Small-Molecule Peptides from Lactiplantibacillus plantarum SCS2 Attenuate H 2 O 2 -Induced Oxidative Damage in INS-1 Cells via Regulating the Keap1-Nrf2 Signaling Pathway

Objective . Tis study aimed to delve into the antioxidant potential of small-molecule peptides derived from Lactiplantibacillus plantarum SCS2 ( L . plantarum SCS2), targeting hydrogen peroxide-(H 2 O 2 -) induced rat insulinoma (INS-1) cells. Methods . INS-1cells were pretreated with small-molecule peptides of distinct molecular weights, specifcally P1 ( ≤ 1kDa), P2 (1–5kDa), and P3 (5–10 kDa), which were isolated from L. plantarum SCS2, followed by H 2 O 2 to induce oxidative damage in INS-1cells. Te oxidative status of the cells was assessed by measuring reactive oxygen species (ROS), malondialdehyde (MDA) concentrations, and antioxidant enzyme activities. In addition, the expression levels of proteins and genes associated with the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway were examined to elucidate the underlying antioxidative mechanisms of the small-molecule peptides from L. plantarum SCS2. Results . Te cellular activity and the activities of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, were higher after pretreatment with P1 than those after pretreatment with P2 and P3, which led to the suppression of ROS and MDA production. In addition, P1 upregulated mRNA and protein expression of Nrf2 and heme oxygenase-1, signifcantly decreased mRNA and protein expression of Keap1, and promoted the entry of Nrf2 into the nucleus compared with that in the model group. Conclusion . P1 from L. plantarum SCS2 could activate the Keap1-Nrf2 signaling pathway, thereby ameliorating oxidative damage in INS-1cells.


Introduction
Oxidative stress, characterized by an imbalance in the pro-/ antioxidant systems, is often associated with various health conditions, including cancer, cardiovascular disease, hypertension, and diabetes [1].It is typically linked to increased intracellular levels of reactive oxygen species (ROS), causing potential harm to lipids, DNA, and proteins [2,3].Consequently, avenues to reduce oxidative stress may provide novel insights for disease treatment and prevention.Studies have shown that the resultant ROS activate various signaling pathways upon cellular stimulation via exogenous or endogenous oxidation [4].In normal physiological states, a dimer is formed by Nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) within the cytoplasm.A dissociation event occurs upon the onset of oxidative stress within the cell whereby Nrf2 translocates to the nucleus, thereby binding to the antioxidant response element (ARE) and subsequently facilitating the transcription of certain cytoprotective proteins such as phase II detoxifcation metabolism and detoxifcation genes [5][6][7].Notably, the Keap1-Nrf2/ARE signaling pathway is considered a paramount endogenous antioxidant pathway, serving as a crucial regulator of antioxidant defense mechanisms [8].Given this signifcance, the efective regulation of the Keap1-Nrf2 signaling pathway, reduction of ROS, and enhancement of antioxidant activity are essential in improving oxidative damage in cells [9,10].
Lactic acid bacteria (LAB), frequently used in probiotic formulations, have a long history of their remarkable roles in food safety [11].LAB exhibit advantageous host interactions, which include modulating intestinal microfora balance, reducing cholesterol level, and demonstrating antioxidant properties [12].Hence, LAB are acknowledged as safe and nontoxic potential antioxidants and have emerged as a prominent research hotspot.Currently, research eforts dedicated to understanding the antioxidant impacts of LAB mainly focus on live LAB, heat-inactivated LAB, LABproduced peptides from fermentation processes, and LAB extracellular polysaccharides.It has been reported that Lactobacillus strains TSP05, TSF331, and TSR332 have been shown to alleviate alcohol-triggered oxidative stress and infammatory responses in mouse liver, primarily by amplifying the activities of antioxidant enzymes and thereby forestalling liver damage [13].Heat-killed Lactobacillus rhamnosus ATCC 7469 has demonstrated the capacity to increase the expression of Nrf2 in the nucleus and superoxide dismutase (SOD) activity, decrease ROS concentrations, and confer protection against oxidative damage induced by ultraviolet radiation in cells [14].Peptides derived from rice fermentation by Lactiplantibacillus plantarum have been observed to modulate oxidative cell damage via modulating the Keap1-Nrf2 pathway [15].In addition, the exopolysaccharides of Lactobacillus reuteri SJ-47 further contribute to the antioxidant defense system by reducing ROS and malondialdehyde (MDA) levels and upregulating antioxidant enzyme expression, thereby exerting a protective efect on human skin fbroblasts under oxidative stress [16].However, Luo et al. [17] identifed intracellular peptides isolated from Lactobacillus rhamnosus that also demonstrated antioxidant capacity.
Meanwhile, natural peptides are recognized for their multifaceted benefcial efects, including antioxidative, antihypertensive, antiobesity, and anticancer properties [18][19][20].Recently, the peptides extracted from food and biological waste have emerged as a prominent focus in antioxidant research.For instance, researchers have successfully extracted peptides from the by-products of skipjack tuna, which have exhibited outstanding antioxidative activity, thereby helping alleviate the cell damage caused by oxidative stress [21][22][23].Similarly, the peptides extracted from the swim bladders of monkfsh have shown signifcant antioxidative potential, reducing the risk of potential diseases triggered by oxidative stress [24].Furthermore, peptides with bioactive properties derived from dandelions and purifed from watermelon seed hydrolysates have demonstrated antioxidative capabilities [25,26].
Initial investigations into Lactiplantibacillus plantarum SCS2 (L.plantarum SCS2) have unveiled the capacity of its intracellular protein to mitigate oxidative damage in hyperglycemic model mice [27].However, the precise constituents of L. plantarum SCS2 intracellular proteins and whether they exert their antioxidant efects via the Keap1-Nrf2 pathway remain unexplored.Terefore, we hope to delve deeper into the molecular mechanisms by which the intracellular small-molecule peptides of L. plantarum SCS2 with diferent molecular weights alleviate oxidative damage in cells.In conclusion, this study aimed to investigate the potential of small-molecule peptides derived from L. plantarum SCS2 to mitigate cellular oxidative damage and provide a scientifc basis for the subsequent development of foods with antioxidant functions.

Isolation of Peptides from Lactiplantibacillus plantarum
SCS2 by Ultrafltration.L. plantarum SCS2, derived from Sichuan fermented sausage, was stored at the Laboratory Center of Public Health Institute of Chengdu University of Traditional Chinese Medicine.Strains were revived in De Man, Rogosa, and Sharpe broth medium (Biosharp, Beijing, China) at a 0.5% volume ratio, followed by a 24 h incubation at 37 °C.After centrifugation at 10,000 rpm and 4 °C for 10 min, the supernatant was decanted, and the bacteria were washed three times with 0.1 mol/L sterile phosphate-bufered saline (PBS) (Solarbio, Beijing, China).Te bacterial content was collected and resuspended in an adequate volume of PBS.Tis solution was subjected to ultrasonic disruption for 20 min, followed by additional centrifugation at 10,000 rpm and 4 °C for 10 min to obtain the supernatant.Te supernatant was subsequently processed using ultrafltration centrifuge tubes (Pall, USA; Sartorius, Germany; Millipore, USA) equipped with membranes of molecular weight cutof values of 1, 5, and 10 kDa.Te collected permeate fractions, such as ≤1 kDa (referred to as P1), 1-5 kDa (referred to as P2), and 5-10 kDa (referred to as P3), were freeze-dried and stored at −20 °C for further analysis [28].

H 2 O 2 -Induced Oxidative Damage Model.
INS-1 cells in their logarithmic growth phase were seeded at a density of 5 × 10 3 cells per well and cultured in a 96-well plate for a duration of 24 h.INS-1 cells were treated with hydrogen peroxide (H 2 O 2 ) dissolved in RPMI 1640 medium at various concentrations (20-100 μmol/L) in an incubator at 37 °C and in the presence of 5% (v/v) CO 2 for 24 h to establish a model of oxidative injury.Simultaneously, the control group cells were incubated with an equivalent volume of the medium.Te medium was discarded after treatment, and the cells in all groups were washed twice with PBS.Subsequently, 10 μL of the cell counting kit-8 (CCK-8) (Biosharp, Beijing, China) solution was introduced into each well, followed by a further 2 h incubation in the dark.Te absorbance of each well was detected at the wavelength of 450 nm using a microplate reader (BioTek Instruments, Inc., USA) [30].

Viability of INS-1 Cells.
INS-1 cells were cultured in 96well plates (5 × 10 3 cells per well) and incubated overnight in a CO 2 incubator.Te cells were then exposed to diferent concentrations of L. plantarum SCS2 small-molecule peptides with a molecular weight of P1, P2, and P3.Tese were dissolved in RPMI 1640 medium at concentrations ranging from 0.025 to 0.4 mg/mL and administered to the cells for 24 h.Te medium was discarded following treatment, and the cells were rinsed twice with PBS.Each well was then treated with 10 μL of the CCK-8 solution and incubated at 37 °C for 2 h.Ten, the cells were pretreated with diferent concentrations of L. plantarum SCS2 small-molecule peptides, followed by a 24 h exposure to the modeling concentration of H 2 O 2 determined in Section 2.3.Te oxidative damage model was established as described earlier.Te medium was removed after the treatment, the cells were washed twice with PBS, and 10 μL of CCK-8 was added to each well, followed by a 2 h incubation at 37 °C.Te absorbance was assessed at a 450 nm wavelength using an enzyme marker [31].

ROS Level Measurement.
ROS levels were assessed using the 2′,7′-dichlorofuorescin diacetate (DCFH-DA) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) method.Prior to analysis, INS-1 cells were pretreated with small-molecule peptides for 24 h and rinsed three times with PBS.Te cell damage was induced by H 2 O 2 .Te cells were incubated with 10 μmol/L DCFH-DA at 37 °C for 30 min in the dark.Te medium containing the DCFH-DA probe was then discarded, and the cells were rinsed with PBS.After collecting cells, perform analysis using a fow cytometer (excitation wavelength: 488 nm, emission wavelength: 525 nm) (Beckman Coulter, Inc., Brea, CA) [32].

Biochemical Analyses. INS-1 cells were inoculated in six-
well plates and damaged by oxidation with the modeling concentration of H 2 O 2 determined in Section 2.3 after pretreatment with small-molecule peptides for 24 h.Tey were washed with PBS, lysed with cell lysis solution (Solarbio, Beijing, China), and centrifuged at 10,000g and 4 °C for 10 min.Te supernatant was collected.MDA, SOD, catalase (CAT), and glutathione peroxidase (GPx) activities were measured using the corresponding diagnostic kits following the manufacturer's protocols (Shanghai Enzyme-Linked Biotechnology Co., Ltd., Shanghai, China) [31].

Real-Time Quantitative Polymerase Chain Reaction.
Te experiments were performed based on the methods used in previous studies [33].Te extraction of total RNA from the cells was accomplished using a Total Cell RNA Isolation Kit (Chengdu Foregene Biological Technology Co., Ltd., Chengdu, China).After confrmation of RNA concentration and purity, the samples were subsequently reverse transcribed into complementary DNA (cDNA) using a Revert Aid First-Strand cDNA Synthesis Kit (Termo Fisher Scientifc, MA, USA).Te quantitative analysis of gene expression was conducted via real-time quantitative polymerase chain reaction (RT-qPCR).Te primer sequences are presented in Table 1 (Chengdu Foregene Biological Technology Co., Ltd., Chengdu, China).Tis procedure was performed using a SYBR Green PCR Master Mix (Chengdu Foregene Biological Technology Co., Ltd., Chengdu, China) and a two-step RT-PCR system (Jena qTOWER 2.0, DE).
2.9.Statistical Analysis.Te data were expressed as mean-± standard deviation of three replicate trials.Te experimental data were statistically analyzed using IBM Statistical Package for the Social Sciences for Windows, version 26.0 (IBM Corp., NY, USA).Te diferences between multiple groups were analyzed using analysis of variance, and the Student t-test was used to analyze the diferences between the two groups.p value < 0.05 indicated a statistically signifcant diference.

Te Efect of H 2 O 2 Concentration on Viability of INS-1 Cells.
In this study, the cellular model of oxidative damage was mainly used to evaluate the antioxidant capacity of the compounds.Typically, H 2 O 2 serves as an oxygen radical generator for cells due to its ability to penetrate the cell membrane and enter the cell interior, subsequently generating free radicals causing cell or tissue damage [35].Hence, H 2 O 2 was selected as the agent to instigate oxidative damage within cells.We observed a decrease in cell viability corresponding to increased H 2 O 2 concentrations (Figure 1).Furthermore, 60 μmol/L H 2 O 2 treatment of cells for 24 h resulted in a signifcant decrease in cell viability to 0.709 ± 0.0221 (p < 0.05).A further increase to 100 μmol/L H 2 O 2 resulted in a substantial decline in cell viability to 0.085 ± 0.008, and the excessive reduction in cell viability may compromise the reliability and interpretability of experimental results.To induce oxidative damage while maintaining cellular viability and drawing insights from relevant literature [32], the oxidative damage model in INS-1 cells was established with 60 μmol/L H 2 O 2 for 24 h.Tis model was then employed as the foundation for subsequent experiments.

Efects of Small-Molecule Peptides from Lactiplantibacillus plantarum SCS2 on the Viability of INS-1 Cells. Te results in
Figure 2 demonstrated that an increase in the concentrations of small-molecule peptides corresponded to a decrease in cellular activity and an elevation in cytotoxicity.A signifcant diminution in cellular activity was observed when the concentration of small-molecule peptides with molecular weights of P1, P2, and P3 reached 0.2 mg/mL (p < 0.05), registering cellular activities of 0.725 ± 0.026, 0.670 ± 0.052, and 0.573 ± 0.027, respectively.Tis result indicated that small-molecule peptides had cytotoxic efects at concentrations greater than or equal to 0.2 mg/mL.Hence, to avoid cytotoxic efects and ensure the safe application of small-molecule peptides in future, our subsequent research opted for employing small-molecule peptides at concentrations less than 0.2 mg/mL.

Efects of Small-Molecule Peptides from Lactiplantibacillus plantarum SCS2 on the Oxidative Damage of INS-1 Cells.
As depicted in the results of Figure 3, a marked reduction was observed in cell viability in the model group compared with the control group (p < 0.05).Small-molecule peptides with molecular weights of P1, P2, and P3 increased cell activity, and the highest cell viability was achieved when the concentration of small-molecule peptides was 0.05 mg/mL.Furthermore, P1 delivered superior protection against H 2 O 2 -induced oxidative damage in INS-1 cells at all tested concentrations compared with P2 and P3.We concluded that the small-molecule peptides of L. plantarum SCS2 had a protective efect against H 2 O 2 -induced oxidative damage in INS-1 cells when the small-molecule peptide concentration was 0.05 mg/mL.Terefore, subsequent experimental procedures were planned to use a small-molecule peptide concentration of 0.05 mg/mL.Results are expressed as mean ± standard deviation; number of parallel experiments n � 3. 0 indicates normal group, and the numbers in the fgure indicate small-molecule peptides doses of 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, and 0.4 mg/mL, respectively.# indicates signifcant diference compared with the normal group (0 mg/mL peptide) (p < 0.05).4 Journal of Food Biochemistry

Efects of Small-Molecule
Peptides from Lactiplantibacillus plantarum SCS2 on the Levels of ROS and MDA.Based on the results of Figure 4, it could be seen that the model group displayed signifcantly elevated levels of ROS and MDA compared with the control group (p < 0.05), indicating that oxidative damage occurred in the cells after H 2 O 2 treatment, alongside a considerable generation of ROS and MDA.However, pretreatment with small-molecule peptides resulted in a reduction of intracellular ROS and MDA concentrations across all three groups compared with the model group, with the lowest ROS and MDA levels observed in the P1 group relative to the P2 and P3 groups.In summary, the evidence suggested that the small-molecule peptides could protect INS-1 cells from H 2 O 2 -induced damage by inhibiting the cellular production of ROS and MDA, and P1 might have stronger antioxidant efects than the other two small-molecule peptides.

Efects of Small-Molecule Peptides from Lactiplantibacillus plantarum SCS2 on the Levels of Antioxidant Enzymes.
From the results in Figure 5, it could be found that the activities of SOD, CAT, and GPx signifcantly reduced in the model group compared with the normal group (p < 0.05).Relative to the model group, the cells pretreated with smallmolecule peptides demonstrated notably enhanced activities of SOD, CAT, and GPx, with the highest levels observed in the P1 group among the three small-molecule peptide categories (p < 0.05).When excessive ROS are generated, the cells have antioxidant defense systems to counteract the resulting damage [36].SOD, CAT, and GPx, as crucial antioxidant enzymes, facilitate the reduction of oxidative damage within cells.P1 could markedly upregulate the activities of important antioxidant enzymes in oxidatively damaged cells, reducing oxidative damage in INS-1 cells (p < 0.05).In addition, combined with the preceding results on the efect of three small-molecule peptides of L. plantarum SCS2 on cellular oxidative damage, we concluded that P1 exhibited superior antioxidant efects compared with P2 and P3.Te rest of this manuscript discusses our exploration of how P1 ameliorates oxidative damage in INS-1 cells by mediating the Keap1-Nrf2 signaling pathway.

Efects of P1 on the Expression of Keap1-Nrf2
Pathway-Related Genes.Our study explored the change in mRNA expression levels for the key constituents of the Keap1-Nrf2 signaling pathway, such as Nrf2, Keap1, and HO-1.Te results in Figures 6(a)-6(c) showed that a marked decrease in the mRNA expression of Nrf2 and HO-1 and an increased expression of Keap1 mRNA were observed in the model group relative to the control group (p < 0.05).Strikingly, P1 promoted the expression of antioxidant genes Nrf2 and HO-1 and decreased the expression of Keap1 mRNA compared with that in the model group (p < 0.05).Tese fndings indicated that the molecular mechanism of P1 alleviating oxidative damage in INS-1 cells might be through the Keap1-Nrf2 signaling pathway.

Efects of P1 on the Expression of Proteins Related to
Keap1-Nrf2 Signaling Pathway.Te results in Figures 7(a)-7(d) indicated that compared with the control group, the expression of Nrf2 protein and its downstream antioxidant enzyme HO-1 protein signifcantly decreased and Keap1 protein expression signifcantly increased in the model group (p < 0.05).Contrastingly, the pretreatment with P1 markedly increased the expression levels of Nrf2 protein and its downstream antioxidant enzyme HO-1 protein while simultaneously curtailing the expression of Keap1 protein in the oxidatively compromised cells relative to the model group (p < 0.05).Tis suggested that P1 could potentially exert antioxidant efects through modulating the Keap1-Nrf2 signaling pathway.(p < 0.05).Interestingly, the level of Nrf2 nuclear translocation signifcantly increased in oxidatively damaged cells after P1 intervention compared with that in the model group.Te Keap1-Nrf2 pathway is a classical signaling pathway that regulates oxidative stress in the body [37], and the translocation of Nrf2 across the membrane into the nucleus serves as a critical marker of the Keap1-Nrf2 pathway activation.Terefore, the results of this study showed that P1 could signifcantly reduce the expression of Keap1 protein and promote the translocation of Nrf2 to the nucleus, thereby activating downstream antioxidant enzymes and ultimately leading to the alleviation of cellular oxidative damage.

Discussion
ROS react with the double bonds of polyunsaturated fatty acids in the event of cellular oxidative damage, resulting in the formation of lipid hydroperoxides.MDA is a primary product of these lipid hydroperoxides [38].Consequently, ROS and MDA serve as critical markers in detecting oxidative stress.It is imperative to efciently and promptly eliminate excess ROS through the endogenous antioxidant defense systems to mitigate the extent of oxidative damage [39].SOD, CAT, and GPx play indispensable roles as the initial line of defense against oxidative stress [40].However, the body's endogenous antioxidant systems may be inadequate to clear the surplus ROS when the degree of cellular damage surpasses the cell's capacity for adaptive regulation.In such cases, exogenous antioxidants can intervene by stimulating specifc intracellular signaling pathways, thereby efectively reducing ROS levels [41].In this study, the antioxidant capacity of small-molecule peptides from L. plantarum SCS2 was explored by examining the antioxidant enzyme activities of cells and the changes in ROS and MDA levels.Te results of the study showed that ROS and MDA levels signifcantly increased and antioxidant enzyme activities signifcantly decreased in INS-1 cells subjected to H 2 O 2 -stimulated cells.Te small-molecule peptides of L. plantarum SCS2 signifcantly reduced ROS and MDA levels and increased antioxidant enzyme activity in oxidatively damaged cells, with the strongest antioxidant capacity of P1 from L. plantarum SCS2.Tese results aligned with the fndings of another study that concluded that L. plantarumHFY09-fermented soymilk improved the activities of SOD, CAT, and GPx and reduced the MDA level in serum, liver, and brain tissues of mice with D-galactoseinduced oxidative aging [42].Unlike the present study in  which small-molecule peptides in L. plantarum SCS2 cells reduced oxidative damage to cells, the main components of L. plantarumHFY09-fermented soymilk having antioxidant efects were low-molecular-weight active peptides and soy isofavones, which together reduced oxidative stress in mice.
Furthermore, the Keap1-Nrf2 pathway is activated during oxidative stress, and Nrf2 disassociates from Keap1, becomes activated, and translocates into the nucleus, consequently initiating the expression of phase II detoxifcation and antioxidant enzymes [43][44][45].HO-1, predominantly regulated by Nrf2, is considered to play a pivotal role in endogenous defense against oxidative stress [46].Terefore, the present study sought to explore the mechanisms through which small-molecule peptides mitigated oxidative stress by examining the changes in the expression levels of Keap1, Nrf2, and the antioxidant enzyme HO-1 in H 2 O 2 -induced oxidatively damaged cells.An increase in Keap1 expression level and a decrease in Nrf2 and HO-1 expression levels were observed after H 2 O 2 induction of oxidative damage in INS-1 cells.However, the pretreatment with P1 efectively reduced the expression of Keap1 and promoted the expression of Nrf2 and HO-1 in these cells.Te results of a previous study showed that the pretreatment with L. plantarum KSFY06 decreased Keap1 expression and increased the expression of Nrf2 and downstream antioxidant enzymes expression, indicating that L. plantarum KSFY06 could inhibit oxidative stress injury in mice through the Keap1-Nrf2 pathway [47].Tis was consistent with the results of our study.On the contrary, Wang et al. [48] argued that L. plantarumY16-fermented soymilk increased the protein expression levels of Keap1, Nrf2, and HO-1 and then enhanced the activities of SOD, CAT, and other antioxidant enzymes related to the Keap1-Nrf2 signaling pathway, thereby protecting HepG2 cells from oxidative damage caused by 2,2′-azobis (2-amidinopropane) dihydrochloride.Tis was inconsistent with the changes in the protein expression levels of Keap1 in our experiments.It was found that the sustained accumulation of Nrf2 might have adverse consequences such as apoptosis and tumorigenesis [49,50].As cells returned to normalcy, Keap1 levels increased to regulate the ongoing accumulation of intracellular Nrf2 [51].Terefore, the increase in Keap1 protein expression in oxidatively damaged cells by L. plantarumY16-fermented soymilk might be associated with this factor.Furthermore, this study revealed that P1 could promote Nrf2 nuclear translocation in H 2 O 2 -induced oxidative damage in INS-1 cells.Tis result further demonstrated that P1 might reduce oxidative damage in cells by activating the Keap1-Nrf2 pathway.Te results of a study concluded that the pretreatment with L. plantarum ZLP001 did not promote Nrf2 entry into the nucleus in oxidatively damaged cells [52], contradicting our results.Wang et al. [52] suggested that this discrepancy might be due to the activation of Nrf2 by L. plantarum ZLP001 prior to their detection time point.Hence, the cells were capable of resisting oxidative damage without requiring sustained Nrf2 accumulation in the nucleus.
Many recent studies investigated the benefcial efects of postbiotics on the host, such as anti-infammatory, immunomodulatory, antiproliferative, and antioxidant efects [53].Te postbiotic components can be broadly classifed into two categories: benefcial microbial cellular components and metabolites.Te latter include extracellular polysaccharides, intracellular polypeptides, and small active molecules such as short-chain fatty acids [54].Tis study focused on the small-molecule peptides in L. plantarum SCS2, microbial cellular components.We examined their potential to exert antioxidant infuence during oxidative stress via regulating the Keap1-Nrf2 pathway.However, this study only confrmed the antioxidant efect of intracellular small-molecule peptides of L. plantarum SCS2 in vitro.Terefore, further studies are needed to verify the in vivo antioxidant efect and investigate the key substances within P1 of L. plantarum SCS2 that exerted antioxidant efects.Tis will facilitate the development and use of postbiotics in functional foods.

Conclusions
P1 from L. plantarum SCS2 ameliorated oxidative damage in INS-1 cells by promoting Nrf2 entry into the nucleus and upregulating antioxidant enzymes, with this efect being mediated through regulation of the Keap1-Nrf2 signaling pathway.Next, the research will focus on isolating and purifying the key components responsible for the antioxidant properties of P1 derived from L. plantarum SCS2.Subsequently, our goal is to investigate the potential antioxidant efects exhibited by these components in vivo and in vitro.

Figure 1 :Figure 2 :
Figure 1: Cell viability of INS-1 cells in response to various concentrations of H 2 O 2 .Results are expressed as mean ± standard deviation; number of parallel experiments n � 3. 0 indicates normal group, and the other numbers in the fgure indicate the dose of H 2 O 2 .# indicates signifcant diference compared to the normal group (0 μmol/L H 2 O 2 ) (p < 0.05).

Figure 7 :
Figure 7: (a-d) Efects of P1 on the protein expression of total Nrf2, Keap1, and HO-1 in H 2 O 2 -induced INS-1 cells.Results are expressed as mean ± standard deviation; number of parallel experiments n � 3. * indicates signifcant diference compared with the model group (p < 0.05).# indicates signifcant diference compared with the normal group (p < 0.05).

Figure 8 :
Figure 8: (a) Validation results of nuclear and cytoplasmic protein extraction experiment.(b-d) Efects of P1 on the protein expression of Nrf2 in the nucleus and cytoplasm of H 2 O 2 -induced INS-1 cells.Results are expressed as mean ± standard deviation; number of parallel experiments n � 3. * indicates signifcant diference compared with the model group (p < 0.05).# indicates signifcant diference compared with the normal group (p < 0.05).

Table 1 :
Sequence of primers used for real-time quantitative PCR.