Crassostrea gigas peptide PEP-1 prevents tert-butyl hydroperoxide (t-BHP) induced oxidative stress in HepG2 cells

Abstract

Exposure to tert-butyl hydroperoxide (t-BHP) leads to cytotoxicity and oxidative stress in various organs and cell types. The bioactive peptides extracted from Oysters exhibit marked antioxidant activity. The impacts of Crassostrea gigas peptides on t-BHP-triggered oxidative stress remain largely unknown. The protective and antioxidant activity of a C.gigas peptide, PEP-1, on t-BHP-treated HepG2 cells, was investigated. PEP-1, this peptide is arginine kinase in oysters. This enzyme functions as a catalyst for the chemical reaction and serves as a phosphate transferase. Since it was the most expressed protein in the adductor muscle of oysters. Our determination showed the lowest level of a toxic concentration of t-BHP (200 µM) and the resting concentration of PEP-1 (0–1000 ng/ml). PEP-1 exerted a protective effect against t-BHP-induced apoptosis by modifying the expression of pro-and anti-apoptotic proteins. PEP-1 administration reduced nitric oxide and ROS levels while restoring levels of antioxidant proteins in t-BHP-induced cells. PEP-1 exhibited the capacity to enhance the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the C. gigas peptide PEP-1 has demonstrated its ability to protect HepG2 cells against oxidative stress induced by t-BHP.

Introduction

Oxidative stress denotes an oxidant and antioxidant imbalance (Miao et al., 2018). The presence of excess free radicals in cells causes this to happen. It is common for an atom or molecule to have one or more unpaired electrons in its outer shell is generally known as a free radical and can exist independently. Because of their odd numbers of electrons, free radicals are highly reactive and cytotoxic. Free radicals can obtain electrons from other molecules. The damaged molecules become free radicals and initiate a chain reaction that damages living cells. Free radicals are generated by cells and tissues during regular metabolic reactions, and these molecules can have a detrimental effect on biomolecules like nucleic acids, lipids, and proteins (Phaniendra et al., 2015). They have an impact on the redox status and promote oxidative stress, which is linked to diabetes mellitus, neurodegenerative disorders, cardiovascular diseases, pulmonary dysfunction, respiratory diseases, rheumatoid arthritis, cataracts, and various cancers that are associated with free radicals. (Ulagesan et al., 2022). Inflammatory, metabolic, and proliferative liver diseases are associated with the redox state. (Cichoz-Lach and Michalak, 2014).

Tert-butyl hydroperoxide (t-BHP) exposure causes cytotoxicity and oxidative stress in different cell types and organs, such as the liver, testes, oocytes, and retina. t-BHP affects hepatocyte viability by increasing membrane leakage and disturbing the intracellular antioxidant machinery (Bhattacharya et al., 2011). There are two metabolic pathways by which t-BHP induces oxidative stress. Initially, cytochrome P450 creates peroxyl and alkoxyl radicals that trigger the lipoperoxidation of membrane phospholipids, which subsequently alters the membrane’s fluidity and permeability. The process of detoxifying t-BHP to tertbutanol is achieved through glutathione peroxidase, which leads to the depletion of glutathione (GSH) by its oxidation to GSSG. This, in turn, is linked to lipoperoxidation, GSH depletion, and the initiation of mitochondrial permeability transition (MPT) as a result of oxidative stress causing cell damage. (Kučera et al., 2014). This is an organic hydroperoxide used as a model for assessing the mechanisms of cellular changes caused by oxidative stress (Fatemi et al., 2014; Kim et al., 2013). Thus, exogenous oxidative stress inducers like t-BHP mimic liver pathologies linked to oxidative stress.

Antioxidants function as scavengers of free radicals and effectively inhibit cellular damage by interrupting the radical chain reaction that forms the basis of lipid peroxidation, by eliminating reactive oxygen species (ROS), which have been linked to a variety of illnesses (Kim et al., 2007; Papachristoforou et al., 2020). The presence of antioxidants in food can help enhance the oxidative state and balance of oxidants and antioxidants in the gut. (Truong et al., 2022; Yan et al., 2022). Ascorbic acid, tocopherols, and catechin are natural antioxidants that are frequently found in medicines, foods and pharmaceuticals and are utilized in various industrial applications. However, the toxicity of synthetic antioxidants, such as tert-butyl hydroquinone, propyl gallate, and butylated hydroxytoluene hinders their usage. (Ulagesan et al., 2022). Food proteins and their constituent peptides can serve as antioxidants and have few side effects. The safety and biological activity of small peptides derived from marine organisms has made them a prime area of investigation. Oysters are the largest shellfish and contain high levels of nutrients like taurine, vitamins, proteins, trace elements, glycogen, and fatty acids. Oysters contain numerous active peptides that are derived from the ocean (Chen et al., 2022). Significant antioxidant activity is displayed by oyster peptides, and oysters have been the source of several bioactive peptides. (Dong et al., 2010; Qian et al., 2020; Wang et al., 2020; Xie et al., 2018; Zhang et al., 2021). Analyzing the amino acid sequences of oyster peptides can be achieved through either solid-phase or liquid-phase synthesis.

PEP-1 (L-G-G-T-L-A-D-C-I-R) is a peptide of the arginine kinase (AK) enzyme that is commonly found in invertebrates. The enzyme AK, known for its high abundance, acts as a phosphagen kinase (PK) and serves a vital role in maintaining cellular homeostasis in situations where energy turnover is either high or variable. It accomplishes this by facilitating the reversible transfer of a phosphate group between ATP and naturally existing guanidine compounds (Jarilla and Agatsuma, 2010). This is achieved by catalyzing the transfer of a high-energy phosphate group from ATP to arginine. This reaction creates a metabolic energy reservoir of phosphoarginine, which can then be used to rapidly generate ATP when needed. AK is therefore essential for maintaining physiological functions and cellular energy metabolism in invertebrates (Jiang et al., 2016; Xia et al. 2007). AK is the well-expressed protein present in the adductor muscle of oyster C. gigas. However, the protective effect of this peptide against oxidative stress is largely unknown. Hence, we analyzed the antioxidant activity of the chemically synthesized oyster peptide PEP-1 and investigated its protective effect in t-BHP-induced HepG2 cells.

Materials and methods

Peptide synthesis

PEP-1 (L-G-G-T-L-A-D-C-I-R) Arginine kinase, Accession No. (NCBI/UniProt) K1PLF9_CRAGI. a peptide sequence derived from Crassostrea gigas, was commercially synthesized by Peptron Co., Ltd. (Daejeon, Republic of Korea) and subsequently purified to a purity level of over 97% using high-performance liquid chromatography (HPLC) with a C18 column (Capcellpak: Japan). PEP-1 was solubilized in a solution of 0.1% trifluoroacetic acid and water, with a 1 mg/mL concentration, followed by 40 µL into the HPLC. The experimental conditions comprised a 3–60% acetonitrile gradient, a 1 ml/min flow rate, room temperature (RT), and UV detection at 220 nm. The molecular weight of PEP-1 was 1017.53 Da as determined by mass spectrometry (Shimadzu LCMS-2020; Shimadzu Corp in ionization mode (+ H, 1.0079 Da; − H, − 1.0079 Da) alongside multiple reaction monitoring (300–2300 m/z). Following synthesis, the peptides were dissolved in water (10 mg/ml) and then placed in storage at a temperature of – 50 °C.

Cell culture

HepG2 human liver cancer cells (HB 8065) were acquired from the ATCC (American Type Culture Collection; Manassas, VA) and subsequently maintained in a minimum essential medium (MEM; Sigma Aldrich, Burlington, MA) containing 10% fetal bovine serum (GenDEPOT, Katy, TX) supplemented with 100 U/ml of penicillin and 100 mg/ml of streptomycin at 37 °C with 5% CO₂. The culture medium was renewed every 2 days.

Cell viability assay

An EZ Cytox Cell Viability Assay Kit (cat. no. EZ-1000; DoGenBio, Seoul, Korea) was employed to estimate cell viability. The 96-well plates were seeded with cells at a concentration of (1 × 10⁴/well in 200 μl of MEM) and incubated at 37 °C for 24 h to facilitate attachment. The PEP-1 (125, 250, 500, 1000, or 2000 ng/ml) in serum-free MEM (SFM) was used to treat the attached cells for 24 h at 37 °C. After treatment, the cells were treated with Cytox solution and then incubated at 37 °C for 1 h. Measurement of absorbance at a wavelength of 450 nm was conducted with a FilterMAX F5 microplate reader. (Molecular Devices LLC, San Jose, CA).

t-BHP toxicity assay

HepG2 cells were cultured at a density of 1 × 10⁴ cells per well in 96-well plates. Following 24 h of incubation, cells were treated with t-BHP (200, 400, 600, 800, or 1000 μM/ml) in an FBS-free medium. Cell viability was assessed at 0–4 h using an EZ Cytox Cell Viability Assay Kit (DoGenBio).

Effect of PEP-1 on t-BHP-treated HepG2 cells

The impact of PEP-1 on cellular viability was assessed using an EZ Cytox Cell Viability Assay Kit (catalog number EZ-1000; DoGenBio). Cells were cultured in 96-well plates (1 × 10⁴/well in 200 μl of medium) and allowed to adhere for 24 h at 37 °C. After treatment with PEP-1 (125, 250, 500, or 1000 ng/ml) in SFM for 24 h at 37 °C and exposure to 200 µM t-BHP for 3 h, the cells were incubated with Cytox solution for 1 h at 37 °C. The absorbance at 450 nm was then measured using a FilterMAX F5 microplate reader (Molecular Devices LLC).

Acridine orange (AO) and PI staining

HepG2 cells were cultured in six-well plates, followed by treatment with PEP-1 and t-BHP, subsequent washing with ice-cold PBS, staining with AO/PI (1 mg/ml each), and further incubation at room temperature for 20 min in the absence of light. The cells were observed under a fluorescence microscope after removing the stain with ice-cold PBS.

Nitric oxide (NO) assay

The concentration of nitrite in the culture medium was measured by spectrophotometry as previously reported (Goshi et al., 2019). Cells were cultured in 48-well plates at a density of 2 × 10⁶ cells per well and incubated at 37 °C for 24 h. Next, the cells were treated with different concentrations of PEP-1 (125, 250, 500, or 1000 ng/ml) and 200 µM t-BHP in SFM for 3 h at 37 °C. Afterward, 100 µl of culture medium was transferred to a 96-well plate, followed by the addition of Griess reagent (G4410; Sigma-Aldrich) in equal volume. The absorbance was measured at 540 nm using a FilterMAX F5 microplate reader (Molecular Devices LLC) after a 10-min incubation at 37 °C.

ROS assay

The quantification of ROS was conducted by utilizing the previously described fluorescent probe DCFH-DA. The seeding of cells was conducted in 96-well plates, with a density of 1 × 10⁴ cells per well. Subsequently, the cells were exposed to different concentrations of PEP-1 (0, 125, 250, 500, or 1000 ng/ml). After a 24 h incubation, an FBS-free medium containing 25 μM DCFH-DA was added to the cells, which were then incubated at 37 °C for 1 h. The cells were washed with PBS and then exposed to 200 μM t-BHP for 3 h, and the fluorescence intensity corresponding to intracellular ROS was measured using a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT) at excitation and emission wavelengths of 485 and 530 nm, respectively (Kim and Hue, 2020).

Quantification of the intracellular ROS concentration

The Muse™ Oxidative Stress Kit (EMD Millipore, Burlington, MA) was utilized to ascertain the intracellular levels of ROS. Cells were harvested and washed with 1 × assay buffer, followed by incubation with the Muse™ Oxidative Stress Reagent Working Solution (MCH100111; EMD Millipore) at 37 °C for 30 min. Flow cytometry was employed to measure the ROS levels in cells treated with or without PEP-1 and treated with 200 µM t-BHP.

Western blot analysis

The HepG2 cells were subjected to a 24-h incubation in serum-free medium (SFM), which was supplemented with different concentrations of PEP-1 (0, 125, 250, 500, or 1000 ng/ml). Following this, the cells were treated with 200 µM t-BHP for 3 h at 37 °C. After rinsing the cells with PBS, they were lysed with RIPA lysis buffer, comprising 50 mM Tris–HCl, 1 mM EDTA, 150 mM sodium chloride, 1% NP-40, and 0.25% sodium deoxycholate; pH 7.4, that contained protease inhibitors, including aprotinin, leupeptin, pepstatin, sodium orthovanadate, sodium fluoride, and phenyl methane-sulfonyl fluoride at 4 °C for 30 min. Centrifugation of extracts was performed at 12,000×g for 10 min at 4 °C followed by the use of the supernatants for Western blot analysis. The quantification of protein concentration was conducted using a Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich) for the analysis. Equal quantities of protein (30 μg) were separated using a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (EMD Millipore). The membranes were then blocked through incubation with 3% bovine serum albumin in TBS-T (10 mM Tris HCl, 150 mM NaCl, and 0.1% Tween 20) at room temperature for 60 min, followed by incubation with primary antibodies (dilution, 1:1000) overnight at 4 °C with gentle agitation. Following three washes with TBS-T for 15 min each, the membranes were incubated for 60 min at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody that had been diluted 1:10000 in TBS-T. Signal detection was carried out using an Enhanced Chemiluminescence Western Blot Analysis Kit (Thermo Fisher Scientific, Inc., Waltham, MA), and the experiments were conducted in triplicate, with densitometry analysis performed using ImageJ software.

The study was conducted using an array of primary antibodies, including anti-Bcl-2 (cat. no. sc-492; rabbit), anti-Bcl-xL (sc-7195; rabbit), anti-Bad (sc-7869; rabbit), anti-Bax (sc-493; rabbit), anti-BH3 interacting domain death agonist (Bid; sc-11423; rabbit), anti-catalase (CAT; OAAB05216; rabbit), anti-superoxide dismutase 1 (SOD1; OASE00355; rabbit), anti-superoxide dismutase 2 (SOD2; OASE00357; rabbit; Aviva Systems Biology Corp.), anti-quinone oxidoreductase 1 (NQO1; sc-16464; goat), anti-heme oxygenase 1 (HO1; sc-1796; goat), and anti-nuclear factor erythroid 2-related factor 2 (Nrf2; sc-722; rabbit). Additionally, anti-β-actin (cat. no. sc-47778; mouse) antibodies were employed as a control. The secondary antibodies utilized in this study were HRP‑conjugated anti‑mouse IgG (cat. no. 7076S; Cell Signaling Technology, Inc., Danvers, MA), anti-rabbit IgG (31460), and anti-goat IgG (31400; Invitrogen, Waltham, MA; Thermo Fisher Scientific, Inc).

Results and discussion

PEP-1 protects against t-BHP-induced toxicity in HepG2 cells

The effect of PEP-1 on HepG2 cell viability was measured in concentrations ranging from 0 to 2000 ng/ml. The administration of PEP-1 alone exhibited no toxicity at any of the concentrations tested. The maximum concentration of 2000 ng/ml demonstrated a notable cell viability of 91.28 ± 1.05% (Fig. 1A). t-BHP is typically employed as an oxidative stress inducer and damages lipids and DNA. To identify a suitable concentration of t-BHP for avoiding acute toxicity, a pilot study was performed using t-BHP at 200, 400, 600, 800, and 1000 μM; cell viability was measured at 0 to 4 h. t-BHP was selected for use in subsequent experiments because it inhibited HepG2 cell viability by 50% at 3 h (Fig. 1B). Cell viability in the t-BHP group was 49.887 ± 2.976% compared to the control (Fig. 1C). Treatment with 200 µM t-BHP and 125, 250, 500, 1000, or 2000 ng/ml of PEP-1 significantly increased cell viability to 52.71 ± 1.69%, 59.06 ± 1.7%, 64.71 ± 2.13%, 67.68 ± 1.81%, and 73.05 ± F1.52%, respectively, compared with the control. Microscopy confirmed these results (Fig. 1D). PEP-1 (Arginine kinase) is a vital enzyme involved in cellular energy metabolism and the regulation of ATP levels in invertebrate cells. Additionally, AK functions as a phosphotransferase, which catalyzes reversible phosphorylation between phosphoarginine and ADP and contributes significantly to physiological processes and cellular energy metabolism (Jiang et al., 2016; Wang et al., 2009). The present study also highlighted that the PEP-1 prevented the oxidative stress-induced death of HepG2 cells without showing any toxicity.

Fig. 1.

PEP-1 enhanced the viability of tBHP-treated HepG2 cells. (A) Viability of HepG2 cells treated with increasing concentrations of PEP-1. (B) tBHP (200–1000 µM) toxicity in HepG2 cells at 0–4 h. (C) Viability of HepG2 cells co-treated with PEP-1 and 200 µM tBHP is shown as the surviving cell percentages in comparison to the control group. (D) Microscopic observations of the viability of HepG2 cells co-treated with PEP-1 and 200 µM tBHP. The data are means ± standard deviation (SD) of three experiments conducted independently. Two‑way analysis of variance (ANOVA); *P < 0.05 vs. control group; #P < 0.05 vs. 200 µM tBHP group. tBHP, tert-butyl hydroperoxide; PEP-1, a Crassostrea gigas peptide

Arginine kinase has been shown to play a vital role in the resistance mechanisms against various stress factors, including reactive oxygen species, trypanocidal drugs, hypoxia, toxicants, pesticides, pH imbalances, and starvation, in numerous unicellular and multicellular organisms (Canonaco et al., 2003; Peraira, 2014; Shofer et al., 1997; Zhang et al., 2022). There are few studies that have been conducted on AK as a chemotherapeutic target against parasites and in pest control (Jarilla and Agatsuma, 2010; Zhang et al., 2022). However, the protective and anti-oxidant activity of AK was not known. In view of this, we investigated the effect of PEP-1 on human HepG2 cells treated with t-BHP, which increases intracellular ROS levels (Yoon et al., 2014). In biological systems, t-BHP degradation causes lipid peroxidation chain reactions, induces cellular toxicity via DNA damage, and depletes cellular GSH and protein thiols, leading to generalized cellular damage and apoptosis (Alía et al., 2005). Because HepG2 cells are derived from hepatoblastomas, they express many plasma proteins absent from primary hepatocytes. Due to the secretion of phase I, II, and antioxidant enzymes, cultured HepG2 cells are used to study xenobiotic and cytoprotective mechanisms of natural and synthetic antioxidants in vitro without using laboratory animals (Yoon et al., 2014). Our results showed that PEP-1 (125–1000 ng) had no adverse effect on cell viability. Consistent with recent reports, t-BHP at 400–1000 µM significantly reduced HepG2 viability, whereas 200 µM t-BHP reduced the viability by 50% (Lee et al., 2018; Liang et al., 2018). However, the effect of t-BHP was restored by PEP-1 pretreatment in a dose-dependent manner. These results suggest that in the presence of t-BHP, PEP-1 exerts a cytoprotective effect in HepG2 cells.

In order to explore the molecular mechanism through which PEP-1 inhibits apoptosis, we assessed the expression levels of pro-apoptotic proteins (Bad and Bax) as well as anti-apoptotic proteins (Bcl-2, Bcl-xL, and Bid) from the Bcl-2 family. These proteins play a crucial role in regulating apoptosis by controlling the permeability of the mitochondrial membrane and the release of cytochrome c (Shamas-din et al., 2013), in t-BHP‑treated HepG2 cells. The PEP-1 co‑treatment groups had lower Bad levels and higher Bcl‑2 and Bid levels than the t-BHP group, and the effect was concentration‑dependent (Fig. 2A and B). PEP-1 pretreatment downregulated the levels of apoptosis-related proteins such as Bad and Bax and upregulated those of the anti-apoptotic proteins Bcl-2, Bcl-xL, and Bid. Bcl-2 family proteins encompass both pro-and anti-apoptotic factors, and their interplay influences cell sensitivity to apoptotic signals. Pro-apoptotic proteins promote cytochrome c release whereas anti-apoptotic Bcl-2 family members inhibit cytochrome c release, thereby preventing apoptosis (Li et al., 2015; Sarkar and Sil, 2010). Bid, a pro-apoptotic Bcl-2 protein, was discovered by binding to both pro-apoptotic Bax and anti-apoptotic Bcl-2. Sequence analysis indicates that Bcl-2 and Bid belong to the BH3-only subgroup; however, structural and phylogenetic analyses suggest that Bid is related to the pro-apoptotic Bax protein rather than to BH3 proteins (Billen et al., 2008). A truncated form of Bid, tBid, reportedly facilitated the release of apoptogenic proteins such as cytochrome c by mitochondria (Esposti, 2002). All types of apoptosis require direct activation of Bax and Bak in mitochondria by Bid family proteins (Ren et al., 2010). Moreover, the anti-apoptotic protein Bcl-2 hinders Bid-triggered apoptosis within the mitochondria by preventing the release of cytochrome c, while not impeding Bid’s processing or activation (Yi et al., 2003). Our data strengthen the hypothesis that t-BHP increases the permeability of the mitochondrial outer membrane, promoting cytochrome c release (Bossy-Wetzel and Green, 1999). PEP-1 prevented t-BHP-induced cytochrome c release and regulated the expression of pro-and anti-apoptotic proteins, including Bcl-2, an inhibitor of the mitochondrial apoptosis pathway.

Fig. 2.

PEP-1 suppressed the tBHP-induced apoptosis of HepG2 cells. (A) HepG2 cells were subjected to a 24-h incubation with or without the specified concentrations of PEP-1, followed by treatment with 200 µM tBHP. The Western blot analysis reveals the expression of apoptosis-associated proteins (Bcl-2, Bcl-xL, Bid, Bad, and Bax). (B) Normalization of the bands to β-actin was performed as the internal control, and the protein levels are depicted. Three independent experiments were conducted, and the data are presented as means ± SD. Statistical analysis using two-way ANOVA revealed significant differences (*P < 0.05) compared to the control group and (#P < 0.05) compared to the 200 µM tBHP group

PEP-1 decreases t-BHP-induced oxidative stress in HepG2 cells

A t-BHP overdose causes liver damage by inducing the production of various cytokines and NO. The t-BHP overdose group had a significantly higher NO level (147.32 ± 1.84%) than the control group. Co-treatment with PEP-1 significantly suppressed the NO level in a concentration-dependent manner (Fig. 3A). In the 1000 ng/ml PEP-1 group, NO production was reduced to 116.046 ± 5.64% compared to 147.32 ± 1.84% in the t-BHP overdose group. t-BHP treated HepG2 cells had a higher NO level, which was modulated by PEP-1 in a dose-dependent manner (Fig. 3A). NO produced by iNOS neutralizes pathogens, but NO in excess is detrimental to immunity and can lead to cell death (Li et al., 2019; Qian et al., 2008). Indeed, an increase in the NO level was inhibited by oyster peptides in RAW264.7 cells (Hwang et al., 2019, 2012).

Fig. 3.

PEP-1 restored tBHP-induced oxidative stress in tBHP-treated HepG2 cells. (A) HepG2 cells were incubated with 200 µM tBHP with or without the indicated concentrations of PEP-1. NO levels were measured by the Griess assay. (B) ROS levels were determined by the 2′,7′-dichlorofluorescein diacetate assay. (C) Fluorescence images of ROS. (D) ROS measurement. (E) Western blotting was employed to determine the levels of oxidative stress-associated proteins (CAT, SOD1, SOD2, and NQO1). (F) Normalizing the bands to β-actin as the internal control, the graph was constructed based on the ratio of total protein. The data represents the means ± SD of three independent experiments. The statistical analysis involved a two-way ANOVA, and *P < 0.05 indicates a significant difference compared to the control group, while #P < 0.05 indicates a significant difference compared to the 200 µM tBHP group. NO nitric oxide, ROS reactive oxygen species, CAT catalase, SOD1 superoxide dismutase 1, SOD2 superoxide dismutase 2, NQO1 quinone oxidoreductase 1

In comparison to the control group, the t-BHP overdose group exhibited a substantially higher ROS level (183.1 ± 3.34%) (Fig. 3B). However, PEP-1 co-treatment concentration-dependently reduced the ROS level compared to the t-BHP overdose group. This was confirmed by fluorescence micrographs (Fig. 3C). The percentage of macrophages (M1) was 84.30% in the control group and 61.79% in the 200 µM t-BHP-treated group. Co-treatment with PEP-1 significantly increased the macrophage (M1) percentage in a dose-dependent manner to 80.03% in the 1000 ng/ml PEP-1-treated group (Fig. 3D). Free radicals, including superoxide anions (O₂⁻) and hydroxyl radicals (OH), form during respiration and are linked to several diseases. Because of their instability, these radicals are highly reactive, causing damage to cells and tissues (Kim et al., 2007). The ROS level increased and decreased in the t-BHP-treated group and the t-BHP + PEP-1-pretreated group, respectively.

The Western blot analysis revealed decreased protein levels of CAT and SOD2 in the t-BHP overdose group compared to the control group. Concurrent administration of PEP-1 demonstrated a concentration-dependent increase in the levels of CAT, SOD2, and NQO1 (Fig. 3E and F). CAT and SOD are key antioxidant enzymes that maintain the oxidant–antioxidant balance (Fernando et al., 2020; Zhang et al., 2019), and their activity is reduced under conditions of oxidative stress (Sarkar and Sil, 2010). The downregulation of SOD by t-BHP demonstrates intracellular oxidative stress resulting in lipid membrane disruption and hepatic cell death via inflammation (Lee et al., 2018). In this study, CAT and SOD2 were downregulated by t-BHP. SOD1 did not show any downregulation after t-BHP treatment. The induction of cytoprotective enzymes by natural compounds has the potential to alleviate oxidative stress. Two major cytoprotective enzymes, NQO1 and HO1, inhibit redox impairment. HO1 has scavenging activity and NQO1 protects against superoxide scavenging (Liang et al., 2018). In this study, PEP-1 exerted a cytoprotective effect against t-BHP-induced hepatotoxicity, in a manner involving the upregulation of NQO1 and HO1.

PEP-1 increases Nrf2 expression in t-BHP‑treated HepG2 cells

The t-BHP overdose group had lower HO1 and Nrf2 levels than the control group (Fig. 4A and B). Nrf2 is an essential transcription factor; its stimulation upregulates downstream antioxidant-related genes, including those encoding HO1 and NQO1. In typical situations, Kelch-like ECH-associated protein-1 (Keap1) is responsible for targeting Nrf2 for proteasomal destruction through ubiquitin-mediated pathways. Keap1 is inactivated in the presence of an external stimulus, resulting in a decrease in the clearance of Nrf2 through ubiquitination and causing the upregulation of nuclear Nrf2. Consequently, Nrf2 binds to antioxidant response factors. This leads to a decrease in the levels of antioxidants and second-stage detoxification enzymes, such as HO1 and NQO1 (Chen et al., 2022). Nrf2 regulates antioxidant responses, and its absence increases susceptibility to oxidative damage (Liang et al., 2018). In this study, the nuclear accumulation of Nrf2 and the levels of detoxification enzymes (HO1 and NQO1) in t-BHP-treated HepG2 cells were dose-dependently increased by PEP-1, suggesting that PEP-1 suppresses ROS generation. A previous study also suggested that the AK might be involved in the detoxification of insecticides (rutin and quercetin) by regulating cellular energy balance (Zhang et al., 2022).

Fig. 4.

Effect of PEP-1 on Nrf2 activity. (A) HepG2 cells were subjected to incubation with 200 µM tBHP, either with or without the indicated concentrations of PEP-1. The levels of Nrf2-associated proteins (HO1, Nrf2) were determined by Western blotting. (B) Normalization of the bands was performed using β-actin as the internal control, and the graph illustrated the ratio of total protein. The data are means ± SD of three independent experiments. The control group exhibited a significant distinction compared to other groups, as indicated by Two-way ANOVA (*P < 0.05 vs. control group; #P < 0.05 vs. 200 µM tBHP group). Notably, the genes HO1 (heme oxygenase 1) and Nrf2 (nuclear factor erythroid 2-related factor 2) played a role in these observed differences

The present study revealed that the Crassostrea gigas peptide PEP-1 exhibited significant antioxidant activity and decreased t-BHP-induced oxidative damage in HepG2 cells. PEP-1 exerted an inhibitory effect on apoptosis through the modulation of pro-apoptotic and anti-apoptotic markers’ levels. PEP-1 administration to t-BHP-induced cells resulted in a decrease in nitric oxide and ROS levels, restoration of antioxidant-associated protein levels, and an increase in the translocation of nuclear factor erythroid 2-related factor 2. More studies are needed to explore the pathways of signal transduction implicated in hepatotoxicity induced by t-BHP.

Declarations

Conflict of interest

The authors declare no conflict of interest.