The BALB/c-derived RAW264.7 macrophages were a generous gift from Professor Chong Yan (Guangdong Medical University, Dongguan, China). RAW264.7 macrophages were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Bovogen, New Zealand) and incubated at 37°C in a 5% CO₂ atmosphere. Cells were seeded in a 6-well plate at a density of 5.0 × 10⁵ cells/well and incubated overnight. Then, cells were harvested after 24 h treatment with different concentrations of BPF (CAS No. 620-92-8, purity 98%, Sigma-Aldrich, St. Louis, MO, United States) or BPA (CAS No.80-05-7, purity 97%, Sigma-Aldrich, St. Louis, MO, United States), which was dissolved in 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, United States). Untreated cells (0 μM BPF) were exposed to 0.1% DMSO.

The cell viability of RAW264.7 macrophages treated with BPF (or BPA) was determined by CCK-8 (Cell Counting Kit-8) (CCK-8, Dojindo Laboratories, Japan). Briefly, RAW264.7 macrophages were seeded into 96-well plates at a density of 1.0 × 10⁴ cells per well. After incubation for 24 h, 0, 1, 5, 10, 20, 50, 100, or 200 μM BPF (or BPA) in DMEM medium was added to 96-well plates and incubated for another 24 h. Then, the CCK-8 solution (10 μl) was added to each well and subsequently cultured for 3 h at 37°C. The absorbance at 450 nm was read by using a microplate reader (ELx808, BioTek Instruments, Inc., Winooski, VT, United States). The cell viability was calculated by the absorbance ratio of the sample well to the control well and expressed as a percentage, assigning the cell viability of the control well as 100%.

Lactate dehydrogenase (LDH) activity released in the culture media was measured by the CytoTox 96^® non-radioactive cytotoxicity assay (Promega corporation, Wis, United States), according to the manufacturer’s instructions. Absorbance at 490 nm was recorded using a microplate reader. LDH release % = (Experimental LDH Release (OD490)/Maximum LDH Release (OD490)) × 100.

Apoptosis assay was measured with an Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China), according to the manufacturer’s instructions. In brief, the RAW264.7 macrophages (5 × 10⁵ cell/well) were seeded in a 6-well plate and exposed to BPF at different concentrations. After 24 h treatment, the cells and cell culture medium were collected and centrifuged at 1,000 g for 5 min. The supernatant was discarded, and the cells were suspended with Annexin V-FITC binding buffer and stained with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature. The apoptosis of RAW264.7 macrophages was detected by flow cytometry (FACScantoII System, BD Biosciences, San Jose, CA, United States). All the experiments were conducted in triplicates.

RAW264.7 macrophages (5 × 10⁵ cell/well) were cultured in 6-well plates and exposed to 20 μM BPF for 24 h. Then, the supernatant was removed, and the cells were washed twice with ice-cold PBS buffer solution. The total RNA was extracted using TRIzol reagent (Life technologies, NY, United States), according to the manufacturer’s protocol. The Bioanalyzer 2100 system (Agilent Technologies, CA, United States) was used to assess the RNA integrity. Samples were sent to Shanghai Jikai Gene Technology Co., Ltd. for whole-transcriptome sequencing analysis. Briefly, a total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, Beijing, China), and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot cluster generation system using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated.

RNA-seq experiments were performed using the Illumina Novaseq platform. Raw data (raw reads) of fastq format were first processed through in-house perl scripts to obtain clean data (clean reads) by removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data. The processed reads were mapped to the reference using HISAT2. The mapped reads of each sample were assembled by StringTie (Pertea et al., 2015), and then, FPKM of each gene was calculated based on the length of the gene and read counts mapped to this gene. FPKM, the expected number of fragments per kilobase of transcript sequence per million’s base pairs sequenced, considers the effect of the sequencing depth and gene length for the read counts at the same time, and it is currently the most used method for estimating gene expression levels. Differential expression analysis of two conditions was performed using the edgeR R package. The p values were adjusted using the Benjamini & Hochberg method. A corrected p-value of 0.05 and |log2 (fold change)| of 0.26 were set as the threshold for significantly differential expressions. The gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify significant pathways of RAW264.7 macrophages altered by BPF. We used the cluster Profiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.

The total RNA of RAW264.7 macrophages was extracted with TRIzol reagent, and then, cDNAs were synthesized by using the reverse transcription kit (Takara Bio, Japan), according to the manufacturer’s instructions. The TB Green Real-time PCR kit (Takara Bio, Japan) was used and detected by PikoReal 96 fluorescence quantitative PCR. qRT-PCR amplification was carried out under the following conditions for 40 cycles: 95°C for 30 s, 95°C for 15 s, and 60°C for 30 s. The primers of H2-T23, Sec61a1, Sec61b, Sec61g, Eea1, Hspa5, Psme2b, Psme2, Canx, Gusb, Hexb, Atp6v0a1, Hyal1, Idua, Mdm2, Cdk1, Cdc20, Bub1b, Dbf4, Atp6v0d2, Snp23, Vamp7, Vps34, Rab7, and β-actin were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China), which are listed in Table 1. The relative quantity of mRNA was calculated using the standard 2^−ΔΔCT method with β-actin serving as an internal standard.

All data were analyzed by GraphPad Prism version 8.0.2. A one-way analysis of variance (ANOVA) with Duncan’s multiple range tests was performed for each response variable. Significance was determined at p < 0.05.

The cytotoxicity of BPF on RAW264.7 macrophages was measured by CCK-8 and LDH releasing assay after 24 h treatment. The result of the CCK-8 assay showed that when the concentration of BPF was lower than 100 μM, the viability of macrophages was not significantly affected (p > 0.05). When the BPF concentration was 100 and 200 μM, it could significantly inhibit the viability of macrophages. The inhibition rate on cell viability of 200 μM BPF was close to 50% compared to the control group (Figure 1A). Under normal conditions, LDH exists in the cytoplasm. When the integrity of the phospholipid bilayer structure of the cell membrane is destroyed, LDH will leak into the culture medium through the incomplete cell membrane. Hence, the degree of cell damage can be reflected by detecting the LDH activity in the cell culture supernatant. The result of LDH releasing assay presented that BPF significantly promoted the release rate of LDH at the concentration of 100 and 200 μM (Figure 1B), which was consistent with the result of CCK-8 assay. The aforementioned results showed that there was no obvious cytotoxicity of BPF on RAW264.7 macrophages when the concentration was lower than 100 μM, but high concentrations of BPF could significantly reduce the survival rate of RAW264.7 macrophages. Furthermore, the cytotoxicity of BPA was detected by CCK-8 and LDH assays (Supplementary Figures S1A,B). It is proved that the cytotoxicity of BPF is lower than that of BPA on RAW 264.7 macrophages.

In this study, flow cytometric measurement (Annexin V/FITC and PI double staining) was used to quantify the extent of apoptosis of RAW264.7 macrophages after BPF exposure. In normal cells, phosphatidylserine (PS) is located on the inner side of the lipid bilayer of the cell membrane, and the cell membrane is intact. When cells undergo early apoptosis, the cell membrane remains intact, while PS everts to the side of the cell membrane and binds with annexin V-FITC with high affinity to emit green fluorescence. In the late stage of apoptosis, the cell membrane structure is damaged. Propidium iodide (PI) enters the cell through the damaged cell membrane, and the cells emit red fluorescence and green fluorescence at the same time. The proportion of cells with early apoptosis and late apoptosis was analyzed by detecting the signals of annexin V-FITC (green fluorescence) and PI (red fluorescence) simultaneously. The total apoptosis rate is the sum of the early apoptosis rate and late apoptosis rate. It was found that the BPF exposure for 24 h triggered macrophage apoptosis in a concentration-dependent manner (Figure 2A). BPF concentrations at 20, 50, 100, and 200 μM induced approximately 7.40, 21.69, 23.66, and 36.16% total apoptosis rate (Figure 2B) and 4.52, 7.36, 8.70, and 13.03% late apoptosis rate, respectively (p < 0.05) (Figure 2D). Moreover, treatment with 50, 100, and 200 μM BPF induced approximately 14.33, 14.97, and 23.13% early apoptosis rate, respectively (p < 0.001) (Figure 2C). These results showed that the apoptosis events of RAW264.7 macrophages treated with non-cytotoxic doses (20–50 μM) of BPF observed in CCK-8 assay were significantly increased. It suggested that although the medium dose of BPF exposure seemed to have no impact on the cell activity of macrophages, it caused the occurrence of apoptosis.

RNA-seq was applied to identify gene expression changes induced by BPF treatment in RAW264.7 macrophages. The heat map (Figure 3A) showed the expression amount of differentially expressed genes between the BPF group and control group. The genes with high expression (red) and low expression (green) were distributed in the opposite direction, indicating that BPF treatment induced significant differences in the gene expression. The volcano map (Figure 3B) revealed that there were 203 differentially expressed genes (DEGs) between the BPF group and control group. Of these genes, 121 genes were upregulated, and 82 genes were downregulated by BPF. The Venn diagram (Figure 3C) could show the overlap of different genes between the BPF group and control group. There were 11,750 genes expressed both in the control group and BPF group, and 194 genes were expressed only in the BPF group.

To understand the implications of these differentially expressed genes and clarify the potential regulatory mechanisms on BPF-treated RAW264.7 macrophages, functional enrichment analysis based on the GO annotation category and KEGG pathway category was performed. The GO annotation category is a comprehensive database describing the gene function, which can be divided into the biological process (BP), cellular component (CC), and molecular function (MF). The adjusted p value less than 0.05 was taken as the threshold value for significant enrichment of GO function, and the most significant 30 terms were shown in the histogram. In this study, the upregulated genes after BPF treatment were enriched in mitosis, cell cycle, chromosome segregation, and so on. However, the downregulated genes were enriched in the immune response and ATPase activity (Figures 4A,B; Supplementary Tables S1 and S2). KEGG is a database resource to understand the advanced functions and utility of biological systems (such as cells, organism, and ecosystems) by using molecular level information such as large-scale molecular data sets generated by genome sequencing and other high-throughput experimental technologies. KEGG pathway enrichment analysis takes the adjusted p value < 0.05 as the threshold of significant enrichment. When RAW264.7 macrophages were treated with BPF for 24 h, the most significant 20 terms of the KEGG pathway were shown in the bubble charts (Figures 4C,D). The upregulated genes were enriched in protein processing in the endoplasmic reticulum, protein export, cell cycle, and so on. However, the downregulated genes were enriched in lysosome, glycosaminoglycan degradation, rheumatoid arthritis, and so on.

In KEGG enrichment analysis, the following terms were significantly enriched in our gene list: protein processing in the endoplasmic reticulum (mmu04141), lysosome (mmu04142), phagosome (mmu04145), cell cycle (mmu04110), antigen processing and presentation (mmu04612), N-Glycan biosynthesis (mmu00510), protein export (mmu03060), and glycosaminoglycan degradation (mmu00531), which are exhibited in Table 2. The upregulated and downregulated DEGs in the top eight most significant results of KEGG enrichment are shown in Table 3. Terms including lysosome (mmu04142), phagosome (mmu04145), and antigen processing and presentation (mmu04612) provided clues that BPF may have effect on immune function of RAW264.7 macrophages.

As shown in Figure 5, the influence of BPF on the cell cycle, phagosome, lysosome, and antigen processing and presentation pathway was depicted by using the path view package in R software according to the results from cluster analyses (Figures 5A–D), and the FPKM relative expressions of the top five genes with significant differences in these four pathways are represented by a histogram. BPF upregulated the expressions of Mam2, Cdk1, Cdc20, Bub1b, and Dbf4 of the cell cycle pathway (Figure 5E), upregulated the expressions of Sec61a1, Sec61b, Sec61g, and Eea1, and downregulated H2-T23 in the phagosome pathway (Figure 5F). At the same time, BPF downregulated the expressions of Gusb, Hexb, Atp6v0a1, Hyal1, and Idua of the lysosome pathway (Figure 5G). In the antigen processing and presentation pathway, the expressions of Hspa5 and Psme2 were upregulated, and the expressions of Psme2b and H2-T23 were downregulated (Figure 5H).

The top five genes with significant differences in these four pathways were re-confirmed by qRT-PCR. The results showed that BPF upregulated the expressions of Mam2 and Cdc20, downregulated those of Cdk1, Bub1b, and Dbf4 of the cell cycle pathway (Figure 5I), upregulated the expressions of Sec61a1, Sec61b, Sec61g, and Eea1, and downregulated H2-T23 in the phagosome pathway (Figure 5J). In addition, BPF downregulated the expressions of Hexb, Hyal1, and Idua and upregulated the expression of Gusb in the lysosome pathway (Figure 5K). In the antigen processing and presentation pathway, the expression of Hspa5 was upregulated, and the expressions of Psme2, H2-T23, and Canx were downregulated (Figure 5L). Although the expression of a few genes differed from the results of RNA-seq, these results still suggested that BPF could affect the cell cycle, phagocytosis, and antigen processing and presentation of macrophages.

The fusion of phagosome and lysosome is the main driving factor of phagosome maturation and is targeted by several adaptive intracellular pathogens. The damage of this process will have a significant impact on microbial infection, tissue inflammation, adaptive immunity, and diseases. Given the importance of phagosome-lysosome fusion to phagocyte function, the genes related to the phagosome-lysosome fusion (Atp6v0d2, Snap23, Rab7, Vps34, and Vamp7) were selected and detected by qRT-PCR. The expressions of Atp6v0d2, Vamp7, and Vsp34 were significantly downregulated, while that of Snap23 was upregulated by BPF (Supplementary Figure S2). It suggested that BPF may affect the phagosome-lysosome fusion of RAW264.7 macrophages.