < 0.05.

Figure 7

PE-NPs docked to IL-6R via binding to Pro94, Pro95, Glu96, Glu97, Gln99 (red asterisk), Arg118, Ser119, Thr120, Pro121 (blue asterisk), Gly193 and Ile194 (violet asterisks), and Glu283 (green asterisks) (A); IL-17R via binding to Ser177, Lys178, Asn179, Leu181, Pro183, Glu186, His187, Ala188, Arg189, Lys191, Asp204, Pr205, Asn206, Ile207 (red asterisks) (B); integrin CD41/CD61 via binding to many amin acids especially, Asp369 and Tyr371 (red arrow), Ser396, Arg400, Arg402, Ser404, Gln405, Val406, Leu407, Asp408, Pro410 (red asterisk) and Lys678 (blue asterisk) (C); and CD47/SIRP via binding to amino acids located within the highest predicted B-cell epitopes on SIRP (Gly55, His56, Val60, Thr61) of chain A and (His56 and Phe57) of chain B (red asterisk) followed by (Phe94, Pro99, Asp100, Thr101, Glu102) of chain B (blue asterisk) (D).

Figure 8

(A) Docking of PE-NPs to BCL-2 isoform 1 by binding to many residues, especially those located within the BH1 and BH4 apoptosis regulator signature (black arrow); (B) Docking of PE-NPs to BCL-2 isoform 2 by binding to many residues, especially those located within the BH4 apoptosis regulator signature (black arrow) and site of phosphorylation by MAPK (red arrow); (C) Docking of PE-NPs to c-MYC by binding to many residues, especially those located within the sulfate binding sites (black arrow) and nucleotide DT (red arrow).

Figure 9

Serine carboxypeptidase docking to PE-NPs.