Digestion and absorption properties of selenium-enriched Pleurotus eryngii peptides and their alleviating effects on lead poisoning
Abstract
The results showed that SePEPs exhibited excellent processing and digestion stability. The apparent permeability coefficient (Papp) of digested SePEPs (D-SePEPs) was greater than 1.0×10−5 cm/s, indicating good bioavailability. D-SePEPs were primarily absorbed by intestinal epithelial cells via endocytosis, and wortmannin significantly inhibited their transport. Following absorption, SePEPs significantly upregulated the expression of Nrf2, HO-1, GCL, GCLM, and NQO1 proteins in the liver and enhanced the activity of antioxidant enzymes, particularly GSH-Px, thereby alleviating lead-induced liver oxidative damage. Furthermore, SePEP treatment significantly reduced TNF-α and IL-6 levels in the liver of mice, thereby alleviating lead-induced liver damage. Molecular docking results further demonstrated that specific sequences from SePEPs, GFSSeMPGLKQDLVLPR and PDGVSeMLVEGK, compete with Nrf2 for the active pocket of Keap1, thereby activating the Keap1-Nrf2 pathway. These results suggest that SePEPs could be considered as a potential selenium-enriched functional food to offset lead toxicity.
Introduction
Lead (Pb) is a well-established neurotoxin that is widely present in the environment due to long-term industrial use, particularly in developing countries. Lead undergoes bioaccumulation and trophic transfer in ecosystems, ultimately posing a threat to human health through dietary exposure. Elevated blood lead levels are associated with a variety of pathological conditions, including cardiovascular disease and cognitive impairment. Notably, even blood lead concentrations below 5 μg/dL can produce significant adverse effects in children, manifested by reduced IQ, decreased academic performance, decreased attention span, and worsening mental health. Studies have shown that oxidative stress is a major pathway of lead-induced toxicity. Lead exposure disrupts cellular redox homeostasis by increasing intracellular reactive oxygen species (ROS) production and impairing antioxidant defenses. Due to its electron-sharing capacity, lead can form covalent bonds with sulfhydryl (-SH) groups of antioxidant enzymes or displace essential cofactors such as zinc and copper ions, thereby inactivating key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Furthermore, lead exhibits preferential long-term accumulation in liver and bone tissues following chronic exposure, leading to persistent immune dysfunction and metabolic disturbances.
Selenium (Se) is an essential trace element that plays a key role in mitigating heavy metal toxicity while enhancing immunity, antioxidant defenses, and antiviral responses. Studies have shown that various crops (including rice, tea, and edible fungi) can absorb inorganic Se from the substrates they grow in and incorporate it into proteins and polysaccharides, converting it into an organic form that is more easily absorbed and utilized by the body. Selenoproteins and selenopeptides are the primary bioactive components of Se-rich foods, synergistically modulating systemic health benefits. Studies have shown that Se-enriched soy peptides enhance the antioxidant capacity of rats by increasing serum glutathione peroxidase (GSH-Px) activity and GSH levels while reducing serum aspartate aminotransferase (AST) and glutamate aminotransferase (ALT) activities. Similarly, the tuna-derived seleno-α-glucosidase inhibitory peptide KPLSeCPK enhances the antioxidant capacity and reduces insulin resistance in IR-HepG2 cells. SeCPPs, another selenium-rich peptide derived from cactus, significantly reduced acetylcholinesterase (AchE) activity and MDA content in the brain tissue of aged mice induced by D-galactose, enhanced GSH-Px activity, downregulated insomnia-inducing cytokines, and improved memory function. Selenium-enriched egg white peptide Se-EWP enhanced the antioxidant capacity of mouse livers and effectively alleviated cyclophosphamide-induced immunosuppression. Mechanistically, Se is incorporated into low-molecular-weight peptides in the form of selenomethionine (SeMet) or selenocysteine (SeCys), leveraging their enhanced intestinal absorption compared to larger proteins. These selenopeptides exhibit diverse biological activities, including broad-spectrum antioxidant, anti-irritant, and immunomodulatory properties, suggesting therapeutic potential for combating heavy metal-induced diseases.
The strong binding affinity between organic selenium and lead enables the formation of stable complexes, effectively reducing lead bioavailability and promoting its excretion in urine or feces, thereby mitigating its cellular and systemic toxicity. Previous studies have shown that selenium not only affects lead absorption and accumulation but also significantly mitigates lead-induced oxidative stress, immune dysfunction, and other toxic effects. Dietary organic selenium supplementation has been shown to reduce lead accumulation and alleviate lead-induced oxidative stress, mitochondrial damage, and apoptosis in chicken liver. Furthermore, Se alleviates lead-induced splenic damage by restoring antioxidant function and inhibiting the MAPK/NF-κB pathway. Specific selenopeptides also promote lead excretion. For example, administration of selenium-enriched soy peptides via drinking water significantly reduced lead levels in various organs of lead-exposed mice. Organic selenium can also effectively reduce lead accumulation in the brain and hippocampus, thereby alleviating lead neurotoxicity. Selenium-enriched rice protein hydrolysate inhibits the activation of the caspase family of proteins, reduces mitochondrial membrane permeability, and prevents the inactivation of matrix metalloproteinases, thereby alleviating lead-induced oxidative stress and apoptosis in PC12 cells. Among the identified selenopeptides, two sequences, TSeMMM and SeMDPGQQ, significantly alleviated lead-induced oxidative damage in HT22 cell line neurons. These findings highlight the potential of selenopeptides as dietary interventions to mitigate lead toxicity. In previous studies, we successfully extracted selenoproteins from selenium-enriched E. rubrum. These proteins demonstrated the ability to mitigate lead toxicity both in vitro and in vivo. Furthermore, selenium-enriched peptides (SePEPs) were obtained by enzymatic hydrolysis and fractionation. In vitro results showed that SePEPs modulated the Keap1-Nrf2 pathway in the NCTC1469 cell line. Furthermore, LC-MS/MS analysis identified the sequences of 12 selenopeptides. Notably, the digestive stability and absorption mechanisms of selenopeptides in vivo are crucial for their bioavailability in humans. Furthermore, the interaction mechanism between key selenopeptide sequences and the Keap1 protein remains to be clearly elucidated. Therefore, in this study, a Caco-2 cell monolayer model was constructed to investigate the transmembrane transport and absorption pathways of SePEPs, employing inhibitors or promoters. A co-culture model of NCTC1469 hepatocytes and Caco-2 cells, as well as a chronic lead exposure mouse model, were also established to explore the mechanisms by which SePEPs mitigate lead-induced oxidative damage in hepatocytes or tissues. In addition, Autodock Vina was used to mimic the binding interaction between specific SePEP sequences and the Keap1 protein. This study aimed to evaluate the digestive stability of SePEPs in mitigating lead toxicity and provide a theoretical basis for the development of functional selenium-enriched peptide products.
Results and Discussion
Processing and Gastrointestinal Stability of Selenium-enriched Peptides
The stability of SePEPs under different temperature and pH conditions is shown in Figure 1. Compared with untreated peptides, heating treatment within the range of 30–80°C had no significant effect on the ability of SePEPs to mitigate lead toxicity in NCTC1469 cells (P>0.05). However, exposure to 100°C significantly reduced the activity of SePEPs (P<0.05). Under acidic conditions (pH 2–6), SePEPs maintained stable activity, while their ability to mitigate lead toxicity was significantly reduced at pH 8–12 (P<0.05), although this remained significantly different from the lead-treated group. These results demonstrate that SePEPs exhibit excellent thermal and acid stability, making them suitable for food processing applications. Based on the results of heat and pH treatment, the stability of SePEPs during simulated in vitro gastrointestinal digestion was further investigated (Figure 1C). Compared to undigested SePEP-treated cells, the viability of SePEP-treated cells collected at 210 and 240 minutes of digestion increased by 13% and 8%, respectively. This study demonstrated that SePEPs maintained significant lead toxicity mitigation activity throughout the in vitro digestion process. Comparative analysis of the efficacy of SePEPs and their digestion product (D-SePEP) revealed a dose-dependent improvement in cell viability within a specific concentration range (Figure 1D). D-SePEP demonstrated superior efficacy compared to SePEP at concentrations of 80 and 160 μg/mL. At a concentration of 320 μg/mL, SePEP and D-SePEP treatment significantly increased cell viability by 28.18% and 25.22%, respectively, compared to the lead-treated group.
Figure 1. Processing and Gastrointestinal Stability of Selenium-Enriched Pleurotus eryngii Peptides
Absorption and Transport Mechanism of D-SePEPs
The effect of D-SePEPs on Caco-2 monolayer integrity was also investigated. As shown in Figure 2A, the TEER of the monolayer slowly decreased during transport of SePEPs over a concentration range of 160 to 640 μg/mL, but remained within the acceptable experimental threshold. The Papp of the SePEPs was quantified by measuring the increasing Se content in the lower chamber. Concentration-dependent transport kinetics of the SePEPs were observed (Figure 2B), with the transmembrane transport rate gradually increasing over 30 to 60 minutes before reaching equilibrium. Furthermore, the Papp values of the SePEPs were calculated, as shown in Figure 2C. Peak Papp values at various concentrations were all above 1.0 × 10−5 cm/s, indicating efficient intestinal absorption of the SePEPs.
Figure 2. In vitro absorption and transport mechanisms of SePEPs in a Caco-2 monolayer model
To investigate the primary transport mechanism of SePEPs in the intestine, various transport inhibitors or enhancers were applied to the monolayer system, as shown in Figure 2D. Specifically, the endocytosis inhibitor wortmannin significantly reduced the Papp of SePEPs (P < 0.05). The transcellular transport inhibitor TF3′G (TF3′G) also reduced transmembrane transport, but the effect was less significant than that of wortmannin (P < 0.05). In contrast, treatment with cytochalasin D showed the opposite trend. Neither the P-glycoprotein inhibitor verapamil nor the multidrug resistance protein inhibitor MK-571 significantly affected the transport levels of SePEPs (P > 0.05). These results suggest that digested SePEPs are primarily transported via endocytosis, with a small amount of small peptides likely being passively transported via the transcellular pathway.
SePEPs and D-SePEPs Ameliorate Lead-Induced Oxidative Damage in Hepatocytes in a Coculture System
A Caco-2/NCTC1469 coculture model was established to evaluate the hepatoprotective effects of SePEPs and their digestive derivative, DSePEPs, against lead toxicity following intestinal absorption (Figure 3). Treatment with SePEPs and D-SePEPs at concentrations of 160 and 320 μg/mL significantly increased cell viability compared to the lead-treated group (P < 0.05). Notably, only D-SePEPs treatment significantly increased the survival rate of lead-exposed cells at higher concentrations (P < 0.05) (Figure 3B). The protective effects of SePEPs and D-SePEPs were consistently inferior to those of the positive control, DMSA, consistent with observations in the NCTC1469 cell model. Further analysis of AST and ALT levels in NCTC1469 cells revealed that all concentrations of SePEPs and D-SePEPs conferred a protective effect relative to the lead-treated group (Figure 3D and E). Specifically, treatment with 320 μg/mL D-SePEPs reduced AST and ALT enzyme activities by 46.29% and 30.23%, respectively. These results suggest that SePEPs and D-SePEPs maintain their significant efficacy in reducing lead toxicity after transport across the intestinal epithelium. Lead accumulation disrupts hepatic redox homeostasis, induces lipid and protein oxidation, and affects the expression of key antioxidant genes. The coculture model was also used to investigate the antioxidant capacity of SePEPs and D-SePEPs after absorption and transport in Caco-2 monolayers. Compared with the lead-treated group, SePEPs and D-SePEPs significantly reduced malondialdehyde (MDA) levels, a marker of lipid peroxidation, after absorption (P < 0.05) (Figure 3F). Lead exposure induced oxidative stress in hepatocytes, with GSH-Px, SOD, and CAT activities decreasing by 45.77%, 28.49%, and 43.36%, respectively, compared with the control group. D-SePEPs at 320 μg/mL significantly increased GSH-Px, SOD, and CAT activities in lead-exposed cells by 160.26%, 14.69%, and 58.47%, respectively (P < 0.05). A similar trend was observed in the SePEPs-treated group. In summary, the digestive fraction, DSePEPs, retained potent antioxidant activity after intestinal epithelial absorption, effectively enhancing intracellular antioxidant enzyme activity, particularly GSH-Px, and attenuating lead-induced oxidative damage in hepatocytes.
Figure 3: Ameliorative effects of SePEPs and D-SePEPs on Pb2+-induced oxidative damage in hepatocytes after transmembrane transport.
Molecular Docking Analysis of Specific SePEPs Sequences with Keap1 Protein
Peptide sequences from SePEPs were analyzed using LC-MS/MS and auto-assembly sequencing. Twelve seleno-peptide sequences were screened and identified with average local identity (ALC) scores greater than 60. These sequences all contain one or more SeMet or SeCys residues, have molecular weights ranging from 900 to 2000 Da, and are rich in hydrophobic amino acids, demonstrating potential antioxidant activity. The mass spectrometric characterization and structural details of these seleno-peptides have been previously reported. In this study, molecular docking simulations were performed using AutoDock Vina to determine the minimum binding energy between the two highest-confidence seleno-peptide sequences from SePEPs and Keap1 protein and identify the active binding sites. The docking binding energies for these sequences were -6.5 and -8.3 kcal/mol, respectively. GFSSeMPGLKQDLVLPR forms one or more hydrogen bonds with Keap1 amino acid residues Val420, Val465, Val467, Val512, Gly423, Thr560, Val608, Val514, Val561, and Gln563 (Figure 4A). PDGVSeMLVEGK forms hydrogen bonds with residues Val467, Val420, Val608, Val561, Thr560, Val604, Val606, Leu365, Ile416, and Leu557 (Figure 4B). Additional hydrophobic interactions and salt bridges were also observed. These findings suggest that seleno-peptide sequences can compete with Nrf2 for the active pocket on the Keap1 protein surface. This binding mechanism may unleash and activate the Nrf2 signaling pathway, thereby promoting the synthesis of antioxidant enzymes in cells.
Figure 4 Overview and details of the optimal position between SePEPs and Keap1 after automated docking of the protein active pocket
Effects of D-SePEPs on the Expression of Nrf2 Pathway-Related Proteins
To investigate the antioxidant mechanism by which D-SePEPs alleviate lead toxicity, Western blot analysis was performed to assess the expression levels of proteins involved in the Keap1-Nrf2 signaling pathway, as shown in Figure 5. Compared with normal cells, lead exposure significantly increased the expression of several Nrf2 pathway-related proteins, particularly HO-1 and NQO1 (P < 0.05). In contrast, cells pretreated with a moderate dose of D-SePEPs exhibited significantly increased expression levels of Nrf2, HO-1, GCL, GCLM, and NQO1 compared with the lead-treated group (P < 0.05). Furthermore, pretreatment with a high dose of D-SePEPs significantly reduced the expression of Keap1, a protein with inhibitory regulatory functions. These findings suggest that digested SePEPs can enhance cellular antioxidant enzyme activity by regulating the expression of proteins in the Nrf2 signaling pathway, thereby alleviating lead-induced cellular oxidative stress.
Figure 5 Effect of D-SePEPs Pretreatment on the Expression Levels of Keap1-Nrf2 Pathway-Related Proteins in NCTC1469 Cells
Protective Effect of SePEPs on Lead-Induced Oxidative Damage in Mouse Liver
SePEPs exhibited significant protective effects against lead-induced oxidative damage in mouse liver. Lead exposure impairs the liver's antioxidant defense system, leading to increased oxidative stress and subsequent tissue damage. The alleviating effect of SePEPs on liver damage was assessed by measuring oxidative markers, enzyme activity, and expression of genes involved in the Nrf2 pathway (Figure 6). Compared with the lead-exposed group, SePEPs treatment significantly reduced serum AST and ALT enzyme activities (P < 0.001), indicating improved liver function in mice (Figure 6B). Furthermore, the activities of key liver antioxidant enzymes, such as GSH-Px, SOD, and CAT, were significantly increased in the SePEPs-treated group (P < 0.01) (Figure 6C-E). These results indicate that SePEPs effectively enhance the liver's oxidative defense system and help alleviate lead-induced liver damage.
Figure 6: Alleviating Effects of SePEPs on Oxidative Damage and Inflammatory Responses in the Liver of Lead-Exposed Mice
The expression of genes associated with the Nrf2 signaling pathway, including Nrf2, HO-1, and NQO1, was measured to assess the molecular responses of SePEPs in alleviating lead-induced oxidative stress (Figure 6F-H). Significant increases in Nrf2 and NQO1 mRNA expression were observed in lead-exposed liver tissue, highlighting their importance in antioxidant responses. Compared with the lead-treated group, high-dose SePEPs treatment significantly upregulated Nrf2, HO-1, and NQO1 mRNA levels (P < 0.01), indicating enhanced Nrf2 pathway activation. This upregulation contributes to increased production of antioxidant enzymes, helping to mitigate lead-induced oxidative damage.
Furthermore, lead exposure significantly enhanced liver inflammation in mice (Figure 6I). SePEPs treatment significantly reduced liver TNF-α and IL-6 concentrations (P < 0.05), suggesting that SePEPs can mitigate lead-induced liver inflammation.
Conclusion
Both in vitro and in vivo models demonstrated that SePEPs effectively mitigated lead toxicity and exhibited excellent stability during processing and digestion. The majority of SePEPs were internalized primarily through endocytosis in intestinal epithelial cells, while low-molecular-weight digested SePEPs entered the systemic circulation via passive paracellular transport. Structural characterization revealed that specific selenopeptide sequences in SePEPs compete with Nrf2 for binding to the active pocket of Keap1, thereby activating the Nrf2 signaling pathway. This activation subsequently upregulated the expression of HO-1, GCL, GCLM, and NQO1 at the mRNA and protein levels, promoting the synthesis of intracellular antioxidant enzymes and alleviating lead-induced liver oxidative damage. Future research will prioritize the development of nanocarrier-based delivery platforms for these selenopeptides to enhance their in vivo stability and tissue-specific targeting efficacy.