Intracellular ethanol‐mediated oxidation and apoptosis in HepG2/CYP2E1 cells impaired by two active peptides from seahorse (Hippocampus kuda bleeler) protein hydrolysates via the Nrf2/HO‐1 and akt pathways

Abstract Seahorse (Hippocampus kuda Bleeler) are representative marine species in aquaculture, with special value of medicine and food. In this study, the protective effects of two peptides from seahorse hydrolysates (SHP‐1 and SHP‐2) against ethanol‐mediated oxidative stress in HepG2/CYP2E1 cells were investigated. Firstly, SHP‐1 and SHP‐2 presented no cytotoxicity. Compared with the ethanol‐treated groups, SHP‐1 and SHP‐2 increased cell viability in a concentration‐dependent manner. Secondly, SHP‐1 and SHP‐2 markedly reduced intracellular reactive oxygen species (ROS) generation, gamma‐glutamyltranspeptidase (GGT) activity, and tumor necrosis factor‐α (TNF‐α) levels and remarkably enhanced superoxide dismutase (SOD) and glutathione (GSH) activities. SHP‐1 and SHP‐2 also down‐regulated the expressions of GGT, bax, c‐caspase‐8/‐9/‐3, p‐Akt, p‐IκB‐α, p‐p65, p‐ERK, and p‐p38 but up‐regulated SOD, GSH, NF‐E2‐related factor 2 (Nrf2), heme oxygenase‐1 (HO‐1), and bcl‐2 levels, as revealed by Western blot analysis. Moreover, SHP‐1 and SHP‐2 increased the mitochondrial membrane potential (MMP), reduced DNA damage, and suppressed the nuclear translocation of p65. These results suggest that two peptides from seahorse hydrolysates can be considered a potential functional biomaterial and further improve the use value of seahorse in aquaculture.

antioxidant defense system (Dou et al., 2013). ROS can be generated by ethanol metabolism. ROS encompass superoxide anions, hydrogen peroxide, and hydroxyl radicals. The intracellular antioxidant defense system includes superoxide dismutase (SOD) and glutathione (GSH), which could further reduce ethanol-induced oxidative stress (Zeng et al., 2013). Gamma-glutamyltranspeptidase (GGT) plays a fundamental role in the maintenance of GSH homoeostasis (Kang, Qian, Ryu, Karadeniz, et al., 2012). Serum (GGT) levels are routinely examined in clinical laboratories, primarily for the diagnosis of ALD . Excessive intake of alcohol can cause an elevated production of ROS. Excessive ROS trigger oxidative stress, leading to mitochondrial dysfunction and DNA damage (Xing et al., 2017;Zhuang et al., 2017). ROS also play an important role in apoptosis (Su et al., 2016). Therefore, ethanol was utilized to induce oxidative injury in HepG2/CYP2E1 cells in this study.
Antioxidants are molecules that often prevent the oxidation of other molecules by being oxidized themselves (Loperena & Harrison, 2017). Antioxidants may contribute directly to decreasing oxidative cellular damage by scavenging free radicals. They may also act indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular ROS-related enzymes (Vieira et al., 2017). Recently, synthetic antioxidants (butylated hydroxytoluene [BHT], butyl hydroxy anisole [BHA], and tertiary butylhydroquinone [tBHQ]) have been widely used in food and pharmaceutical products, but these antioxidants still have some toxic and carcinogenetic effects (Finley et al., 2011;Liu et al., 2018). Thus, safer and more effective antioxidants containing natural compounds from marine biological resources are needed. Bioactive peptides are specific protein fragments that have a positive impact on a bodily function or condition and may ultimately influence human health . They have a range of physiological and biochemical functions, including antimicrobial, antifatigue, antitumor, antioxidant, antihypertensive, and immunomodulatory activities (Tonolo et al., 2019). For instance, Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu and Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu from the protein hydrolysate of the microalgae Navicula incerta exert antioxidant activities in HepG2/CYP2E1 cells against ethanol-induced oxidative stress (Kang, Qian, Ryu, Karadeniz, et al., 2012). Tyr-Gly-Asp-Glu-Tyr from tilapia fish skin gelatin hydrolysates prevents HepG2 cells from experiencing alcohol-induced damage .
The seahorse (Hippocampus), belonging to the Syngnathidae family, is a marine teleost fish widely distributed all over the world and usually found in a broad range of shallow-water habitats, such as seagrass beds Lin et al., 2008;Oh et al., 2018).
Seahorse is a high-quality material for the preparation of proteins and related products in aquaculture. Hippocampus is rich in proteins and essential amino acids. Previous studies have reported that a high ratio of heterocyclic or aromatic (His, Pro, Tyr, and Phe) and acidic (Glu and Asp) amino acids account for 16.14% and 20.09% of the total amino groups in Hippocampus, respectively .
Moreover, recent studies have shown that amino acids, trace elements, unsaturated fatty acids, and other functional components in Hippocampus contribute to its hormone-like, hematopoiesis, antiaging, antifatigue, and Ca 2+ -blocking functions (Ryu et al., 2010a).
Hippocampus has long been one of the essential materials in traditional Chinese medicine (Muthuramalingam et al., 2017). Because of the heavy demands for seahorses in the traditional medicine market, at least an additional 250 tons of seahorses (dry weight) are imported from Vietnam, Thailand, the Philippines, and Malaysia into China each year to produce products like seahorse wine, capsules, pills, and seahorse soup for human consumption (Lin et al., 2008). However, customers are forced to limit their utilization of seahorse products due to their high cost. It has been reported that Hippocampus exert a variety of biological activities, such as antioxidant, neuro-protective, antiarthritis, and antithrombosis functions (Oh et al., 2018). For example, Hippocampus hydrolysates can quench free radicals and chelate metals, supporting their use as antioxidant agents (Muthuramalingam et al., 2017). Seahorse peptides exert hydroxyl free radical and DPPH scavenging activities, and have an antifatigue effect, in mice . The Leu-Glu-Asp-Pro-Phe-Asp-Lys-Asp-Asp-Trp-Asp-Asn-Trp-Lys from seahorses has inhibitory effects on the collagen release in arthritis (Ryu et al., 2010b). A novel antimicrobial peptide, His-Lys-Pro-Leu-Pro, derived from Hippocampus kuda Bleeker, has been identified (Sun et al., 2012). However, the effects of peptides from seahorse hydrolysates (SHP) on ethanol-induced oxidative injury in HepG2/ CYP2E1 cells have not been studied.

| Materials
The two peptides from seahorse (H. kuda Bleeler) hydrolysates peptide (SHP) were taken from previous studies (Ryu et al., 2010a(Ryu et al., , 2010b. The HepG2/CYP2E1 cell line (HepG2 cell transfected with human CYP2E1 cDNA) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The Dulbecco's modified eagle's medium (DMEM) with high glucose, fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), and penicillin/streptomycin were purchased from Invitrogen Corporations. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. SOD and GSH detection kits were obtained from the Beyotime Institute of Biotechnology. GGT and mitochondrial membrane potential (MMP) detection kits were provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein assay kit was provided by Thermo Scientific, USA. The information on the primary and secondary antibodies used in the Western blot is shown in Table 1. The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Xinbosheng Biotechnology Co., Ltd. All other reagents were of analytical grade and were obtained from commercial sources.

| Protective effects of SHP against ethanolinduced oxidative stress
HepG2/CYP2E1 cells were grown in 96-well plates (5 × 10 4 cells/ well), followed by incubation for 24 hr. SHP-1 and SHP-2 (10, 20, 50, and 100 μM) were then added to each well. After 2-hr incubation, 0.75 M ethanol was added to each well; 24 hr later, MTT (1 mg/ml, 100 μl) was added to each well and reacted in a CO 2 incubator for 4 hr. A total of 100 μl of DMSO was added to each well, and the absorbance was detected at 570 nm. Each experiment was repeated three times.

| Measurement of intracellular ROS
The intracellular formation of ROS was assessed as described previously using the oxidation-sensitive dye DCFH-DA as the substrate. HepG2/CYP2E1 cells were grown in 24-well plates (5 × 10 3 cells/well). The cells' treatment was the same as outlined in Section 2.3. Twenty-four hours later, DCFH-DA (10 μM, 300 μl) was added to each well and reacted for 20 min. The cells were washed with PBS three times and observed under a fluorescent inverted microscope.
The cells' treatment was the same as that outlined in Section 2.3.
SOD, GSH, and GGT activities were conducted according to the manufacturer's instructions. In brief, for the SOD assay, the sample well (20 μl of sample), the blank1 well (20 μl of SOD detection buffers), and the blank2 well (40 μl of SOD detection buffers) were added to each well. Then, 160 μl of WST-8/enzyme working fluid and 20 μl of reaction start working fluid (except for the blank2 well) were added to each well, mixed, and incubated for 30 min at 37°C. The absorbance was measured at 450 nm. SOD activity was calculated using the following equation: For the GSH assay, the sample well (10 μl of sample) and the blank well (10 μl of a protein removal reagent M solution) were added to each well. Then, 150 μl of the total glutathione test solution was added. After incubation for 5 min at 25°C, NADPH (0.5 mg/ml, 50 μl) was added to each well and mixed. The absorbance was measured at 412 nm.
For the GGT assay, cell culture supernatants were collected and centrifuged. The sample well (50 μl of sample) and blank well (50 μl of ddH 2 O) were added to each well, respectively. Next, 1 ml of reagent 1 was added and reacted (37°C, 30 min). Reagent 2 was then added to each well, and reagent 1 was added to the blank well. After reacting for 5 min, the absorbance was measured at 410 nm. GGT activity was calculated using the following equation:

| Western blot analysis
HepG2/CYP2E1 cells were cultured in 6-well plates (5 × 10 5 cells/ ml). After treatment, the cells were washed three times with PBS and lysed with RIPA containing 1% phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice. Lysates were centrifuged (4°C, 6,037.2(xg), 20 min), and supernatant protein concentrations were determined with a BCA assay. Equal amounts (25 μg) of protein from each sample and a known molecular weight marker were loaded onto 10%, 12%, and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and the proteins were separated by electrophoresis. The gels were then transferred onto nitrocellulose (NC) membranes. NC membranes were incubated with 5% skimmed milk dissolved in TBST for 4 hr. Then, the membranes were incubated with primary antibodies overnight at 4°C. After washing with TBST three times, the membranes were incubated with appropriate secondary antibodies for 2 hr and washed with TBST. The bands were visualized with an ECL system, and the band densities were optically scanned with ImageJ software.
After treatment, the cells were fixed with 4% paraformaldehyde (4°C, 30 min) and washed with PBS three times. The cells were then permeabilized using 0.2% Triton X-100 for 10 min on ice and blocked in 5% BSA for 1 hr. Then, the cells were incubated with a p65 antibody dis-

| Alkaline comet assay
HepG2/CYP2E1 cells were cultured in 6-well plates. After treatment, the supernatants were removed. The cells were rinsed three times with DMEM, detached by 0.25% trypsin containing EDTA, and suspended in PBS (1 × 10 5 cells/ml). Following the previous description , the first layer gel was prepared with a 0.8% normal
After treatment, cell supernatants were collected. The TNF-α contents were determined by ELISA, which was operated according to the manufacturer's protocol.

| Molecular docking
To further verify the interaction between SHP-1 or SHP-2 and SOD, bcl-2, and bax, molecular docking was performed using C The types of interactions between the docked proteins and the ligand were analyzed with molecular docking.

| Statistical analysis
All experiments were performed in triplicate. Data were expressed as the mean ± SD (standard deviation) and were analyzed using the GraphPad Prism 5.0 software (GraphPad Prism Software Inc.).
Statistical significances were determined using a one-way ANOVA followed by Dunnett's Multiple Comparison Test. Differences were considered to be significant when p < .05.

| Effects of SHP on HepG2/CYP2E1 cells
The cytotoxicity of ethanol, SHP-1, and SHP-2 in HepG2 cells was evaluated by MTT assay. As shown in Figure 1a, the cell viability was decreased by approximately 50% in 0.75 M ethanol.
Compared with the blank group, SHP-1 and SHP-2 (10, 20, 50, and 100 μM) showed no cytotoxicity (Figure 1b,c). The results in Figure 1d,e indicate that SHP-1 or SHP-2 increased cell viability in a concentration-dependent manner in comparison with the control group.

| Effects of SHP on intracellular ROS and SOD, GSH, and GGT
Exposure to ethanol in HepG2/CYP2E1 cells led to ROS generation.
It can be seen from Figure 2a,b that ethanol induced a significant increase in intracellular ROS generation compared with the blank group. However, SHP-1 and SHP-2 markedly attenuated the production of ROS (as seen by observing the fluorescence density), with respect to the control group. Moreover, as depicted in Figure 2c-h, the activities of SOD and GSH were decreased, while the activity of GGT increased after only ethanol treatment in HepG2/CYP2E1 cells.
Nonetheless, the administration of SHP-1 or SHP-2 for 2 hr significantly enhanced SOD and GSH activities and reduced the activity of GGT. Figure 2i,j consistently showed that expressions of SOD and GSH were down-regulated and the level of GGT was increased (but was down-regulated in the control group). SOD and GSH levels were markedly up-regulated, whereas the expression of GGT was significantly down-regulated when HepG2/CYP2E1 cells were pretreated with SHP-1 or SHP-2.

| Effects of SHP on the expressions of proteins in apoptosis
The levels of apoptosis factors were evaluated using Western blot. As depicted in Figure

| Effects of SHP on MMP and DNA damage
JC-1, a fluorescence probe, has been widely used to detect MMP.

| Effects of SHP on the expressions of Nrf2, Keap1, and HO-1
As described in Figure 4a

| Effects of SHP on the Akt, NF-κB, and MAPK signaling pathways
As depicted in Figure 4c,d, the levels of p-Akt, p-IκB-α, and p-p65 were up-regulated in the control group, whereas SHP-1 or SHP-2 reduced the expressions of p-Akt, p-IκB-α, and p-p65. The levels

| Immunofluorescence analysis
As shown in Figure 4e,f, the fluorescence intensity of p65 increased in the cytosol of the ethanol-treated cells compared with the blank group. Nevertheless, SHP-1 or SHP-2 treatment reduced the fluorescence of p65 in the cytoplasm, which further demonstrated that SHP can suppress the nuclear translocation of p65.

| D ISCUSS I ON
The toxic effect of ethanol in the liver has been widely studied.
Human hepatocellular carcinoma (HepG2) cells are known to metabolize ethanol nonoxidatively to fatty acid ethyl esters (FAEEs). Also, due to their many genotypic and phenotypic similarities to human hepatocytes, HepG2 cells, such as hepatic alcohol dehydrogenase (ADH) and CYP2E1, are often used for a variety of drug metabolism and toxicity studies (Kang, Qian, Ryu, Karadeniz, et al., 2012). In the present study, HepG2/CYP2E1 cells were treated with ethanol to cause oxidative stress-induced injury and to evaluate the protective effects of SHP-1 and SHP-2. In general, cell viability represents the toxicity degree of toxicants, which is the most important index in a cytotoxicity test . The MTT assay showed that ethanol treatment decreased cell viability (Figure 1a), which agrees with the previous study (Neuman et al., 1993). SHP-1 or SHP-2 (10-100 μM) presented no toxicity in HepG2/CYP2E1 cells and dose-dependently enhanced cell viability compared with the group treated only with ethanol (Figure 1b-e). These results suggest that SHP-1 and SHP-2 can protect HepG2/CYP2E1 cells from oxidative damage. However, the mechanism remains unclear.
Alcohol has been shown to promote liver injury through an increased formation of ROS (Chandrasekaran et al., 2012). ROS play a complex role in ALD. Low levels of ROS normally stimulate cell proliferation, while excessive ROS promote irreparable cell damage leading to cell death (Rodenak-Kladniew et al., 2018). In this study, pretreatment with SHP-1 or SHP-2 for 2 hr reduced the production of ROS in HepG2/CYP2E1 cells (Figure 2a,b). The hepatocellular antioxidant defense system mainly includes SOD and GSH, which play a critical role in regulating cellular ROS to maintain a dynamic balance of ROS production and elimination . As mentioned previously, SOD converts superoxide radicals to hydrogen peroxide and oxygen (Zhuang et al., 2017).
GSH, a polypeptide antioxidant, is mainly created and metabolized in the liver and plays an important role in protecting against injuries by scavenging ROS in several tissues and cell lines (Yarahmadi et al., 2017). The decrease in GSH levels has been suggested as one of the primary mechanisms of t-BHP-induced toxicity in liver cells (Kang, Qian, Ryu, Karadeniz, et al., 2012). GGT levels increase in response to an exposure to a variety of drugs and alcohol, contributing to the diagnosis of ALD . This may be mediated via oxidative stress, with resultant reductions in GSH levels. The previous report showed that the treatment of seahorse H. abdominalia hydrolysates by alcalase (SHAH) protected HUVECs against oxidative stress-mediated injury (Oh et al., 2018). It was observed that SHP-1 and SHP-2 increased the activities of SOD and GSH and decreased the activity of GGT in Figure 2c-h. Meanwhile, the expressions of SOD and GSH were up-regulated, and the level of GGT was down-regulated  High levels of ROS trigger tumor cell apoptosis (Dasgupta et al., 2019). Apoptosis is a process regulated by a series of enzymes and genes under physiological or pathological conditions . It is characterized by specific morphological changes, such as a condensation of chromatin, a loss of microvilli, blebbing formation, and the appearance of apoptotic bodies . Apoptosis may be activated by an intrinsic or extrinsic pathway. Apoptosis is regulated by various molecules, such as the bcl-2 family and cysteinyl aspartate-specific proteinase (caspase) family proteins Ye et al., 2016). The Bcl-2 family includes the antiapoptosis protein, bcl-2, and the proapoptosis protein, bax. The change in the expression ratio of bax and bcl-2 determines F I G U R E 5 (Continued) whether apoptosis occurs (Wu et al., 2015). Caspases play essential roles in regulating the apoptosis induced by oxidative stress.
Caspase-3 is an important effector in the apoptotic process. Cleavedcaspase-3 is the least common of the apoptotic cells. During the process of induced apoptosis, activation of the initiator caspase-8 can transmit death signals through the direct activation of the effector caspase-3, and caspase-9 is an initiator of caspase-3 in the mitochondria-dependent pathway (Hwang et al., 2006;Li et al., 2015).
Therefore, cleaved-caspase-3/-8/-9 can be used as a reliable indicator to determine the severity of apoptosis Li, Lin, et al., 2017). In the current study, compared with the blank group, ethanol increased the expression of bax and c-caspase-3/-8/-9 but reduced the expression of bcl-2. In addition, SHP-1 and SHP-2 could markedly induce a decrease in bax and c-caspase-3/-8/-9 levels and an increase in bcl-2 expression compared with the control group ( Figure 3a,b). In short, SHP inhibits caspase-dependent apoptosis.
The mitochondria are responsible for most ATP production in the cell (Loperena & Harrison, 2017). Excessive ROS may result in changes of MMP and cause apoptosis (Rodenak-Kladniew et al., 2018). In this study, ethanol treatment for 24 hr reduced the MMP, whereas SHP-1 and SHP-2 prevented the loss of MMP ( Figure 3c,d). These results reveal that a decrease in MMP is likely involved in ethanol-induced apoptosis.
DNA fragmentation is a hallmark of apoptosis (Han et al., 2015).
A single-cell gel electrophoresis (SCGE) assay, also known as comet assay, has been widely used to detect DNA damage (Bai et al., 2015).
A comet assay is convenient for quantitatively studying concentration-response relationships in vitro (Zhang et al., 2011). Furthermore, the length of the comet tail represents the extent of DNA damage (Ghazi et al., 2017). As demonstrated in Figure 3e,f, a significant increase in the length of the comet tail was observed in the HepG2/ CYP2E1 cells exposed to ethanol. The length of the comet tails decreased with increasing concentrations of SHP-1 and SHP-2 compared with the control group. The results indicate that SHP reduced DNA damage.
NF-E2-related factor 2 (Nrf2), a transcription factor, regulates important antioxidant proteins, including heme oxygenase-1 (HO-1; Roh et al., 2018). Nrf2 exists in the cytoplasm by combining with the kelch-like ECH-associated protein 1 (keap1) under normal circumstances (Kašuba et al., 2017). In an oxidative stress environment, keap1's active site cysteine residues are oxidized, preventing keap1 from interacting with Nrf2 . Subsequently, Nrf2 binds to the antioxidant response element (ARE) to suppress oxidative stress . Furthermore, HO-1 may confer cytoprotection and preserve anti-inflammatory, antiproliferative, and antiapoptotic functions by regulating oxidative stress (Schipper et al., 2019). In this study, the expressions of Nrf2 and HO-1 were up-regulated after pretreatment with SHP-1 and SHP-2 compared with the control group (Figure 4a,b). The results suggested that SHP could protect HepG2 cells against ethanol-induced oxidative damage via the activation of Nrf2/HO-1, which is consistent with the previous study Li, Lin, et al., 2017).
A series of studies have demonstrated that the Akt signaling pathway is associated with apoptosis in various cell types (Wang et al., 2014). Akt can also accelerate the activation of downstream nuclear factor-κB (NF-κB) and then regulate the apoptosis-related kinase, bax/bcl-2 . NF-κB is involved in proliferation, differentiation, and apoptosis. In most cell types, NF-κB is in the cytoplasm bound to IκB-α, which is its inhibitory protein. IκB-α may induce phosphorylation and degradation via the trimeric IκB kinase (IKK). The degradation of IκB-α can free NF-κB to enter the cell's nucleus and activate transcription of the target genes . In this study, Figure 4c-f shows that SHP had an antiapoptosis effect through inhibition of the Akt/NF-κB pathways and the nuclear translocation of p65.
Apoptosis can be activated by signaling of mitogen-activated protein kinases (MAPK; Wang et al., 2017). MAPKs contain extracellular signal-related kinases (ERKs), stress-activated p38 kinases, and c-jun NH2-terminal kinases (JNKs; Li, Lin, et al., 2017 (Zhang et al., 2018). In this study, Figure 4g Antioxidant capacity is related to the molecular size of the peptides, their amino acid composition, sequence, structure, and hydrophobic character. Hydrophobic amino acid residues (Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, and Gly) have an antioxidant capacity because they can quench free radicals due to the abundance of their electrons (Coelho et al., 2019). Moreover, Gly residue can make the peptide skeleton more flexible, and its single hydrogen atom can serve as a proton-donating to neutralize free radicals (Yang et al., 2019). In this study, SHP-1 contained hydrophobic amino acids residues (Ala, Val, and Ile), and SHP-2 contained hydrophobic amino acids residues (Ala, Gly, and Pro), which contributed to their antioxidant capacity. Aromatic amino acid residues (Phe and Tyr) can donate protons to electron-deficient radicals to keep their ROS stable and enhance the potency of their radical scavenging capacity (Yang et al., 2019). Peptides containing amino acids with a phenolic hydroxyl group are efficient radical scavengers (Coelho et al., 2019). Furthermore, polar amino acid residues (Glu, Asp, and Lys) played a critical role in antioxidant activity, including metal ion chelating and hydroxyl radical scavenging activities. Therefore, polar amino acid residues, including Asp and Lys in SHP-1 and Glu in SHP-2 could play a critical role in antioxidant capacity. According to the above analysis and the results obtained from this study, the protective effects of SHP-1 on ethanol-induced oxidative stress in HepG2/CYP2E1 cells may be better than those of SHP-2 from the aspect of an antioxidant. Based on the Western blot results and the molecular docking study, compared with SHP-1, SHP-2 may have a more obvious antiapoptosis effect.

| CON CLUS IONS
In conclusion, this study demonstrated that ethanol can cause oxidative damage and apoptosis to HepG2/CYP2E1 cells. SHP-1 and SHP-2 decreased the production of ROS, the activity of GGT, and TNF-α level. They also enhanced the activities of SOD and GSH and the MMP and reduced DNA damage. The protective effects of two SHPs against ethanol-induced oxidative stress in HepG2 cells may be associated with the Nrf2/HO-1, Akt/NF-κB, and MAPK pathways. The administration of SHP protected cells against oxidative damage through antioxidant and antiapoptosis in two ways. These novel and interesting findings suggest that SHP could be beneficial for the prevention and treatment of ALD.

ACK N OWLED G M ENTS
The research was funded by 2020 Shenzhen International Scientific and Technological Cooperation R&D Project and the Natural Science Foundation of Guangdong Province (2020A1515011075).

The supported by Development Project about Marine Economy
Demonstration of Zhanjiang City (XM-202008-01B1) and Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang, ZJW-2019-07).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.