Food-grade Dictyophora was provided by Zhijin Sifang Hongye (Zhijin City, Guizhou Province, China). Sodium arsenite (NaAsO₂) was obtained from Sigma Chemical Corp (St. Louis, MO, United States). Human normal hepatocytes (L-02 cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 cell culture medium, trypsin, and fetal bovine serum (FBS) were purchased from Gibco Company (California, United States). Phosphate buffer saline (PBS) was purchased from Zhongsha Jinqiao Biotechnology Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was obtained from Sigma (St. Louis, MO, United States). The BCA protein detection kit, protein sample buffer, and Western blot analysis gel preparation kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Protein molecular weight markers were obtained from Fermentas (Burlington, Canada). Polyvinylidene fluoride (PVDF) film and enhanced chemiluminescence (ECL) kit were purchased from Bio-Rad (California, United States). Cell Counting Kit-8 (CCK-8), RIPA lysis buffer, rabbit anti-human Bcl-2, Bax, β-actin, GAPDH, ribosomal protein S5 (Rps5), and 14-3-3 protein sigma (SFN) antibodies and horseradish peroxidase (HRP) labeled secondary antibodies were purchased from Boster Biological Technology, Ltd. (Boster, Wuhan, China).

The separation and purification of DIP was performed as previously described with minor modifications (Liao et al., 2015; Zhang et al., 2016; Yu et al., 2017; Hu et al., 2020). Briefly, the fruit body of Dictyophora was dried at 45°C for 2 h, ground into powder, extracted in boiling water for 3 h at a water-to-material ratio (1:20), and then centrifuged at 4,000 × g for 15 min. The supernatant was extracted and concentrated at 50°C using a rotary evaporator (R-215, Buchi, Switzerland). The concentrated extract was collected and proteins were removed using the Sevage method. The crude polysaccharides of Dictyophora were obtained by freeze-drying after overnight precipitation with 4 volumes of 100% ethanol. The content of sugar was determined by using phenol-sulfuric acid colorimetric assay. The content was 84.13%. According to our experimental data, 10 mg DIP can be obtained from 80 mg of dried Dictyophora powder.

The dry product of 400 mg DIP was dissolved in 20 ml serum-free DMEM medium to prepare a final concentration of 20 mg/ml mother liquor, filtered and stored at −20°C. When in use, it is diluted to the corresponding concentration with serum-free DMEM medium.

L-02 cells were cultured in a 5% CO₂ incubator at 37°C. The control group used DMEM high glucose medium containing 10% FBS and 1% penicillin/streptomycin, and the treatment group was pretreated with NaAsO₂ or DIP for 4 h and then exposed to NaAsO₂. All experiments were performed 24 h after cell inoculation.

The cell viability of L-02 cells was measured by CCK-8 assay. Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well, and were treated with different concentration of NaAsO₂ for 24 h. In order to explore the intervention effect of DIP, L-02 cells were pretreated with different concentration DIP solution for 4 h, and then treated with of 10 μM NaAsO₂ for 24 h. In addition, the viability of cells pretreated with DIP (80 μg/ml) and then exposed to different concentrations sodium arsenate was also investigated. CCK-8 reagent was added to each well and incubated at 37°C for 2 h according to the manufacturer protocol. Microplate Reader (Thermo Fisher Scientific) was used to measure the absorbance at 450 nm to determine cell viability.

The percentage of apoptotic cells was detected by Annexin V-FITC apoptosis detection kit (Beyotime, Shanghai, China). After NaAsO₂ (10 μM) treatment or DIP (80 μg/ml) pretreatment followed by exposure to NaAsO₂, the L-02 cells were collected and washed with PBS, and re-suspended by adding 100 μL binding buffer, and then incubated with 5 µL Annexin V-FITC, and 10 µL PI at room temperature. The apoptosis rate was detected by flow cytometry (ACEA NovoCyte, United States).

After treatment with NaAsO₂ (10 μM) or DIP (80 μg/ml) pretreatment and then exposed to 10 μM NaAsO₂, L-02 cells were rinsed twice with ice-cold PBS. The cells were harvested and resuspended in lysis buffer (8 M urea, 2 mM EDTA, 10 mM DTT, and 1% protease inhibitor cocktail). After sonicated and centrifuged at 13,000 g at 4°C for 10 min to remove debris, the protein in supernatant was precipitated with cold acetone for 2 h at −20°C. After centrifugation at 4°C at 12,000 × g for 10 min, the protein deposit was redissolved by urea buffer [8 M urea, 100 mM TEAB (triethylammonium bicarbonate)]. The protein concentration was detected using Bradford protein assay kit (Beyotime).

For trypsin digestion, 100 μg protein of each sample was first reduced with 10 mM DTT at 37°C for 60 min and then alkylated with 55 mM iodoacetamide (IAM) at room temperature for 30 min in darkness. The urea content of protein extract was diluted by adding 100 mM TEAB less than 2 M. The protein pool of each sample was digested with trypsin with the ratio of protein: trypsin = 50:1 mass ratio at 37°C overnight and 100:1 for a second digestion at 4 h.

After trypsin digestion, the peptides were desalted by Strata X SPE column and vacuum-dried. The digested peptides were then labeled with the iTRAQ reagents (AB Sciex), as follows: the control group was labeled with iTRAQ 113 and 114, while NaAsO₂ treatment group with iTRAQ 115 and 116, and DIP pretreatment and NaAsO₂ treatment group with iTRAQ 117 and 118. Briefly, peptides were reconstituted in 20 μl 500 mM TEAB and processed according to the manufacturer’s protocol for 8-plex iTRAQ kit (AB Sciex, Foster City, CA, United States). One unit of iTRAQ reagent was applied to the peptide solution after thawed and dissolved in 50 μL isopropanol. The peptide mixtures were incubated for 2 h at room temperature, then pooled and dried by vacuum centrifugation.

The dried and labeled peptide was reconstituted with HPLC solution A [2% ACN (acetonitrile), pH 10] and then fractionated into high pH reverse-phase HPLC fractions using Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 × 250 mm). Peptides were first separated by a gradient of 2–98% acetonitrile in pH 10 at a speed of 0.6 ml/min over 88 min into 48 fractions. The peptides were then mixed into 15 fractions and dried by vacuum centrifugation. The peptide fractions were desalted using Ziptip C18 (Millipore, MA, United States). Samples were finally dried under vacuum and kept at −20°C until they were analyzed by MS (mass spectrometry).

The experiment was then performed by NanoLC 1000 LC-MS/MS using a Proxeon EAsY-nLC 1000 coupled to Q-Exactive mass spectrometer (Thermo Fisher Scientific, United States). Trypsin digestion fractions were reconstituted in 0.1% FA (formic acid) and immediately charged to the reversed-phase pre-column (Acclaim PepMap®100 C18, 3 μm, 100 Å, 75 μm × 2 cm) at 5 μl/min in 100% solvent A (0.1 M acetic acid in water). Next, peptides eluted from the trap column were loaded into a reversed-phase analytical column (Acclaim PepMap® RSLC C18, 2 μm, 100 Å, 50 μm × 15 cm). The gradient was comprised of an increase from 0 to 8% solvent B 0.1% FA in 98% ACN over 5 min, 8–25% solvent B over 35 min, 25–98% solvent B during 10 min and keep in 98% in 8 min at a constant flow rate of 300 nL/min at EAsY-nLC 1000 system. The eluent was sprayed from an NSI source at an electrospray voltage of 2.5 kV and then analyzed by tandem mass spectrometry (MS/MS) in Q Exactive. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra (from m/z 300–2000) were acquired in the Orbitrap with a resolution of 70,000. Ion fragments were detected in the Orbitrap at a resolution of 17,500. The 15 most intense precursors were selected for subsequent decision tree-based ion trap HCD fragmentation at the collision energy of 32% in the MS survey scan with 10.0 s dynamic exclusion.

The resulting MS/MS raw data was searched against the transcriptome database using Sequest software integration in Proteome Discoverer (version 1.3, Thermo Scientific). The quest parameters were as follows: trypsin as a digestion enzyme, two missing cleavages, oxidized methionine, acetylation in N-Term, iTRAQ modification at the N-terminus of the peptide and iTRAQ 8-plex (K, Y) as the variable modification, fixed modifications like carbamidomethyl (C). The peptide mass tolerance and fragment mass tolerance were set to 20 ppm and 0.05 Da, respectively. A decoy database search strategy was adopted to estimate the false discovery rate (FDR) for peptide identification. For this study, a high peptide confidence (1% FDR) was selected. The cut-off values of 1.2-fold for up-regulated and 0.83-fold for down-regulated proteins, p-value < 0.05, were set as differentially expressed proteins (DEPs) (Shen et al., 2021).

Bioinformatics analysis was performed by using OMICSBEAN online tools (http://www.omicsbean.cn/) and String (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.1, http://string-db.org/) database. DEPs were analyzed by GO (gene ontology) annotation, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, and protein-protein interaction (PPI) networks (Iqbal et al., 2018; Wei et al., 2018). Functional interaction network analysis was conducted using the ClueGO Cytoscape plugin (Bindea et al., 2009). The GO categories and pathways searched include biological processes (BP), cellular components (CC), molecular function (MF), KEGG (Kyoto Encyclopedia of Genes and Genomes), REACTOME, and Wiki pathway.

L-02 cells were incubated in 6-well plates. After being treated with NaAsO₂ or DIP as indicated, cells were collected and lysed with RIPA buffer containing 1 mM PMSF and 1% protease inhibitor cocktail. The protein concentration was measured using the BCA kit. After SDS-PAGE electrophoresis transformation, the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk at room temperature for 2 h and incubated with the indicated primary antibodies overnight at 4°C. After incubated with HRP-conjugated second antibody at room temperature for 1 h, the blotting signal was generated by chemiluminescence using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific). The gray value of protein band was analyzed by Image Labe software, and the ratio of target protein to internal reference gray value was used to reflect the expression of protein.

Statistical analysis was carried out with SPSS Version 20.0 (SPSS Software, Chicago, IL, United States). The experimental data is shown as the means ± SD. Single factor analysis of variance (ANOVA) was used to detect the different distribution of various groups. The distribution of biometric values is normalized by logarithmic transformation. The statistically significant level was p < 0.05.

The HPLC detection showed that the monosaccharides in the Dictyophora polysaccharides (DIP) were D-mannose, D-glucose, D-galactose, D-xylose, L-fucose (Figure 1A), and their contents were 15.27, 523.57, 27.80, 10.01, and 17.84 mg/L respectively, of which D-glucose accounted for 88.07% (Supplementary Table S1).

To investigate the cytotoxicity of sodium arsenite to L-02 cells, L-02 cells were treated with NaAsO₂ treatment. We found that NaAsO₂ reduced the viability of L-02 cells in a dose-dependent manner. The IC₅₀ value was 39.89 ± 3.20 μM (Figure 1B). However, DIP pretreatment significantly improved L-02 cells viability, even when exposed to 1/8 of IC₅₀ (5 μM) and 1/4 of IC₅₀ (10 μM) NaAsO₂. Pretreatment with 80 μg/ml DIP enabled L-02 cells to tolerate 10 μM sodium arsenite (Figures 1C,D). The results indicated that DIP has the potential to antagonize sodium arsenite cytotoxicity.

Considering sodium arsenite is recognized as an apoptosis inducer (Bashir et al., 2006; Chen et al., 2012; Sun et al., 2017), we then investigated whether NaAsO₂ could induce apoptosis in L-02 cells. Flow cytometry analysis showed that the proportion of apoptotic cells increased significantly after treatment with 10 μM NaAsO₂ (Figure 1E). Furthermore, with the increasing dosage of NaAsO₂ treatment, pro-apoptotic protein Bax was up-regulated, while anti-apoptotic Bcl-2 protein was down-regulated in L-02 cells (Figure 1F), suggesting NaAsO₂ can induce the apoptosis of L-02 cells. However, DIP pretreatment not only significantly reduced the apoptosis induced by NaAsO₂ (Figure 1E) but also reversed the expression of Bax and Bcl-2 (Figure 1G), revealing the protective role of DIP in resisting sodium arsenite-induced apoptosis of L-02 cells.

The iTRAQ analysis was further performed to explore the molecular mechanism of DIP against sodium arsenite-induced cytotoxicity in L-02 cells. We identified the protein expression profile in L-02 cells treated with 10 μM NaAsO₂ compared with the control group (As/Ctrl group). After DIP pretreatment, the protein expression characteristics of arsenic treated group were also analyzed (DIP + As/As group). A total of 2,876 proteins were identified. Among them, 60, 71, and 13 proteins were identified as DEPs in As/Ctrl group, DIP + As/As group, and DIP + As/Ctrl groups (Figures 2A–C; Table 1), respectively. Of these, 14 DEPs were found to be common between the As/Ctrl group and DIP + As/As group, with the opposite expression trend in these two groups (named as reversed proteins; Figure 2D and Table 1). Cluster analysis showed that the protein expression characteristics of DIP + As/As group and DIP + As/Ctrl were more similar, but almost opposite to As/Ctrl group (Figure 2E).

By bioinformatics analysis, the BP, CC, MF, and KEGG pathways associated with these 60 DEPs in As/Ctrl group are presented in Figures 3A–D and Supplementary Table S2–5. The BP related to DEPs in As/Ctrl group involved mainly in the generation of precursor metabolites and energy, glycolytic process, ATP generation from ADP, pyruvate biosynthesis process, and NADH regeneration, etc. (Figure 3A). In particular, 14 DEPs were related to regulation of apoptotic process including ALB, ANXA1, ATPIF1, CLU, FAM129B, HSPE1, LDHA, MFF, MIF, PPIF, PPP1R10, SFN, SIN3A, and STAT5B (Supplemenntary Table S2). Four DEPs (CLU, MFF, PPIF, and SFN) were associated with apoptotic mitochondrial changes, 3 DEPs (MFF, POLDIP2, and SLIRP) were related to mitochondrion morphogenesis, and 3 DEPs (CLU, MFF, and SFN) were involved in the release of cytochrome c from mitochondria (Supplementary Table S2). The DEPs were mainly distributed in the cytoplasm, cytoplasmic parts, mitochondrion, and mitochondrial matrix, etc. (Figure 3B; Supplementary Table S3). The MF of these DEPs included protein binding, calcium-dependent protein binding, alkaline phosphatase activity, and protein phosphatase inhibitor activity, etc. (Supplementary Table S4).

After NaAsO₂ treatment, KEGG pathway analysis showed that most DEPs were involved in metabolic pathways, including glycolysis, carbon metabolism, pyruvate metabolism, amino acid biosynthesis and metabolic thiamine, folic acid biosynthesis, etc. (Figure 3C). Seven (7) DEPs were involved in these metabolism-related pathways, including ACAT1, ALDOA, IDH3B, LDHA, PGK1, PKM, and TPI1. Among them, 5 DEPs were down-regulated in L-02 cells, including ALDOA, LDHA, PGK1, PKM, TPI1 (Table 1; Supplementary Table S5). Other signal transduction pathways included the HIF-1 signaling pathway. The DEPs associated with this pathway include ALDOA, LDHA, and PGK1, which have also been down-regulated (Table 1; Supplementary Table S5). The PPI network associated with the DEPs is shown in Figure 3D. Consistently, these DEPs were enriched into metabolism-related pathways, including glycolysis/gluconeogenesis, glycosylcarbon metabolism, pyruvate metabolism, and amino acid biosynthesis, etc. The proteins include ALDOA, LDHA, PGK1, PKM, TPI1 are a key note in the PPI network. HIF-1 pathway is also shown in PPI network.

By using the ClueGO for functional enrichment, the DEPs were mainly related to the pathway of aerobic glycolysis, the release of mitochondrial cytochrome C, and RAC1/PAK1/MMP2 pathway (Figures 3E,F; Supplementary Figure S1). Aerobic glycolysis contains the above metabolic pathways such as glycolysis, gluconeogenesis, pyruvate metabolism, and HIF-1 related pathways (Supplementary Figure S1). The results showed that NaAsO₂ treatment caused significant changes in metabolism-related pathways in L-02 cells.

Seventy-one (71) DEPs were identified in the DIP + As/As group. By GO analysis, BP-associated DEPs involve primarily SRP-dependent co-translational protein targeting and membrane, translation, cytoplasmic translation, and mitochondrial gene expression (Figure 4A; Supplementary Table S6). They were widely distributed in cells (Figure 4B,C; Supplementary Table S7) and MF is presented in Supplementary Table S8. Of note, combined GO analysis with UniProt annotation, 21 DEPs were ribosomal proteins (RPs), including DAP3, MRPL11, MRPL39, MRPL43, MRPS28, RPL13, RPL17, RPL19, RPL23A, RPL24, RPL26, RPL27, RPL31, RPL6, RPL7, RPL34, RPL35A, RPS13, RPS15A, RPS19, and RPS5. Among them, 5 DEPs were mitochondrial ribosomal proteins, including DAP3, MRPL11, MRPL39, MRPL43, and MRPS28 (Supplementary Table S7). Based on GO analysis and UniProt annotation, the other 16 were classified as cytoplasmic RPs in the present study. KEGG analysis showed the enrichment of ribosome-related protein translation pathways, nucleotides, and protein metabolism pathways (Figure 4C; Supplementary Table S9), which also are consistent with the GO analysis. In the PPI network analysis, a notable feature was that the DEPs associated with ribosomes were enriched. TCA cycle and amino acid-related biosynthesis pathways were also involved (Figure 4D). Similarly, by using ClueGO for functional enrichment analysis, the DEPs in the DIP + As/As group were highly correlated with the ribosomes (Figures 4E,F; Supplementary Figure S2).

Subsequently, we analyzed the protein expression profiles of DIP + As-treated group compared with the control group. Thirteen (13) DEPs were identified in this group. The relatively small number of DEPs suggested that DIP pre-intervention altered the protein expression profile of L-02 cells exposed to As, making it similar to normal growth L-02 cells. In addition, a total of 14 DEPs overlapped between As/Ctrl group and DIP + As/As group. Among these, 9 proteins were up-regulated and 5 proteins were down-regulated in As/Ctrl group, while their expression trend was reversed in DIP + As/As group. They were named as revered proteins (Supplementary Figure S3A).

GO analysis showed that these 14 revered proteins were widely distributed in cells, and KEGG analysis identified that these reversed proteins were not only primarily involved in the regulation of metabolic pathways and other biological processes, including: 2-oxygen carboxylic acid metabolism, amino acid biosynthesis, citric acid cycle (TCA cycle), but also related to the p53 signaling pathway (Supplementary Figure S3B,C; Supplementary Table S10). The PPI network showed that those proteins were correlated with carbon metabolism, amino acid biosynthetic, TCA cycle, 2-oxycarboxylic acid metabolism, cell cycle, and P53 signaling pathways (Supplementary Figure S3D).

The hub genes in As/Ctrl group and DIP + As/As group were analyzed. As shown in Figure 5A,B, 8 DEPs (PKM, TPI1, ALDOA, PGK1, ALPI, ALPL, MIF, and ANPEP) were identified as hub genes in As/Ctrl group. Twenty-three (23) DEPs were identified as hub genes in DIP + As/As group. Of these, 16 were ribosomal proteins.

We also analyzed the expression of DEPs in different groups in the key pathways, i.e., ribosomal protein, apoptosis, mitochondria, and metabolism-related protein (Figures 5C–F). The results showed that DIP pretreatment reversed or restored their expression.

Based on the above analysis, two hub proteins (RPS5 and SFN), for their largest weight in biological pathways, were selected to be confirmed by Western blot analysis (Figures 5A,B). As shown in Figures 5G,H, consistent with the proteomic results, they were down-regulated in the As/Ctrl group, while DIP pretreatment increased their levels in the DIP + As/As group.