Oxidative stress relief to longevity: mulberry-derived anthocyanins enhanced antioxidant defense via Nrf2/SKN-1 activation

Oxidative stress relief to longevity: mulberry-derived anthocyanins enhanced antioxidant defense via Nrf2/SKN-1 activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Oxidative stress relief to longevity: mulberry-derived anthocyanins enhanced antioxidant defense via Nrf2/SKN-1 activation Yuqi LI, Jianglong HE, Xueping JIANG, Haoran HUANG, Yue ZHANG, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7024360/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anthocyanin has been demonstrated that potential health-promoting and anti-aging through antioxidant activities, although the underlying mechanisms remain unclear. In this study, we identified cyanin-3-O-glucoside (61.7%) and cyanin-3-O-rutinoside (25.1%) as the primary components of mulberry fruit anthocyanin extracts (MFAE) using LC-MS/MS. Two experimental models including a hydrogen peroxide-induced hepatocyte oxidative damage and an age-synchronized Caenorhabditis elegans were established. These models were co-cultured with MFAE for varying time periods followed by comprehensive analyses of subcellular structures and comparisons of oxidative stress and senescence-related parameters across treatment groups. The expression levels of Nrf2/SKN-1 pathway-related genes and proteins predicted by molecular docking were investigated. The results demonstrate that MFAE treatment significantly alleviated H2O2-induced cellular oxidative damage, extended lifespan in C. elegans, and improved multiple health indices. Furthermore, MFAE activated the evolutionarily conserved Nrf2/SKN-1 pathway while modulating the expression of various upstream and downstream genes (gst-4, gcs-1, sod-3) and proteins (HO-1, P62, Keap1, BAX). These findings indicate that MFAE exerts anti-aging effects via antioxidant-driven activation of the Nrf2/SKN-1 pathway. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Molecular biology Biological sciences/Plant sciences Anthocyanin Oxidative stress Anti-aging Molecular docking Nrf2/SKN-1 pathway LO2 cells C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Anthocyanins, a class of water-soluble flavonoids found in leaves and fruits of several plants 1 , exert various biological activities, including antioxidant, anticancer, inhibition of inflammation, hypoglycemic, and visual improvement effects 2 . Due to their safety and non-toxicity, anthocyanins are commonly used in food, health supplements, medicine, and cosmetic industries 3 . Therefore, as natural antioxidants, food- and pharmaceutical-grade anthocyanins comprise considerable value in diseases cures and suboptimal health improvement because of oxidative processes. Mulberry fruits attracted attention for their abundant anthocyanin content, and they are commonly consumed in processed beverages and foods such as juices, vines, and jams 3 . In traditional Chinese medicine, mulberries have been used effectively to treat symptoms such as liver and kidney deficiencies, diabetes, and constipation. Modern research also suggests that long-term consumption of edible mulberries may reduce the risk of diabetes and obesity 4 , protect the nervous system 5 , improve cardiovascular health 6 , inhibit inflammation and tumor formation, and promote positive effects on human health 7 . First occurred in 1970, "oxidative stress" refers to an imbalance where oxidants overwhelm antioxidants, disrupting redox signaling and causing molecular damage 8 . Excessive oxidative stress can elicit inflammatory responses and subsequent apoptosis. Furthermore, oxidative stress drives various diseases in organs and tissues 9 . With aging and unhealthy lifestyles and dietary habits, the human body is increasingly exposed to oxidative stress for extended periods, directly or indirectly leading to various chronic afflictions such as cardiovascular diseases, diabetes, obesity, and cancer 10, 11 . Aging is defined as the progressive deterioration of physiological functions, and it manifests through several distinct characteristics: a decline in reproductive capacity, typically culminating in infertility; a reduction in mobility; and atrophy of muscle tissue. At the cellular level, aging is marked by excessive generation of reactive oxygen species (ROS), lipofuscin accumulation, and mitochondrial dysfunction 12 , 13 . Concurrently, as immune system function wanes, the capacity of the organism to withstand stress diminishes, rendering cells and tissues increasingly vulnerable to damage and thereby accelerating the onset and progression of various chronic diseases 14 . Oxidative stress and aging are commonly assumed to be intimately interconnected and mutually reinforcing phenomena, and their effective mitigation is likely instrumental in delaying the onset of age-related changes 15 . Cerro et al. (2025) 16 demonstrated that natural bioactive compounds may serve as a beneficial strategy for promoting healthier aging in postmenopausal women, with mechanisms including enhancing immune function, reducing biological age, improving redox balance, and regulating stress hormones. Natural foods containing active antioxidant components are frequently used as antioxidants to prevent oxidative damage caused by free radicals 17 . The mulberry fruit is a natural source of anthocyanins, and its antioxidant properties were confirmed. Yan et al. (2017) 18 demonstrates that mulberry-derived anthocyanins effectively enhance the cellular antioxidase defense system, showing significant protective effects against hyperglycemia-induced oxidative damage across both in vitro and in vivo experimental models. However, the precise active constituents, underlying mechanisms, and specific molecular targets involved in the anti-aging mechanisms by which mulberry anthocyanins protect against oxidative stress remain unclear. To improve our understanding of the pharmacological pathways underlying therapeutic interventions for disease, researchers are increasingly turning to advanced computational approaches, such as network pharmacology, molecular docking, and molecular dynamics simulations 19 . Molecular docking is used to predict the binding modes and affinity dynamics between small molecules and protein receptors (targets) by computational techniques 20 . To elucidate the mechanistic interplay between mulberry-derived anthocyanins and oxidative stress-mediated aging processes, anthocyanins-derived components from mulberry fruits were identified using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). Two experimental models of H 2 O 2 -induced oxidative damage in LO2 cells and age-synchronized C. elegans were established. The effects of MFAE on cellular structures and the expression levels of antioxidant-related proteins in H 2 O 2 -induced LO2 cells and aging-related parameters in C. elegans were evaluated. Based on molecular docking predictions, the expression levels of Nrf2/SKN-1 pathway-related genes and proteins were investigated using Western blot, immunofluorescence staining, green fluorescent protein (GFP) labeling, and gene editing techniques. Our results provide novel insights and a theoretical foundation to improve the understanding of anthocyanin-mediated anti-aging mechanisms. Materials and methods Materials and reagents Fresh mulberry fruits ( Morus alba L.) were collected from a mulberry plantation at the Sericultural Research Institute of Jiangsu University of Science and Technology. Normal LO2 cells were procured from the American Type Culture Collection (ATCC, VA, USA). Escherichia coli strain OP50 was provided by BioSci (Hangzhou, China). Five C. elegans strains of N2 (wild-type), LD-1 ( skn-1 ::GFP), CF1553 ( sod-3 ::GFP), LD1171 ( gcs-1 ::GFP), CL2166 ( gst-4 ::GFP) and EU1 ( skn-1 mutants) were provided by Caenorhabditis elegans Genetics Centre (MN, USA). Cyanin-3- O -glucoside (C3G) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Thiazole blue (MTT) and Hoechst 33342 were purchased from Shanghai Shentech Co., Ltd. Critical biochemical reagents including RPMI-1640 basal medium (Gibco formulation), RIPA lysate buffer, SDS-PAGE gel preparation kits, and 4´Laemmli sample loading buffer were commercially sourced from Shanghai Solaibao Biotechnology Co., Ltd (Shanghai, China). Oil Red O staining reagent and AB-8 macroporous adsorption resin were commercially acquired from Shanghai YuanYe Biotechnology Co., Ltd. Essential biochemical reagents and detection kits were commercially sourced from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The procurement included, fluorescent probes: DCFH-DA for ROS quantification, DAF-FMDA for NO detection, Lyso-Tracker Red for lysosomal pH monitoring; functional assay kits: JC-1 mitochondrial membrane potential assay kit, catalase activity colorimetric assay kit, WST-8 based total SOD detection system, lipid oxidation (MDA) assay kit, glutathione (GSH/GSSG) quantification kit; immunochemical reagents: RIPA lysis buffer, Cy3-conjugated affiniPure goat anti-rabbit IgG anti-rabbit, complete immunofluorescence staining kit. Monoclonal antibodies, such as Nrf2, β-actin, Keap1, HO-1, and tubulin were purchased from Proteintech Inc. (Wuhan, China). MFAE extraction and compound identification MFAE was prepared following previously work 21 . Following liquid-liquid partitioning of the supernatant with ethyl acetate, the aqueous phase using AB-8 macroporous adsorption resin. Gradient elution was performed with acidified with 70% ethanol (1% formic acid, pH 2.8) at 2 BV/h flow rate, followed by vacuum rotary evaporation to yield anthocyanin-rich fractions. The elution part was vacuum-evaporated and freeze-dried at -80 ℃. Anthocyanins extracts were analyzed with a UHPLC-QTOF-MS/MS system (ExionLC™ AD UPLC coupled to an Applied Biosystems 6500 Triple Quadrupole MS) 22, 23 . High-fidelity spectral data acquisition was conducted via Analyst software (v1.6.3, Sciex, MA, USA), followed by targeted metabolomic quantification employing MultiQuant 3.0.3 (Sciex). Serial dilutions of standard solutions were prepared, and chromatographic peak intensities were quantified for each calibration level. Standard curves were subsequently generated for individual analytes. The integrated peak areas of MFAE samples were interpolated into the linear regression equation derived from the calibration curve, enabling accurate quantification of the target analyte concentrations. LO2 cell culture and cell viability LO2 cells were maintained under standardized culture conditions at 37 ℃ with 5% CO 2 and 95% humidity incubator (Thermo Fisher Scientific, Waltham, MA, USA). A cell culture medium was prepared using RPMI 1640 as the base formulation, supplemented with 10% (v/v) fetal bovine serum, 1% non-essential amino acid mixture, and streptomycin at 100 μg/mL concentration, with routine medium replacement performed every 2–3 days. Cellular metabolic activity was quantified using MTT colorimetric assay. Namely, 20 µL MTT solution (5 mg/mL) was added to each culture followed by incubation for 4 h. After removing the cell medium, 200 µL dimethyl sulfoxide was added to solubilize the formazan crystal. Then the medium was replaced with dimethyl sulfoxide (200 μL) and the absorbance was tested by a microplate reader (TECAN Company, Switzerland) at λ = 570 nm. Establishment of oxidative stress cell model and MFAE treatment To establish an oxidative stress cell model, 2 × 10 3 cells/well LO2 cells were cultured in 96-well plates for 24 h. Then, 100 μL of 0.1, 0.2, 0.4, 1.0, 2.5, or 5.0 mM H 2 O 2 was added, followed by incubation for 2, 4, 6, or 8 h. The oxidative stress cell model was estimated to use the H 2 O 2 concentration and culture time for 50% of cell viability, which was obtained using 1.0 mM H 2 O 2 and incubation for 4 h (Fig. 1A). Different MFAE concentrations (0.2–1.0 mg/mL) were used to pretreat LO2 cells (1 × 10 5 cells/well) for 24 h. After multiple washes of PBS, 100 μL 1.0 mM H 2 O 2 fresh cell medium was replaced. The cells were incubated at 37 ℃ for 4 h for follow-up analytical procedures. The blank control group was a cell medium culture without MFAE or H 2 O 2 , and an H 2 O 2 control group was established using 1.0 mM H 2 O 2 in the cell medium. All treatment groups were incubated at 37 ℃ for 4 h. Assessment of intracellular ROS and reactive nitrogen species (RNS) levels LO2 cells were washed with phosphate-buffered saline (PBS) and added 10 μM/L DCFH-DA or DAF-FMDA for 30 min incubation at 37 ℃. After washing thrice with DMEM, the stained cells were visualized using a fluorescence microscope (Shanghai Changfang Optical Instrument Co., Ltd.) 24 . Hoechst 33342 staining LO2 cells were rinsed with ice-cold PBS twice, dyed with Hoechst 33342 (1 μg/mL) at 37 ℃ for 15 min in the dark. The samples were rinsed with PBS and pictured using a fluorescence microscope 25 . Subcellular localization in LO2 cells treated with MFAE Mitochondrial membrane potential LO2 cells were rinsed using ice-cool PBS, and a mixture of RPMI-1640 medium and JC-1 dye solution was added to a 6-well plate for incubation at 37 ℃ for 20 min in the dark. The medium and dye solution were removed, the cells were further washed using JC-1 staining buffer and the results were photographed with a fluorescence microscope 26 . Lipid droplet fluorescence staining A BODIPY 493/503 fluorescent probe was used to observe the distribution, quantity, and morphology of lipid droplets in LO2 cells. LO2 cells were initially rinsed with PBS and subsequently immersed in 4% paraformaldehyde solution for 10 min fixation period. Following the aspiration of the fixture, two sequential PBS washing steps were carried out. Following PBS treatment, 1 mL of BODIPY 493/503 fluorescence dye working solution lipid droplet solution was applied to the cells for lipid droplet visualization. Cellular samples were maintained at ambient temperature during the 15-min staining protocol. Fluorescence signals were captured using an inverted fluorescence microscopy equipped with a FITC filter set. Lysosome fluorescent staining Lysosomal compartments were specifically labeled using Lyso-Tracker Red fluorescent probe. The LO2 cells were incubated with Lyso-Tracker Red staining working solution at 37 ℃ for 30 min to achieve lysosome staining. After removing the Lyso-Tracker Red staining solution, fresh culture medium was added to the plate. Staining images of cells were obtained using fluorescence microscopy. Anthocyanin antioxidant target protein Nrf2 Assessing interactions of MFAE antioxidant and Nrf2 protein by Molecular docking Following acquisition of stereolithography formatted ligand architectures for anthocyanin derivatives through the PubChem database (Accession CID:44256857), and the tertiary conformation of the Nrf2 protein was obtained from UniProt’s structural repository with PDB ID:5FMV) serving as the crystallographic source 27 . Molecular docking simulations were conducted using AutoDock Vina 1.2.5 28 , with subsequent analysis and visualization of the docking results performed through Discovery Studio 2019 software. Western blotting Proteins were prepared using a lysis buffer, and the lysate was maintained at -20 ℃. BCA Kit (Solarbio) was employed to quantify the protein concentrations, and 10% SDS-PAGE was used to separate the proteins. The wet transfer method was employed to transfer protein samples onto a polyvinylidene difluoride membrane. The membranes were then incubated with non-fat milk (5%) for 4 h, cultured with first antibodies targeting Nrf2, Keap1, BAX, P62, or HO-1 at 4℃ for 12 h, washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and treated with the second antibodies conjugated with horseradish peroxidase for another 4 h at 25℃. All bands were rinsed with TBST, and the results were recorded with chemiluminescence imaging analysis system. Immunofluorescence staining LO2 cells were rinsed with PBS, then fixed using a 4% formaldehyde solution for 10 min following buffer aspiration, and subjected to three thorough PBS washes. The membranes were treated with a protein-based blocking buffer through a 60 min incubation overnight at 4℃ with gentle shaking in Nrf2-targeting primary antibodies. Then, the membranes were subjected to subsequent incubation with Cy3-conjugated anti-rabbit secondary antibodies for 60 min. After three sequential washes with labeled secondary antibody, an aliquot anti-fluorescence-quenching mounting medium containing 4’,6-diamidin-2-phenylindole (DAPI) was added. Cells were visualized and pictured using fluorescence microscopy. Anthocyanins anti-aging in C. elegans C. elegans preparation C. elegans were cultured under axenic conditions at 20 ℃ on conventional nematode growth medium (NGM) agar plates, sustained by a monoxenic diet of viable Escherichia coli OP50 strain as the exclusive nutritional substrate. Age-synchronized C. elegans were obtained by treating the adult worms with a lysis solution (5 M NaOH and 5% NaCl) for 5 min, and L4 stage worms were selected for the next experiment. Age-synchronized adult worms were grown in NGM containing various concentrations of MFAE (0.2, 1.0, and 5 mg/mL), C3G (0.2 mg/mL), and H 2 O 2 (10 mM), with Escherichia coli OP50 pretreated at 65 ℃ for 30 min 29 . To separate adult worms from their progeny, worms were transferred to fresh medium each day. C. elegans lifespan was analyzed as described earlier 30 . Three replicates were used in all experiments. ROS production in C. elegans The C. elegans oxidative stress experiment was executed as described previously with slight modifications 31 . Quantification of intracellular ROS level in C. elegans was performed via fluorescence microscopy using an H2DCF-DA probe following established methods by Zhu et al. (2015) 32 . After anesthetizing with 10 mM levamisole, worms were examined using a fluorescence inversion microscope (Ex/Em: 485/530 nm). Quantitative evaluation of fluorescence signals was assessed using Image-J software. Key gene expression assessed by green fluorescent protein (GFP) label The four GFP labeled transgenic strains LD-1 ( skn-1 ::GFP), CF1553 ( sod-3 ::GFP), LD1171 ( gcs-1 ::GFP) and CL2166 ( gst-4 ::GFP) were used to assess key gene expressions. Age-synchronized L4 worms of the transgenic strains were reared on NGM plates with different treatments at 20 ℃ for 6 days. After anesthetizing with 10 mM levamisole, worms were placed on 2% agar glass slides and photographed using a fluorescence inversion microscope as described above. Statistical analysis Quantitative data derived from three independent experiment replicates were expressed as arithmetic means ± standard deviation (n=3) and subjected to parametric analysis via one-way analysis of variance (ANOVA) implemented in GraphPad Prism software (9.0). Statistical significance was defined as p < 0.05 following Bonferroni correction for multiple comparisons. Results Anthocyanin composition of MFAE Individual anthocyanin compounds identified by UPLC-ESI-MS/MS are listed in Table 1, and the ion mass spectrum of MFAE is shown in Fig. S1. A total of 69 compounds were identified, The proportions of various types of anthocyanins in the total anthocyanins are as follows. Including cyanidin-3- O -glucoside (61.73%), cyanidin-3- O -rutinoside (25.11%), pelargonidin-3- O -coumaroyl-5- O -galactoside (2.34%), pelargonidin-3- O -rutinoside (2.10%), pelargonidin-3- O -glucoside (2.08%), delphinidin-3- O -rutinoside-5- O -glucoside (1.78%), and delphinidin-3- O -(coumaroyl) glucoside-5- O -galactoside (1.11%). H 2 O 2 -induced oxidative stress and protective effects of MFAE To construct an oxidative stress cell model, LO2 cells were used and treated with H 2 O 2 at different concentrations (0.1–5.0 mM) for 2, 4, 6, and 8 h, respectively. The cellular viability of LO2 cells decreased gradually with increasing H 2 O 2 concentrations. Low-concentration (0.1–1.0 mM) of H 2 O 2 , the cellular viability shows a time-dependent and recovery gradually later. But, at high-concentrations (2.5–5.0 mM) of H 2 O 2 , cells lose their viability to repair themselves (Fig. 1A). The IC 50 value was determined under culturing conditions of 1.0 mM H 2 O 2 for 4h, which was used as an oxidative stress cell model to identify the protective effects of MFAE. To investigate the effects of MFAE on normal cells, LO2 cells were treated with different concentrations of MFAE (0.1 - 2.0 mg/mL) for 24 h. MFAE at 0.3 – 2.0 mg/mL positively affected LO2 cell viability (Fig. 1B). Therefore, MFAE concentration of 0.2 mg/mL and 1.0 mg/mL were selected for not effect significantly and effect significantly on cell viability, respectively. To examine the effects of MFAE on the oxidative stress model cells, LO2 cells were preincubated with MFAE (0.2 and 1.0 mg/mL) and C3G (0.2 mg/mL, as positive control) for 24 h and then stimulated with 1.0 mM H 2 O 2 during incubation for an additional 4 h. Pharmacological preconditioning with 1.0 mg/mL MFAE significantly enhanced cellular viability from a baseline of 37.8% to 47.6% under oxidative stress conditions. The cell survival rate was 64.1% in the C3G-positive control (Fig. 1C). This indicated that MFAE improved the survival rate of cells after oxidative damage and reduced the effect of oxidative damage on cells. Reduction of ROS and RNS levels in H 2 O 2 -injured LO2 cells by MFAE DCFH-DA and DAF-FMDA fluorescent probes were used to stain LO2 cells and investigate the effects of MFAE on ROS and RNS levels. LO2 cells were preincubated with MFAE (0.2 and 1.0 mg/mL), C3G (0.2 mg/mL), and oxidative stress inhibitor (N-acetylcysteine [NAC], 100 mM/L) were used as a positive control for 24 h and then treated with H 2 O 2 (1 mM) for an additional 4 h. The fluorescence in the blank control group (normal cells) was relatively weak after fluorescence staining, and the strongest green fluorescence was visualized in the H 2 O 2 -treated positive control group. The MFAE, C3G, and NAC intervention groups demonstrated significantly reduced fluorescence intensity relative to the H 2 O 2 control group (p < 0.05), indicative of diminished ROS accumulation. Quantitative analysis for fluorescence intensity showed similar trends (Figs. 2A-B). Overproduction of ROS and RNS resulted in decreased GSH, SOD, and CAT activity, and increased MDA content (Fig. S2), which indicated that MFAE reduced the levels of ROS and RNS caused by oxidative stress, thereby reducing cell oxidative damage. Alleviat ion of lysosome and nuclear damage in H 2 O 2 -injured LO2 cells by MFAE LO2 cell structure morphology induced by H 2 O 2 in the different treatment groups was observed after Hoechst 33342 fluorescent probe staining (Fig. 3). The nucleus appeared light blue with uniform chromatin distribution in the blank control, whereas in the H 2 O 2 -treated group, the nucleus was of a brighter blue, denser, and more shrunken. The nuclear blue color was weaker in the H 2 O 2 -induced group compared with that in the MFAE, C3G, and NAC groups, and thus MFAE reduced nuclear damage induced by H 2 O 2 (Fig. 3A). Changes in mitochondrial membrane potential induced by JC-1 fluorescence staining occurred in different groups (Fig. 3B). The mitochondria showed strong bright red fluorescence in the blank control and bright red fluorescence in the MFAE, C3G, and NAC groups; fluorescence in the H 2 O 2 group was green. Thus, MFAE effectively reduced the depolarization of the mitochondrial membrane potential caused by H 2 O 2 . Fig. 3C shows distinct green lipid droplets in the control group, and loss of lipid droplet structure attributable to oxidative damage in the H 2 O 2 -induced group. Contrastingly, the extent of lipid droplet damage was prominently mitigated in the C3G, NAC, and MFAE groups when compared with that in the H 2 O 2 -induced group. MFAE thus alleviated intracellular lipid droplet damage caused by H 2 O 2 and protected the lipid metabolism function of cells. To understand the interactions between oxidative stress and lysosomes, LO2 cells were stained with Lyso-Tracker Red. Strong and weak red fluorescence was observed in the H 2 O 2 and MFAE, C3G, and NAC groups, respectively (Fig. 3D); almost no red fluorescence was observed in the blank control. These results suggested that MFAE alleviated H 2 O 2 -induced lysosome damage and inhibited cell apoptosis. Interactions of MFAE antioxidant and Nrf2 protein as assessed by molecular docking Molecular docking is a computational approach employed to elucidate protein-ligand interactions and is extensively used in drug discovery and the mechanistic exploration of natural compounds 33 . The binding affinity between the 25 anthocyanin compounds selected in MFAE with the Nrf2 was assessed using AutoDock Vina 1.2.2. The docking scores ranged from -7.44 kcal/mol to -16.82 kcal/mol among those compounds (Table 2). The most abundant compounds cyanidin-3- O -glucoside and cyanidin-3- O -rutinoside in MFAE exhibited binding energies with Nrf2 of -14.64 and -15.47 kcal/mol, respectively. Eight hydrogen bonds were formed by cyanidin-3- O -glucoside with the amino acid residues LYS (A:719), ARG (A:686), THR (A:690), GLU (A:738, A:441), and SER (A: 689) in Nrf2, and nine hydrogen bonds were formed by cyanidin-3- O -rutinoside with the amino acid residues ASN (A:720, A:499), LYS(A:719), GLU (A:487, A:490, A:738), and TYR(A:489) in Nrf2 (Fig. 4). Cyanidin-3- O -glucoside and cyanidin-3- O -rutinoside exhibited stronger binding affinity to Nrf2 compared to other components. This indicated that Nrf2 is a potential target for MFAE anti-oxidation. Activation of Nrf2 pathway-related proteins in H 2 O 2 -injured LO2 cells by MFAE To investigate the oxidative stress-mediated effect of the Nrf2 signaling pathway, LO2 cells exposed to H 2 O 2 -induced oxidative stress were quantitatively analyzed for expression profiles of Nrf2 and its downstream HO1 protein. The expression of HO-1 and Nrf2 proteins in LO2 cells with H 2 O 2 -induced oxidative stress depended on the H 2 O 2 dosage (Fig. 5). High expression of the two proteins occurred at H 2 O 2 dosages of 0.2 mM and 0.3 mM, confirming that these concentrations can induce high expression of HO-1 and Nrf2 in LO2 cells. To explore whether MFAE alleviated cellular oxidative damage and apoptosis by activating the Nrf2 pathway, ML385 (i.e., an Nrf2 inhibitor) was used as a negative control. Western bolt analysis was performed to quantify the protein expression profiles of key Nrf2 pathway components, including of BAX, P62, Nrf2, Keap1, and HO-1. Nrf2 expression was significantly downregulated in response to H 2 O 2 or ML385-induced stress but was significantly upregulated following pretreatment with MFAE or C3G. HO-1 expression showed a consistent trend with that of Nrf2, whereas Keap1 expression showed an inverse pattern. Additionally, expression of the pro-apoptotic protein BAX was significantly increased when Nrf2 expression was suppressed. P62 expression was upregulated when LO2 cells were stressed by H 2 O 2 , with higher expression induced by MFAE or C3G pretreatment (Fig. 6). The Nrf2 protein in LO2 cells was stained by immunofluorescence to further validate Nrf2 pathway activation by oxidative stress (Fig. 7). Staining with DAPI (i.e., a fluorescent dye strongly binding to DNA) showed a nuclear outline. Fluorescence of the Nrf2 protein was weaker in the control group and was distributed in the cytoplasm. H 2 O 2, MFAE, and C3G groups showed varying degrees of enhancement in red fluorescence and aggregation towards the nucleus (Fig. 7 Merge). The findings indicate that MFAE protects LO2 cells from H 2 O 2 -induced oxidative damage by triggering Nrf2 nuclear translocation and upregulating its expression, thereby activating the Nrf2 antioxidant pathway. Extending of C. elegans lifespan by MFAE To systematically evaluate the longevity-modulating effects of MFAE in C. elegans , age-synchronized N2 wild-type populations were chronically treated with MFAE at graded concentrations (0.2, 1.0, and 5.0 mg/mL) and C3G (0.2 mg/mL) as a reference antioxidant control. Longitudinal survival analysis in Fig. 8A demonstrated significant chronological lifespan extension in C. elegans populations, with 1.0 mg/mL MFAE, 5.0 mg/mL MFAE and 0.2 mg/mL C3G supplementation conferring mean lifespan increases of 4.64% ( p =0.037), 8.35% ( p =0.008), and 8.47% ( p =0.006) respectively, compared with that of the control group. In acute oxidative stress assays, pretreatment with MFAE (1.0 mg/mL and 5.0 mg/mL) and C3G (0.2 mg/mL) prior to H 2 O 2 exposure significantly attenuated mortality rates, yielding 10%, 26.7%, and 33.3% survival rates at the 2 h endpoint (Fig. 8B). Reproductive phenotyping revealed conserved total fecundity across treatment groups but delayed oviposition kinetics in high-dose MFAE (5.0 mg/mL) and C3G cohorts, with peak egg-laying occurring stage, respectively (Fig. S3). This temporal deceleration in reproductive maturation suggests MFAE modulates longevity through developmental retardation of germline-associated aging pathways. For further validation of the above results, skn-1 mutant C. elegans were exposed to MFAE (5.0 mg/mL) or C3G (0.2 mg/mL) to record their lifespans. Fig. 8C shows that the lifespan did not differ among skn-1 mutant (CK), MFAE (5.0 mg/mL) and C3G (0.2 mg/mL) groups, demonstrating that skn-1 gene is the key gene for the effect of MFAE on the lifespan of C. elegans. Mediation of ROS levels in C. elegans by MFAE In LO2 cells, MFAE showed the reduction of ROS and RNS levels and alleviated lysosome and nuclear damage caused by H 2 O 2 oxidative stress. A similar procedure was conducted using C. elegans (N2). Fig. 9A shows that significant attenuation of intracellular ROS accumulation in C. elegans treated with 5.0 mg/mL MFAE and 0.2mg/mL C3G, demonstrating respective fluorescence intensity reduction of 42.3% and 38.9% compared to untreated controls. Dose-response analysis further identified a concentration-dependent ROS modulatory effect of MFAE, with maximal suppression (68.5±5.2 AU) observed at the 5.0 mg/ml dose, paralleling the efficacy of the C3G positive control (71.2±4.8 AU) (Fig. 9C). These findings mechanistically align with the oxidative stress theory of aging, suggesting MFAE-mediated ROS scavenging contributes to delayed senescence phenotypes in nematodes. Controlling of the expression of skn-1 downstream genes sod-3, gcs-1, and gst-4 by MFAE The SKN-1 transcription factor in C. elegans serves as a functional ortholog to mammalian Nrf2, orchestrating the transcriptional activation of sod-3 , gcs-1 , and gst-4 . This evolutionarily conserved regulatory axis mediates oxidative stress resistance by enhancing free radical scavenging capacity and maintaining redox homeostasis through phase Ⅱ detoxification pathways 34 . MFAE facilitated SKN-1 nuclear translocation in the nematode intestine (Fig. 10A), thereby significantly activating the expression of downstream target genes gcs-1 and gst-4 , and oxidative stress-related transcription factor sod-3 (Fig. 10B). Integrating phenotypic data from Fig. 8C, these findings mechanistically establish that MFAE induces prolonged geroprotective effects in C. elegans through pharmacological activation of the evolutionarily conserved the Nrf2/SKN-1 signal axis, as evidenced by coordinated upregulation of downstream antioxidant response elements and stress-resistance biomarkers. Discussion Anthocyanins, a type of flavonoids, exert significant antioxidant properties. To understand the antioxidant mechanism, MFAE was successfully prepared, and its anthocyanin components were characterized using UPLC-ESI-MS/MS. The extract was then evaluated for antioxidant and anti-aging activities. Table 1 shows that six types of anthocyanins were present in mulberry fruit: cyanidin (89.487%), pelargonidin (6.605%), delphinidin (3.703%), peonidin (0.182%), malvidin (0.011%) and petunidin (0.011%). Among all detected anthocyanins, cyanidin-3- O -glucoside dominated at 61.727%, followed by cyanidin-3- O -rutinoside at 25.113%, together comprising the bulk of the anthocyanin profile. Approximately 99.7% of all aglycones in mulberry fruits were cyanidin and delphinidin. Kong et al. (2003) 35 reported that natural anthocyanins originate from six major anthocyanidins: cyanidin, pelargonidin, malvidin, peonidin, delphinidin, and petunidin. The major anthocyanin found in mulberry fruit is cyanidin-3- O -glucoside accounting for approximately 53.94%–78.23%, and anthocyanins in plants vary among plant varieties and tissues 36 . Oxidative stress refers to the state of cell damage caused by oxidants. The antioxidant defense system becomes overwhelmed and unable to effectively eliminate free radicals and their metabolic byproducts. Natural antioxidants inhibit the occurrence and development of oxidative stress, thereby reducing the effect of free radicals on cells. Zhao et al. (2023) 37 demonstrated that plant-derived anthocyanins exhibit potent antiproliferative effects on cancer cells and suppress tumor growth. Anthocyanin antioxidative action subsequently hinders malignant cell initiation and proliferation pathways. Here, an H 2 O 2 -induced oxidative stress LO2 model was constructed. Several H 2 O 2 -induced oxidative stress cell models were established using mammary epithelial 38 , human lens epithelial 39 , fibroblast 40 , and gastric epithelial 41 cells. Fig. 1A shows that LO2 cells were protected against the injury at low H 2 O 2 doses with increasing culture time. The survival rate of H 2 O 2 -injured LO2 cells increased significantly after pretreatment with MFAE. Further, MFAE significantly improved cell viability and mitigated the detrimental effects of H 2 O 2 -induced oxidative stress. LO2 cells were pretreated with 0.2 mg/mL MFAE for 24 h prior to exposure to 1 mM H 2 O 2 for 4 h to establish the oxidative stress model (Fig. 1B). MFAE improved the survival rate of cells after oxidative damage and reduced the effect of oxidative damage on cells (Fig. 1C). Generally, ROS and RNS are signaling molecules inevitably produced during aerobic metabolic processes in liver cells and regulate several physiological activities. Under healthy conditions, the production and clearance of two free radicals are maintained in a dynamic balance within cells. However, when this balance is disrupted by oxidative stress, it can lead to structural and functional cellular damage, such as protein degradation, lipid peroxidation, and DNA damage, ultimately resulting in programmed cell death. Our results showed that LO2 cells injured by H 2 O 2 stress were recovered by MFAE pretreatment (Fig.1B-C, Fig.2, and Fig.3). Plant-derived phytochemicals, such as flavonoids 42 , polysaccharides 43 , and peptides 44 are effective hepatoprotective agents that counteract oxidative damage by scavenging ROS. Molecular docking represents a computational approach that simulates and analyzes the interaction between ligand molecules and target protein, predicting their most favorable binding orientations and association strengths. The molecular binding affinity, quantified as binding energy (kcal/mol), demonstrates an inverse correlation with numerical values: lower binding energies indicate stronger thermodynamic stabilization of ligand-target complexes, whereas higher values reflect weaker intermolecular interactions. The data in Table 2 show that cyanidin-3- O -glucoside (-14.64 Kcal/mol) and cyanidin-3- O -rutinside (-15.47 Kcal/mol) comprised lower binding energies, indicating the high affinity to Nrf2 protein which is intricately linked to oxidative stress and anti-aging processes. The molecular dynamics simulations revealed the detailed binding mechanisms of the ligands with their target protein receptors 45 . Fig. 4 shows key antioxidant constituents in MFAE interactions with the Nrf2 target protein via a network of hydrogen bonds and hydrophobic groups. Through integrated network pharmacology and molecular docking approaches, eight pivotal antioxidant compounds in red wine were identified to interact with multiple metabolic pathways, notably the PI3K/AKT signaling axis 46 . Free radicals cause irreparable structural damage (protein, lipids, DNA, organelles), activating apoptosis and programmed cell death 47 . Nrf2 regulates cellular redox status and oxidative stress sensing, processes implicated in lifespan extension 48, 49 . Results of the cell experiments showed that Nrf2 expression in cells increased and decreased under low and high oxidative stresses, respectively (Fig. 5). This trend was consistent with that of the effects of the H 2 O 2 concentration on cell viability. The Nrf2 inhibitor ML385 was employed to verify that MFAE’s antioxidant protection operates specifically through the Nrf2 pathway. MFAE upregulated the expression of the multifunctional protein P62 and Nrf2 downstream regulated protein HO-1 and downregulated expression of BAX, a pro-apoptotic protein, and Keap1, the primary negative regulator of the Nrf2 antioxidant pathway, thereby inhibiting apoptosis. HO-1 is a protein downstream of Nrf2, and its expression pattern is similar to that of Nrf2; furthermore, in the current study, changes in Keap1 protein expression showed the opposite pattern to those of Nrf2 protein (Fig. 6). Oxidative stress induces conformational changes in Keap1, leading to Nrf2 dissociation and stabilization. The liberated Nrf2 accumulates in the nucleus where it dimerizes with small Maf proteins, and this complex specifically recognizes ARE sequences in promoter regions to upregulate antioxidant gene expression 50 . Additionally, paeoniflorin attenuates intracellular ROS accumulation in senescent MRC-5 cells through Nrf2 pathway activation, as evidenced by enhanced unclear translocation of this transcription factor 51 . Our results suggest that MFAE attenuates oxidative cellular damage through Nrf2 pathway activation, resulting in enhanced endogenous antioxidant defense systems. Nematode C. elegans serves as an exemplary model system for investigating the molecular and genetic underpinning of aging 52 . Aging represents a biologically unidirectional process governed by evolutionary conserved genetic and molecular mechanisms 13 . Natural polyphenolic compounds including anthocyanins counteract oxidative stress to effectively extend the lifespan of C. elegans . Anthocyanins extracted from Lycium ruthenicum (black goji berries) extend C. elegans lifespan and augment antioxidant defense via activation of the JNK-1 and DAF-16/FOXO signaling cascades 53 . Herbenya et al. (2016) 54 demonstrated that anthocyanins derived from acai ( Euterpe precatoria Mart.) improve stress resistance and delay aging in C. elegans by modulating longevity-associated markers, including sod-3 and hsp-16 . We revealed that mulberry-derived anthocyanins mitigate exogenous oxidative stress and extend C. elegans lifespan through Nrf2 pathway activation, resulting in enhanced cellular antioxidant defense (Fig. 8B). The gene skn-1 of C. elegans shares high homology with human Nrf2, serving as a central regulator of oxidative stress resistance and lifespan extension 55 . Lee et al. (2024) 56 demonstrated that sodium benzoate promotes lipid accumulation and reduces longevity in C. elegans through suppression of the evolutionarily conserved Nrf2/SKN-1 pathway. Natural polyphenolic compounds alleviate oxidative stress to prolong lifespan modulation by several transcription factors, including sod3 , cat1 , gst4 , ctl1 , sek1 , skn1 , clk1 , mev1 , and isp-1 57 . Nrf2/SKN-1 mediates critical cellular functions including redox regulation, hemeostasis preservation, and longevity modulation 34 . An Immunofluorescence analysis reveals that both Nrf2 and SKN-1 in C. elegans show predominant cytoplasmic localization within intestinal cells. Upon occurrence of stress, SKN-1 was activated by phosphorylation and translocated to the nucleus. The downstream target genes, gcs-1 and gst-4 , were expressed subsequently, which affected metabolism, oxidative stress responses, and aging 58 . Skn-1 null mutant reduces C. elegans lifespan, whereas skn-1 overexpression compensates for this reduction 59 . MFAE/C3G extended lifespan only in skn-1 -competent N2 worms via skn-1 upregulation, with no effect in skn-1 loss-of-function mutants (Fig. 10). Furthermore, MFAE enhanced SKN-1 nuclear localization and upregulated downstream targets gcs-1 , gst-4 , and sod-3 , confirming SKN-1 pathway activation (Fig. 10). Collectively, these results demonstrate that SKN-1 mediates both the oxidative stress resistance and lifespan extension induced by MFAE in C. elegans . Conclusion This study reveals that MFAE exerts its antioxidant and anti-aging effects through two complementary models: protecting LO2 cell against H 2 O 2 -induced oxidative damage and extended longevity in C. elegans are shown in Fig. 11. H 2 O 2 stress enhanced intracellular ROS and RNS levels, which resulted in decreased GSH, SOD, and CAT activity, increased MDA content, reduced mitochondrial membrane potential and lipid droplets, and induction of apoptosis. However, pretreatment with MFAE reversed the changes in these indicators and extended lifespan in C. elegans . Collectively, the evidence suggests MFAE combats aging through coordinated mechanisms involving oxidative stress regulation, enhanced cellular antioxidant capacity, and reduced apoptosis. Mechanistically, our data indicate that MFAE-mediated activation pathway provides the molecular foundation for its biological effects. Declarations CRediT authorship contribution statement Yuqi LI : Writing – original draft, Methodology, Investigation, Conceptualization. Jianglong HE : Investigation, Resources, Software. Xueping JIANG : Investigation, Validation. Haoran HUANG : Investigation, Validation. Yue ZHANG : Conceptualization, Data analysis. Ping WU : Data analysis, Formal analysis. Ran ZHANG : Data curation, Visualization. Hao LI : Validation, Conceptualization. Gaiqun HUANG and Gang LIU : Resources, Investigation. Kwang Sik LEE and Byung Rae JIN : Supervision, Formal analysis. Zhongzheng GUI : Writing–review & editing, Supervision, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was funded by National Natural Science Foundation of China (No.32302687), Graduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX24_4145), Jiangsu Provincial Agriculture Science and Technology Independent Innovation Fund (CX (24) 3084). Data availability Data will be made available on request. References Bell, L., Lamport, D. J., Butler, L. T., & Williams, C. M. A study of glycaemic effects following acute anthocyanin-rich blueberry supplementation in healthy young adults. Food Funct. 8(9) , 3104-3110 (2017). Lila, M. A., Burton-Freeman, B., Grace, M., & Kalt, W. Unraveling anthocyanin bioavailability for human health. Annu. Rev. Food Sci. Technol. 7 , 375-393 (2016). Ayvaz, H. et al. Anthocyanins: metabolic digestion, bioavailability, therapeutic effects, current pharmaceutical/industrial use, and innovation potential. Antioxidants-Basel 12(1) , 48 (2022). Min, A. Y., Yoo, J. M., Sok, D. E., & Kim, M. R. 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Tables Table 1 is available in the Supplementary Files section. Table 2. Nrf2 binding energy with MFAE predicted by molecular docking. Compounds Nrf2 binding energy （ Kcal·mol -1 ） Compounds Nrf2 binding energy （ Kcal·mol -1 ） Cyanidin-3- O -glucoside -14.64 Cyanidin-3- O -sophoroside -12.64 Cyanidin-3- O -rutinoside -15.47 Pelargonidin-3,5- O -diglucoside -16.51 Pelargonidin-3- O -rutinoside -7.44 Delphinidin-3- O -sophoroside -13.68 Pelargonidin-3- O -glucoside -14.15 Delphinidin-3- O -rhamnoside -16.82 Cyanidin-3-gentiobioside -12.47 Malvidin-3- O -rutinoside -10.04 Cyanidin-3,5- O -diglucoside -13.94 Cyanidin-3- O -arabinoside -15.15 Cyanidin-3- O -sophoroside -13.79 Peonidin-3,5- O -diglucoside -16.58 Delphinidin-3- O -galactoside -15.07 Delphinidin -10.52 Delphinidin-3- O -glucoside -14.79 Delphinidin-3,5,3-Triglucoside -10.64 Cyanidin-3- O -xyloside -14.08 Peonidin-3- O -sophoroside -10.13 Cyanidin-3- O -(6- O -malonyl-beta-D-glucoside) -16.08 Procyanidin A2 -10.23 Peonidin-3- O -glucoside -14.08 Petunidin-3- O -arabinoside -15.40 Naringenin -9.32 Additional Declarations No competing interests reported. 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Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhongzheng","middleName":"","lastName":"GUI","suffix":""}],"badges":[],"createdAt":"2025-07-02 02:23:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7024360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7024360/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88457731,"identity":"78593319-0c24-42a4-bcb8-cda8693fd289","added_by":"auto","created_at":"2025-08-06 15:44:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":429045,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7024360/v1/837a15e81ad2f41cc9be9e44.png"},{"id":88457939,"identity":"2ec365b2-c858-4f3d-adaf-92421736bbae","added_by":"auto","created_at":"2025-08-06 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10","display":"","copyAsset":false,"role":"figure","size":811771,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-7024360/v1/577a3b4de2aa6530296ceafb.png"},{"id":88457941,"identity":"7a4ab076-2248-4fd8-adf8-3cb0b9cd299e","added_by":"auto","created_at":"2025-08-06 15:52:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2629157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the possible antioxidant mechanisms of MFAE\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-7024360/v1/5647e4ffd11398259defd31f.png"},{"id":92600468,"identity":"436e4090-5182-44b9-b45d-9d8803d76e8d","added_by":"auto","created_at":"2025-10-01 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15:52:41","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":633791,"visible":true,"origin":"","legend":"","description":"","filename":"supplmentarymaterial.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7024360/v1/39ea6f328111e855ead044d7.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oxidative stress relief to longevity: mulberry-derived anthocyanins enhanced antioxidant defense via Nrf2/SKN-1 activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnthocyanins, a class of water-soluble flavonoids found in leaves and fruits of several plants\u003csup\u003e1\u003c/sup\u003e, exert various biological activities, including antioxidant, anticancer, inhibition of inflammation, hypoglycemic, and visual improvement effects\u003csup\u003e2\u003c/sup\u003e. Due to their safety and non-toxicity, anthocyanins are commonly used in food, health supplements, medicine, and cosmetic industries\u003csup\u003e3\u003c/sup\u003e. Therefore, as natural antioxidants, food- and pharmaceutical-grade anthocyanins comprise considerable value in diseases cures and suboptimal health improvement because of oxidative processes.\u003c/p\u003e\n\u003cp\u003eMulberry fruits attracted attention for their abundant anthocyanin content, and they are commonly consumed in processed beverages and foods such as juices, vines, and jams\u003csup\u003e3\u003c/sup\u003e. In traditional Chinese medicine, mulberries have been used effectively to treat symptoms such as liver and kidney deficiencies, diabetes, and constipation. Modern research also suggests that long-term consumption of edible mulberries may reduce the risk of diabetes and obesity\u003csup\u003e4\u003c/sup\u003e, protect the nervous system\u003csup\u003e5\u003c/sup\u003e, improve cardiovascular health\u003csup\u003e6\u003c/sup\u003e, inhibit inflammation and tumor formation, and promote positive effects on human health\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFirst occurred in 1970, \u0026quot;oxidative stress\u0026quot; refers to an imbalance where oxidants overwhelm antioxidants, disrupting redox signaling and causing molecular damage\u003csup\u003e8\u003c/sup\u003e. Excessive oxidative stress can elicit inflammatory responses and subsequent apoptosis. Furthermore, oxidative stress drives various diseases in organs and tissues\u003csup\u003e9\u003c/sup\u003e. With aging and unhealthy lifestyles and dietary habits, the human body is increasingly exposed to oxidative stress for extended periods, directly or indirectly leading to various chronic afflictions such as cardiovascular diseases, diabetes, obesity, and cancer\u003csup\u003e10,\u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAging is defined as the progressive deterioration of physiological functions, and it manifests through several distinct characteristics: a decline in reproductive capacity, typically culminating in infertility; a reduction in mobility; and atrophy of muscle tissue. At the cellular level, aging is marked by excessive generation of reactive oxygen species (ROS), lipofuscin accumulation, and mitochondrial dysfunction\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e. Concurrently, as immune system function wanes, the capacity of the organism to withstand stress diminishes, rendering cells and tissues increasingly vulnerable to damage and thereby accelerating the onset and progression of various chronic diseases\u003csup\u003e14\u003c/sup\u003e. Oxidative stress and aging are commonly assumed to be intimately interconnected and mutually reinforcing phenomena, and their effective mitigation is likely instrumental in delaying the onset of age-related changes\u003csup\u003e15\u003c/sup\u003e. Cerro et al. (2025)\u003csup\u003e16\u003c/sup\u003e demonstrated that natural bioactive compounds may serve as a beneficial strategy for promoting healthier aging in postmenopausal women, with mechanisms including enhancing immune function, reducing biological age, improving redox balance, and regulating stress hormones. Natural foods containing active antioxidant components are frequently used as antioxidants to prevent oxidative damage caused by free radicals\u003csup\u003e17\u003c/sup\u003e. The mulberry fruit is a natural source of anthocyanins, and its antioxidant properties were confirmed. Yan et al. (2017)\u003csup\u003e18\u003c/sup\u003e demonstrates that mulberry-derived anthocyanins effectively enhance the cellular antioxidase defense system, showing significant protective effects against hyperglycemia-induced oxidative damage across both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experimental models. However, the precise active constituents, underlying mechanisms, and specific molecular targets involved in the anti-aging mechanisms by which mulberry anthocyanins protect against oxidative stress remain unclear.\u003c/p\u003e\n\u003cp\u003eTo improve our understanding of the pharmacological pathways underlying therapeutic interventions for disease, researchers are increasingly turning to advanced computational approaches, such as network pharmacology, molecular docking, and molecular dynamics simulations\u003csup\u003e19\u003c/sup\u003e. Molecular docking is used to predict the binding modes and affinity dynamics between small molecules and protein receptors (targets) by computational techniques\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanistic interplay between mulberry-derived anthocyanins and oxidative stress-mediated aging processes, anthocyanins-derived components from mulberry fruits were identified using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). Two experimental models of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage in LO2 cells and age-synchronized \u003cem\u003eC. elegans\u003c/em\u003e were established. The effects of MFAE on cellular structures and the expression levels of antioxidant-related proteins in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced LO2 cells and aging-related parameters in \u003cem\u003eC. elegans\u003c/em\u003e were evaluated. Based on molecular docking predictions, the expression levels of Nrf2/SKN-1 pathway-related genes and proteins were investigated using Western blot, immunofluorescence staining, green fluorescent protein (GFP) labeling, and gene editing techniques. Our results provide novel insights and a theoretical foundation to improve the understanding of anthocyanin-mediated anti-aging mechanisms.\u003c/p\u003e\n"},{"header":"Materials and methods","content":"\u003ch3\u003e\u003cstrong\u003eMaterials and reagents\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFresh mulberry fruits (\u003cem\u003eMorus alba\u003c/em\u003e L.) were collected from a mulberry plantation at the Sericultural Research Institute of Jiangsu University of Science and Technology.\u003c/p\u003e\n\u003cp\u003eNormal LO2 cells were procured from the American Type Culture Collection (ATCC, VA, USA). \u003cem\u003eEscherichia coli\u003c/em\u003e strain OP50 was provided by BioSci (Hangzhou, China). Five \u003cem\u003eC. elegans\u003c/em\u003e strains of N2 (wild-type), LD-1 (\u003cem\u003eskn-1\u003c/em\u003e::GFP), CF1553 (\u003cem\u003esod-3\u003c/em\u003e::GFP), LD1171 (\u003cem\u003egcs-1\u003c/em\u003e::GFP), CL2166 (\u003cem\u003egst-4\u003c/em\u003e::GFP) and EU1 (\u003cem\u003eskn-1\u003c/em\u003e mutants) were provided by \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e Genetics Centre (MN, USA). \u003c/p\u003e\n\u003cp\u003eCyanin-3-\u003cem\u003eO\u003c/em\u003e-glucoside (C3G) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Thiazole blue (MTT) and Hoechst 33342 were purchased from Shanghai Shentech Co., Ltd. Critical biochemical reagents including RPMI-1640 basal medium (Gibco formulation), RIPA lysate buffer, SDS-PAGE gel preparation kits, and 4\u0026acute;Laemmli sample loading buffer were commercially sourced from Shanghai Solaibao Biotechnology Co., Ltd (Shanghai, China). Oil Red O staining reagent and AB-8 macroporous adsorption resin were commercially acquired from Shanghai YuanYe Biotechnology Co., Ltd.\u003c/p\u003e\n\u003cp\u003eEssential biochemical reagents and detection kits were commercially sourced from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The procurement included, fluorescent probes: DCFH-DA for ROS quantification, DAF-FMDA for NO detection, Lyso-Tracker Red for lysosomal pH monitoring; functional assay kits: JC-1 mitochondrial membrane potential assay kit, catalase activity colorimetric assay kit, WST-8 based total SOD detection system, lipid oxidation (MDA) assay kit, glutathione (GSH/GSSG) quantification kit; immunochemical reagents: RIPA lysis buffer, Cy3-conjugated affiniPure goat anti-rabbit IgG anti-rabbit, complete immunofluorescence staining kit. Monoclonal antibodies, such as Nrf2, \u0026beta;-actin, Keap1, HO-1, and tubulin were purchased from Proteintech Inc. (Wuhan, China).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMFAE extraction and compound identification\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMFAE was prepared following previously work\u003csup\u003e21\u003c/sup\u003e. Following liquid-liquid partitioning of the supernatant with ethyl acetate, the aqueous phase using AB-8 macroporous adsorption resin. Gradient elution was performed with acidified with 70% ethanol (1% formic acid, pH 2.8) at 2 BV/h flow rate, followed by vacuum rotary evaporation to yield anthocyanin-rich fractions. The elution part was vacuum-evaporated and freeze-dried at -80 ℃.\u003c/p\u003e\n\u003cp\u003eAnthocyanins extracts were analyzed with a UHPLC-QTOF-MS/MS system (ExionLC\u0026trade; AD UPLC coupled to an Applied Biosystems 6500 Triple Quadrupole MS)\u003csup\u003e22,\u003c/sup\u003e\u003csup\u003e23\u003c/sup\u003e. High-fidelity spectral data acquisition was conducted via Analyst software (v1.6.3, Sciex, MA, USA), followed by targeted metabolomic quantification employing MultiQuant 3.0.3 (Sciex). Serial dilutions of standard solutions were prepared, and chromatographic peak intensities were quantified for each calibration level. Standard curves were subsequently generated for individual analytes. The integrated peak areas of MFAE samples were interpolated into the linear regression equation derived from the calibration curve, enabling accurate quantification of the target analyte concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLO2 cell culture and cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLO2 cells were maintained under standardized culture conditions at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity incubator (Thermo Fisher Scientific, Waltham, MA, USA). A cell culture medium was prepared using RPMI 1640 as the base formulation, supplemented with 10% (v/v) fetal bovine serum, 1% non-essential amino acid mixture, and streptomycin at 100 \u0026mu;g/mL concentration, with routine medium replacement performed every 2\u0026ndash;3 days. Cellular metabolic activity was quantified using MTT colorimetric assay. Namely, 20 \u0026micro;L MTT solution (5 mg/mL) was added to each culture followed by incubation for 4 h. After removing the cell medium, 200 \u0026micro;L dimethyl sulfoxide was added to solubilize the formazan crystal. Then the medium was replaced with dimethyl sulfoxide (200 \u0026mu;L) and the absorbance was tested by a microplate reader (TECAN Company, Switzerland) at \u0026lambda; = 570 nm.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eEstablishment of oxidative stress cell model and MFAE treatment\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo establish an oxidative stress cell model, 2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well LO2 cells were cultured in 96-well plates for 24 h. Then, 100 \u0026mu;L of 0.1, 0.2, 0.4, 1.0, 2.5, or 5.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added, followed by incubation for 2, 4, 6, or 8 h. The oxidative stress cell model was estimated to use the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003econcentration and culture time for 50% of cell viability, which was obtained using 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and incubation for 4 h (Fig. 1A).\u003c/p\u003e\n\u003cp\u003eDifferent MFAE concentrations (0.2\u0026ndash;1.0 mg/mL) were used to pretreat LO2 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) for 24 h. After multiple washes of PBS, 100 \u0026mu;L 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e fresh cell medium was replaced. The cells were incubated at 37 ℃ for 4 h for follow-up analytical procedures. The blank control group was a cell medium culture without MFAE or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003econtrol group was established using 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the cell medium. All treatment groups were incubated at 37 ℃ for 4 h.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAssessment of intracellular ROS and reactive nitrogen species (RNS) levels\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLO2 cells were washed with phosphate-buffered saline (PBS) and added 10 \u0026mu;M/L DCFH-DA or DAF-FMDA for 30 min incubation at 37 ℃. After washing thrice with DMEM, the stained cells were visualized using a fluorescence microscope (Shanghai Changfang Optical Instrument Co., Ltd.)\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eHoechst 33342 staining\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLO2 cells were rinsed with ice-cold PBS twice, dyed with Hoechst 33342 (1 \u0026mu;g/mL) at 37 ℃ for 15 min in the dark. The samples were rinsed with PBS and pictured using a fluorescence microscope\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSubcellular localization in LO2 cells treated with MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003ch4\u003e\u003cstrong\u003eMitochondrial membrane potential\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eLO2 cells were rinsed using ice-cool PBS, and a mixture of RPMI-1640 medium and JC-1 dye solution was added to a 6-well plate for incubation at 37 ℃ for 20 min in the dark. The medium and dye solution were removed, the cells were further washed using JC-1 staining buffer and the results were photographed with a fluorescence microscope\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eLipid droplet fluorescence staining\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eA BODIPY 493/503 fluorescent probe was used to observe the distribution, quantity, and morphology of lipid droplets in LO2 cells. LO2 cells were initially rinsed with PBS and subsequently immersed in 4% paraformaldehyde solution for 10 min fixation period. Following the aspiration of the fixture, two sequential PBS washing steps were carried out. Following PBS treatment, 1 mL of BODIPY 493/503 fluorescence dye working solution lipid droplet solution was applied to the cells for lipid droplet visualization. Cellular samples were maintained at ambient temperature during the 15-min staining protocol. Fluorescence signals were captured using an inverted fluorescence microscopy equipped with a FITC filter set.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eLysosome fluorescent staining\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eLysosomal compartments were specifically labeled using Lyso-Tracker Red fluorescent probe. The LO2 cells were incubated with Lyso-Tracker Red staining working solution at 37 ℃ for 30 min to achieve lysosome staining. After removing the Lyso-Tracker Red staining solution, fresh culture medium was added to the plate. Staining images of cells were obtained using fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnthocyanin antioxidant target protein Nrf2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessing interactions of MFAE antioxidant and Nrf2 protein by Molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing acquisition of stereolithography formatted ligand architectures for anthocyanin derivatives through the PubChem database (Accession CID:44256857), and the tertiary conformation of the Nrf2 protein was obtained from UniProt\u0026rsquo;s structural repository with PDB ID:5FMV) serving as the crystallographic source\u003csup\u003e27\u003c/sup\u003e. Molecular docking simulations were conducted using AutoDock Vina 1.2.5\u003csup\u003e28\u003c/sup\u003e, with subsequent analysis and visualization of the docking results performed through Discovery Studio 2019 software.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eProteins were prepared using a lysis buffer, and the lysate was maintained at -20 ℃. BCA Kit (Solarbio) was employed to quantify the protein concentrations, and 10% SDS-PAGE was used to separate the proteins. The wet transfer method was employed to transfer protein samples onto a polyvinylidene difluoride membrane. The membranes were then incubated with non-fat milk (5%) for 4 h, cultured with first antibodies targeting Nrf2, Keap1, BAX, P62, or HO-1 at 4℃ for 12 h, washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and treated with the second antibodies conjugated with horseradish peroxidase for another 4 h at 25℃. All bands were rinsed with TBST, and the results were recorded with chemiluminescence imaging analysis system.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eLO2 cells were rinsed with PBS, then fixed using a 4% formaldehyde solution for 10 min following buffer aspiration, and subjected to three thorough PBS washes. The membranes were treated with a protein-based blocking buffer through a 60 min incubation overnight at 4℃ with gentle shaking in Nrf2-targeting primary antibodies. Then, the membranes were subjected to subsequent incubation with Cy3-conjugated anti-rabbit secondary antibodies for 60 min. After three sequential washes with labeled secondary antibody, an aliquot anti-fluorescence-quenching mounting medium containing 4\u0026rsquo;,6-diamidin-2-phenylindole (DAPI) was added. Cells were visualized and pictured using fluorescence microscopy.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAnthocyanins anti-aging in C. elegans\u003c/strong\u003e\u003c/h3\u003e\n\u003ch4\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003cstrong\u003e preparation\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e were cultured under axenic conditions at 20 ℃ on conventional nematode growth medium (NGM) agar plates, sustained by a monoxenic diet of viable \u003cem\u003eEscherichia coli\u003c/em\u003e OP50 strain as the exclusive nutritional substrate. Age-synchronized \u003cem\u003eC. elegans\u003c/em\u003e were obtained by treating the adult worms with a lysis solution (5 M NaOH and 5% NaCl) for 5 min, and L4 stage worms were selected for the next experiment. Age-synchronized adult worms were grown in NGM containing various concentrations of MFAE (0.2, 1.0, and 5 mg/mL), C3G (0.2 mg/mL), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM), with \u003cem\u003eEscherichia coli\u003c/em\u003e OP50 pretreated at 65 ℃ for 30 min\u003csup\u003e29\u003c/sup\u003e. To separate adult worms from their progeny, worms were transferred to fresh medium each day. \u003cem\u003eC. elegans\u003c/em\u003e lifespan was analyzed as described earlier\u003csup\u003e30\u003c/sup\u003e. Three replicates were used in all experiments.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eROS production in C. elegans\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe \u003cem\u003eC. elegans\u003c/em\u003e oxidative stress experiment was executed as described previously with slight modifications\u003csup\u003e31\u003c/sup\u003e. Quantification of intracellular ROS level in \u003cem\u003eC. elegans\u003c/em\u003e was performed via fluorescence microscopy using an H2DCF-DA probe following established methods by Zhu et al. (2015)\u003csup\u003e32\u003c/sup\u003e. After anesthetizing with 10 mM levamisole, worms were examined using a fluorescence inversion microscope (Ex/Em: 485/530 nm). Quantitative evaluation of fluorescence signals was assessed using Image-J software.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eKey gene expression assessed by green fluorescent protein (GFP) label\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe four GFP labeled transgenic strains LD-1 (\u003cem\u003eskn-1\u003c/em\u003e::GFP), CF1553 (\u003cem\u003esod-3\u003c/em\u003e::GFP), LD1171 (\u003cem\u003egcs-1\u003c/em\u003e::GFP) and CL2166 (\u003cem\u003egst-4\u003c/em\u003e::GFP) were used to assess key gene expressions. Age-synchronized L4 worms of the transgenic strains were reared on NGM plates with different treatments at 20 ℃ for 6 days. After anesthetizing with 10 mM levamisole, worms were placed on 2% agar glass slides and photographed using a fluorescence inversion microscope as described above.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eQuantitative data derived from three independent experiment replicates were expressed as arithmetic means \u0026plusmn; standard deviation (n=3) and subjected to parametric analysis via one-way analysis of variance (ANOVA) implemented in GraphPad Prism software (9.0). Statistical significance was defined as p \u0026lt; 0.05 following Bonferroni correction for multiple comparisons. \u003c/p\u003e\n"},{"header":"Results","content":"\u003ch3\u003e\u003cstrong\u003eAnthocyanin composition of MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eIndividual anthocyanin compounds identified by UPLC-ESI-MS/MS are listed in Table 1, and the ion mass spectrum of MFAE is shown in Fig. S1. A total of 69 compounds were identified, The proportions of various types of anthocyanins in the total anthocyanins are as follows. Including cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside (61.73%), cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside (25.11%), pelargonidin-3-\u003cem\u003eO\u003c/em\u003e-coumaroyl-5-\u003cem\u003eO\u003c/em\u003e-galactoside (2.34%), pelargonidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside (2.10%), pelargonidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside (2.08%), delphinidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside-5-\u003cem\u003eO\u003c/em\u003e-glucoside (1.78%), and delphinidin-3-\u003cem\u003eO\u003c/em\u003e-(coumaroyl) glucoside-5-\u003cem\u003eO\u003c/em\u003e-galactoside (1.11%). \u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress \u003c/strong\u003e\u003cstrong\u003eand protective effects\u003c/strong\u003e\u003cstrong\u003e of MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo construct an oxidative stress cell model, LO2 cells were used and treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eat different concentrations (0.1\u0026ndash;5.0 mM) for 2, 4, 6, and 8 h, respectively. The cellular viability of LO2 cells decreased gradually with increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003econcentrations. Low-concentration (0.1\u0026ndash;1.0 mM) of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the cellular viability shows a time-dependent and recovery gradually later. But, at high-concentrations (2.5\u0026ndash;5.0 mM) of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells lose their viability to repair themselves (Fig. 1A). The IC\u003csub\u003e50\u003c/sub\u003e value was determined under culturing conditions of 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4h, which was used as an oxidative stress cell model to identify the protective effects of MFAE.\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of MFAE on normal cells, LO2 cells were treated with different concentrations of MFAE (0.1 - 2.0 mg/mL) for 24 h. MFAE at 0.3 \u0026ndash; 2.0 mg/mL positively affected LO2 cell viability (Fig. 1B). Therefore, MFAE concentration of 0.2 mg/mL and 1.0 mg/mL were selected for not effect significantly and effect significantly on cell viability, respectively. To examine the effects of MFAE on the oxidative stress model cells, LO2 cells were preincubated with MFAE (0.2 and 1.0 mg/mL) and C3G (0.2 mg/mL, as positive control) for 24 h and then stimulated with 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e during incubation for an additional 4 h. Pharmacological preconditioning with 1.0 mg/mL MFAE significantly enhanced cellular viability from a baseline of 37.8% to 47.6% under oxidative stress conditions. The cell survival rate was 64.1% in the C3G-positive control (Fig. 1C). This indicated that MFAE improved the survival rate of cells after oxidative damage and reduced the effect of oxidative damage on cells.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eReduction of ROS and RNS levels in \u003c/strong\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-injured LO2 cells\u003c/strong\u003e\u003cstrong\u003e by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eDCFH-DA and DAF-FMDA fluorescent probes were used to stain LO2 cells and investigate the effects of MFAE on ROS and RNS levels. LO2 cells were preincubated with MFAE (0.2 and 1.0 mg/mL), C3G (0.2 mg/mL), and oxidative stress inhibitor (N-acetylcysteine [NAC], 100 mM/L) were used as a positive control for 24 h and then treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1 mM) for an additional 4 h. The fluorescence in the blank control group (normal cells) was relatively weak after fluorescence staining, and the strongest green fluorescence was visualized in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated positive control group. The MFAE, C3G, and NAC intervention groups demonstrated significantly reduced fluorescence intensity relative to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e control group (p \u0026lt; 0.05), indicative of diminished ROS accumulation. Quantitative analysis for fluorescence intensity showed similar trends (Figs. 2A-B). Overproduction of ROS and RNS resulted in decreased GSH, SOD, and CAT activity, and increased MDA content (Fig. S2), which indicated that MFAE reduced the levels of ROS and RNS caused by oxidative stress, thereby reducing cell oxidative damage.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAlleviat\u003c/strong\u003e\u003cstrong\u003eion of lysosome and nuclear damage in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-injured LO2 cells by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLO2 cell structure morphology induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the different treatment groups was observed after Hoechst 33342 fluorescent probe staining (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe nucleus appeared light blue with uniform chromatin distribution in the blank control, whereas in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group, the nucleus was of a brighter blue, denser, and more shrunken. The nuclear blue color was weaker in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced group compared with that in the MFAE, C3G, and NAC groups, and thus MFAE reduced nuclear damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csub\u003e \u003c/sub\u003e(Fig. 3A). Changes in mitochondrial membrane potential induced by JC-1 fluorescence staining occurred in different groups (Fig. 3B). The mitochondria showed strong bright red fluorescence in the blank control and bright red fluorescence in the MFAE, C3G, and NAC groups; fluorescence in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group was green. Thus, MFAE effectively reduced the depolarization of the mitochondrial membrane potential caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Fig. 3C shows distinct green lipid droplets in the control group, and loss of lipid droplet structure attributable to oxidative damage in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced group. Contrastingly, the extent of lipid droplet damage was prominently mitigated in the C3G, NAC, and MFAE groups when compared with that in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced group. MFAE thus alleviated intracellular lipid droplet damage caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and protected the lipid metabolism function of cells. To understand the interactions between oxidative stress and lysosomes, LO2 cells were stained with Lyso-Tracker Red. Strong and weak red fluorescence was observed in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MFAE, C3G, and NAC groups, respectively (Fig. 3D); almost no red fluorescence was observed in the blank control. These results suggested that MFAE alleviated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced lysosome damage and inhibited cell apoptosis.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eInteractions of MFAE antioxidant and Nrf2 protein as assessed by molecular docking\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMolecular docking is a computational approach employed to elucidate protein-ligand interactions and is extensively used in drug discovery and the mechanistic exploration of natural compounds\u003csup\u003e33\u003c/sup\u003e. The binding affinity between the 25 anthocyanin compounds selected in MFAE with the Nrf2 was assessed using AutoDock Vina 1.2.2. The docking scores ranged from -7.44 kcal/mol to -16.82 kcal/mol among those compounds (Table 2). The most abundant compounds cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside and cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside in MFAE exhibited binding energies with Nrf2 of -14.64 and -15.47 kcal/mol, respectively. Eight hydrogen bonds were formed by cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside with the amino acid residues LYS (A:719), ARG (A:686), THR (A:690), GLU (A:738, A:441), and SER (A: 689) in Nrf2, and nine hydrogen bonds were formed by cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside with the amino acid residues ASN (A:720, A:499), LYS(A:719), GLU (A:487, A:490, A:738), and TYR(A:489) in Nrf2 (Fig. 4). Cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside and cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside exhibited stronger binding affinity to Nrf2 compared to other components. This indicated that Nrf2 is a potential target for MFAE anti-oxidation.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eActivation of Nrf2 pathway-related proteins in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-injured LO2 cells by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo investigate the oxidative stress-mediated effect of the Nrf2 signaling pathway, LO2 cells exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress were quantitatively analyzed for expression profiles of Nrf2 and its downstream HO1 protein. The expression of HO-1 and Nrf2 proteins in LO2 cells with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress depended on the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e dosage (Fig. 5). High expression of the two proteins occurred at H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e dosages of 0.2 mM and 0.3 mM, confirming that these concentrations can induce high expression of HO-1 and Nrf2 in LO2 cells.\u003c/p\u003e\n\u003cp\u003eTo explore whether MFAE alleviated cellular oxidative damage and apoptosis by activating the Nrf2 pathway, ML385 (i.e., an Nrf2 inhibitor) was used as a negative control. Western bolt analysis was performed to quantify the protein expression profiles of key Nrf2 pathway components, including of BAX, P62, Nrf2, Keap1, and HO-1. Nrf2 expression was significantly downregulated in response to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eor ML385-induced stress but was significantly upregulated following pretreatment with MFAE or C3G. HO-1 expression showed a consistent trend with that of Nrf2, whereas Keap1 expression showed an inverse pattern. Additionally, expression of the pro-apoptotic protein BAX was significantly increased when Nrf2 expression was suppressed. P62 expression was upregulated when LO2 cells were stressed by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, with higher expression induced by MFAE or C3G pretreatment (Fig. 6).\u003c/p\u003e\n\u003cp\u003eThe Nrf2 protein in LO2 cells was stained by immunofluorescence to further validate Nrf2 pathway activation by oxidative stress (Fig. 7). Staining with DAPI (i.e., a fluorescent dye strongly binding to DNA) showed a nuclear outline. Fluorescence of the Nrf2 protein was weaker in the control group and was distributed in the cytoplasm. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2, \u003c/sub\u003eMFAE, and C3G groups showed varying degrees of enhancement in red fluorescence and aggregation towards the nucleus (Fig. 7 Merge). The findings indicate that MFAE protects LO2 cells from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage by triggering Nrf2 nuclear translocation and upregulating its expression, thereby activating the Nrf2 antioxidant pathway.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eExtending of C. elegans\u003c/strong\u003e\u003cstrong\u003e lifespan by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo systematically evaluate the longevity-modulating effects of MFAE in \u003cem\u003eC. elegans\u003c/em\u003e, age-synchronized N2 wild-type populations were chronically treated with MFAE at graded concentrations (0.2, 1.0, and 5.0 mg/mL) and C3G (0.2 mg/mL) as a reference antioxidant control. Longitudinal survival analysis in Fig. 8A demonstrated significant chronological lifespan extension in \u003cem\u003eC. elegans\u003c/em\u003e populations, with 1.0 mg/mL MFAE, 5.0 mg/mL MFAE and 0.2 mg/mL C3G supplementation conferring mean lifespan increases of 4.64% (\u003cem\u003ep\u003c/em\u003e=0.037), 8.35% (\u003cem\u003ep\u003c/em\u003e=0.008), and 8.47% (\u003cem\u003ep\u003c/em\u003e=0.006) respectively, compared with that of the control group. In acute oxidative stress assays, pretreatment with MFAE (1.0 mg/mL and 5.0 mg/mL) and C3G (0.2 mg/mL) prior to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure significantly attenuated mortality rates, yielding 10%, 26.7%, and 33.3% survival rates at the 2 h endpoint (Fig. 8B). Reproductive phenotyping revealed conserved total fecundity across treatment groups but delayed oviposition kinetics in high-dose MFAE (5.0 mg/mL) and C3G cohorts, with peak egg-laying occurring stage, respectively (Fig. S3). This temporal deceleration in reproductive maturation suggests MFAE modulates longevity through developmental retardation of germline-associated aging pathways. \u003c/p\u003e\n\u003cp\u003eFor further validation of the above results, \u003cem\u003eskn-1\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e were exposed to MFAE (5.0 mg/mL) or C3G (0.2 mg/mL) to record their lifespans. Fig. 8C shows that the lifespan did not differ among \u003cem\u003eskn-1\u003c/em\u003e mutant (CK), MFAE (5.0 mg/mL) and C3G (0.2 mg/mL) groups, demonstrating that \u003cem\u003eskn-1\u003c/em\u003e gene is the key gene for the effect of MFAE on the lifespan of \u003cem\u003eC. elegans.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMediation of ROS levels in C. elegans by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eIn LO2 cells, MFAE showed the reduction of ROS and RNS levels and alleviated lysosome and nuclear damage caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eoxidative stress. A similar procedure was conducted using \u003cem\u003eC. elegans\u003c/em\u003e (N2). Fig. 9A shows that significant attenuation of intracellular ROS accumulation in\u003cem\u003e C. elegans \u003c/em\u003etreated with 5.0 mg/mL MFAE and 0.2mg/mL C3G, demonstrating respective fluorescence intensity reduction of 42.3% and 38.9% compared to untreated controls. Dose-response analysis further identified a concentration-dependent ROS modulatory effect of MFAE, with maximal suppression (68.5\u0026plusmn;5.2 AU) observed at the 5.0 mg/ml dose, paralleling the efficacy of the C3G positive control (71.2\u0026plusmn;4.8 AU) (Fig. 9C). These findings mechanistically align with the oxidative stress theory of aging, suggesting MFAE-mediated ROS scavenging contributes to delayed senescence phenotypes in nematodes. \u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eControlling of the expression of skn-1 downstream genes sod-3, gcs-1, and gst-4 by MFAE\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe SKN-1 transcription factor in \u003cem\u003eC. elegans\u003c/em\u003e serves as a functional ortholog to mammalian Nrf2, orchestrating the transcriptional activation of \u003cem\u003esod-3\u003c/em\u003e, \u003cem\u003egcs-1\u003c/em\u003e, and\u003cem\u003e gst-4\u003c/em\u003e. This evolutionarily conserved regulatory axis mediates oxidative stress resistance by enhancing free radical scavenging capacity and maintaining redox homeostasis through phase Ⅱ detoxification pathways\u003csup\u003e34\u003c/sup\u003e. MFAE facilitated SKN-1 nuclear translocation in the nematode intestine (Fig. 10A), thereby significantly activating the expression of downstream target genes \u003cem\u003egcs-1\u003c/em\u003e and\u003cem\u003e gst-4\u003c/em\u003e, and oxidative stress-related transcription factor \u003cem\u003esod-3\u003c/em\u003e (Fig. 10B). Integrating phenotypic data from Fig. 8C, these findings mechanistically establish that MFAE induces prolonged geroprotective effects in \u003cem\u003eC. elegans\u003c/em\u003e through pharmacological activation of the evolutionarily conserved the Nrf2/SKN-1 signal axis, as evidenced by coordinated upregulation of downstream antioxidant response elements and stress-resistance biomarkers.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnthocyanins, a type of flavonoids, exert significant antioxidant properties. To understand the antioxidant mechanism, MFAE was successfully prepared, and its anthocyanin components were characterized using UPLC-ESI-MS/MS. The extract was then evaluated for antioxidant and anti-aging activities. Table 1 shows that six types of anthocyanins were present in mulberry fruit: cyanidin (89.487%), pelargonidin (6.605%), delphinidin (3.703%), peonidin (0.182%), malvidin (0.011%) and petunidin (0.011%). Among all detected anthocyanins, cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside dominated at 61.727%, followed by cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside at 25.113%, together comprising the bulk of the anthocyanin profile. Approximately 99.7% of all aglycones in mulberry fruits were cyanidin and delphinidin. Kong et al. (2003)\u003csup\u003e35\u003c/sup\u003ereported that natural anthocyanins originate from six major anthocyanidins: cyanidin, pelargonidin, malvidin, peonidin, delphinidin, and petunidin. The major anthocyanin found in mulberry fruit is cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside accounting for approximately 53.94%\u0026ndash;78.23%, and anthocyanins in plants vary among plant varieties and tissues\u003csup\u003e36\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eOxidative stress refers to the state of cell damage caused by oxidants. The antioxidant defense system becomes overwhelmed and unable to effectively eliminate free radicals and their metabolic byproducts. Natural antioxidants inhibit the occurrence and development of oxidative stress, thereby reducing the effect of free radicals on cells. Zhao et al. (2023)\u003csup\u003e37\u003c/sup\u003e demonstrated that plant-derived anthocyanins exhibit potent antiproliferative effects on cancer cells and suppress tumor growth. Anthocyanin antioxidative action subsequently hinders malignant cell initiation and proliferation pathways. Here, an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress LO2 model was constructed. Several H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress cell models were established using mammary epithelial\u003csup\u003e38\u003c/sup\u003e, human lens epithelial\u003csup\u003e39\u003c/sup\u003e, fibroblast\u003csup\u003e40\u003c/sup\u003e, and gastric epithelial\u003csup\u003e41\u003c/sup\u003ecells. Fig. 1A shows that LO2 cells were protected against the injury at low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e doses with increasing culture time. The survival rate of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-injured LO2 cells increased significantly after pretreatment with MFAE. Further, MFAE significantly improved cell viability and mitigated the detrimental effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress. LO2 cells were pretreated with 0.2 mg/mL MFAE for 24 h prior to exposure to 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h to establish the oxidative stress model (Fig. 1B). MFAE improved the survival rate of cells after oxidative damage and reduced the effect of oxidative damage on cells (Fig. 1C).\u003c/p\u003e\n\u003cp\u003eGenerally, ROS and RNS are signaling molecules inevitably produced during aerobic metabolic processes in liver cells and regulate several physiological activities. Under healthy conditions, the production and clearance of two free radicals are maintained in a dynamic balance within cells. However, when this balance is disrupted by oxidative stress, it can lead to structural and functional cellular damage, such as protein degradation, lipid peroxidation, and DNA damage, ultimately resulting in programmed cell death. Our results showed that LO2 cells injured by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003estress were recovered by MFAE pretreatment (Fig.1B-C, Fig.2, and Fig.3). Plant-derived phytochemicals, such as flavonoids\u003csup\u003e42\u003c/sup\u003e, polysaccharides\u003csup\u003e43\u003c/sup\u003e, and peptides\u003csup\u003e44\u003c/sup\u003e are effective hepatoprotective agents that counteract oxidative damage by scavenging ROS.\u003c/p\u003e\n\u003cp\u003eMolecular docking represents a computational approach that simulates and analyzes the interaction between ligand molecules and target protein, predicting their most favorable binding orientations and association strengths. The molecular binding affinity, quantified as binding energy (kcal/mol), demonstrates an inverse correlation with numerical values: lower binding energies indicate stronger thermodynamic stabilization of ligand-target complexes, whereas higher values reflect weaker intermolecular interactions. The data in Table 2 show that cyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside (-14.64 Kcal/mol) and cyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinside (-15.47 Kcal/mol) comprised lower binding energies, indicating the high affinity to Nrf2 protein which is intricately linked to oxidative stress and anti-aging processes. The molecular dynamics simulations revealed the detailed binding mechanisms of the ligands with their target protein receptors\u003csup\u003e45\u003c/sup\u003e. Fig. 4 shows key antioxidant constituents in MFAE interactions with the Nrf2 target protein via a network of hydrogen bonds and hydrophobic groups. Through integrated network pharmacology and molecular docking approaches, eight pivotal antioxidant compounds in red wine were identified to interact with multiple metabolic pathways, notably the PI3K/AKT signaling axis\u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFree radicals cause irreparable structural damage (protein, lipids, DNA, organelles), activating apoptosis and programmed cell death\u003csup\u003e47\u003c/sup\u003e. Nrf2 regulates cellular redox status and oxidative stress sensing, processes implicated in lifespan extension\u003csup\u003e48,\u003c/sup\u003e\u003csup\u003e49\u003c/sup\u003e. Results of the cell experiments showed that Nrf2 expression in cells increased and decreased under low and high oxidative stresses, respectively (Fig. 5). This trend was consistent with that of the effects of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration on cell viability.\u003c/p\u003e\n\u003cp\u003eThe Nrf2 inhibitor ML385 was employed to verify that MFAE\u0026rsquo;s antioxidant protection operates specifically through the Nrf2 pathway. MFAE upregulated the expression of the multifunctional protein P62 and Nrf2 downstream regulated protein HO-1 and downregulated expression of BAX, a pro-apoptotic protein, and Keap1, the primary negative regulator of the Nrf2 antioxidant pathway, thereby inhibiting apoptosis. HO-1 is a protein downstream of Nrf2, and its expression pattern is similar to that of Nrf2; furthermore, in the current study, changes in Keap1 protein expression showed the opposite pattern to those of Nrf2 protein (Fig. 6). Oxidative stress induces conformational changes in Keap1, leading to Nrf2 dissociation and stabilization. The liberated Nrf2 accumulates in the nucleus where it dimerizes with small Maf proteins, and this complex specifically recognizes ARE sequences in promoter regions to upregulate antioxidant gene expression\u003csup\u003e50\u003c/sup\u003e. Additionally, paeoniflorin attenuates intracellular ROS accumulation in senescent MRC-5 cells through Nrf2 pathway activation, as evidenced by enhanced unclear translocation of this transcription factor\u003csup\u003e51\u003c/sup\u003e. Our results suggest that MFAE attenuates oxidative cellular damage through Nrf2 pathway activation, resulting in enhanced endogenous antioxidant defense systems.\u003c/p\u003e\n\u003cp\u003eNematode \u003cem\u003eC. elegans\u003c/em\u003e serves as an exemplary model system for investigating the molecular and genetic underpinning of aging\u003csup\u003e52\u003c/sup\u003e. Aging represents a biologically unidirectional process governed by evolutionary conserved genetic and molecular mechanisms\u003csup\u003e13\u003c/sup\u003e. Natural polyphenolic compounds including anthocyanins counteract oxidative stress to effectively extend the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e. Anthocyanins extracted from \u003cem\u003eLycium ruthenicum\u003c/em\u003e (black goji berries) extend \u003cem\u003eC. elegans\u003c/em\u003e lifespan and augment antioxidant defense via activation of the JNK-1 and DAF-16/FOXO signaling cascades\u003csup\u003e53\u003c/sup\u003e. Herbenya et al. (2016)\u003csup\u003e54\u003c/sup\u003e demonstrated that anthocyanins derived from acai (\u003cem\u003eEuterpe precatoria\u003c/em\u003e Mart.) improve stress resistance and delay aging in \u003cem\u003eC. elegans\u003c/em\u003e by modulating longevity-associated markers, including \u003cem\u003esod-3\u003c/em\u003e and \u003cem\u003ehsp-16\u003c/em\u003e. We revealed that mulberry-derived anthocyanins mitigate exogenous oxidative stress and extend \u003cem\u003eC. elegans\u003c/em\u003e lifespan through Nrf2 pathway activation, resulting in enhanced cellular antioxidant defense (Fig. 8B). The gene\u003cem\u003e skn-1\u003c/em\u003e of \u003cem\u003eC. elegans\u003c/em\u003e shares high homology with human Nrf2, serving as a central regulator of oxidative stress resistance and lifespan extension\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eLee et al. (2024)\u003csup\u003e56\u003c/sup\u003e demonstrated that sodium benzoate promotes lipid accumulation and reduces longevity in \u003cem\u003eC. elegans\u003c/em\u003e through suppression of the evolutionarily conserved Nrf2/SKN-1 pathway. Natural polyphenolic compounds alleviate oxidative stress to prolong lifespan modulation by several transcription factors, including \u003cem\u003esod3\u003c/em\u003e, \u003cem\u003ecat1\u003c/em\u003e, \u003cem\u003egst4\u003c/em\u003e, \u003cem\u003ectl1\u003c/em\u003e, \u003cem\u003esek1\u003c/em\u003e, \u003cem\u003eskn1\u003c/em\u003e, \u003cem\u003eclk1\u003c/em\u003e, \u003cem\u003emev1\u003c/em\u003e, and \u003cem\u003eisp-1\u003c/em\u003e\u003csup\u003e57\u003c/sup\u003e. Nrf2/SKN-1 mediates critical cellular functions including redox regulation, hemeostasis preservation, and longevity modulation\u003csup\u003e34\u003c/sup\u003e. An Immunofluorescence analysis reveals that both Nrf2 and SKN-1 in \u003cem\u003eC. elegans\u003c/em\u003e show predominant cytoplasmic localization within intestinal cells. Upon occurrence of stress, SKN-1 was activated by phosphorylation and translocated to the nucleus. The downstream target genes, \u003cem\u003egcs-1\u003c/em\u003e and \u003cem\u003egst-4\u003c/em\u003e, were expressed subsequently, which affected metabolism, oxidative stress responses, and aging\u003csup\u003e58\u003c/sup\u003e. \u003cem\u003eSkn-1 \u003c/em\u003enull mutant reduces \u003cem\u003eC. elegans\u003c/em\u003e lifespan, whereas \u003cem\u003eskn-1\u003c/em\u003e overexpression compensates for this reduction\u003csup\u003e59\u003c/sup\u003e. MFAE/C3G extended lifespan only in \u003cem\u003eskn-1\u003c/em\u003e-competent N2 worms via\u003cem\u003e skn-1\u003c/em\u003e upregulation, with no effect in \u003cem\u003eskn-1\u003c/em\u003e loss-of-function mutants (Fig. 10). Furthermore, MFAE enhanced SKN-1 nuclear localization and upregulated downstream targets \u003cem\u003egcs-1\u003c/em\u003e, \u003cem\u003egst-4\u003c/em\u003e, and\u003cem\u003e sod-3\u003c/em\u003e, confirming SKN-1 pathway activation (Fig. 10). Collectively, these results demonstrate that SKN-1 mediates both the oxidative stress resistance and lifespan extension induced by MFAE in \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e\n"},{"header":"Conclusion","content":"\u003cp\u003eThis study reveals that MFAE exerts its antioxidant and anti-aging effects through two complementary models: protecting LO2 cell against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage and extended longevity in \u003cem\u003eC. elegans\u003c/em\u003e are shown in Fig.\u0026nbsp;11. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stress enhanced intracellular ROS and RNS levels, which resulted in decreased GSH, SOD, and CAT activity, increased MDA content, reduced mitochondrial membrane potential and lipid droplets, and induction of apoptosis. However, pretreatment with MFAE reversed the changes in these indicators and extended lifespan in \u003cem\u003eC. elegans\u003c/em\u003e. Collectively, the evidence suggests\u0026nbsp;MFAE combats aging through coordinated mechanisms involving oxidative stress regulation, enhanced cellular antioxidant capacity, and reduced apoptosis. Mechanistically, our data indicate that MFAE-mediated activation pathway provides the molecular foundation for its biological effects. \u0026nbsp;\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuqi LI\u003c/strong\u003e: Writing \u0026ndash; original draft, Methodology, Investigation, Conceptualization. \u003cstrong\u003eJianglong HE\u003c/strong\u003e: Investigation, Resources, Software. \u003cstrong\u003eXueping JIANG\u003c/strong\u003e: Investigation, Validation. \u003cstrong\u003eHaoran HUANG\u003c/strong\u003e: Investigation, Validation. \u003cstrong\u003eYue ZHANG\u003c/strong\u003e: Conceptualization, Data analysis. \u003cstrong\u003ePing WU\u003c/strong\u003e: Data analysis, Formal analysis. \u003cstrong\u003eRan ZHANG\u003c/strong\u003e: Data curation, Visualization. \u003cstrong\u003eHao LI\u003c/strong\u003e: Validation, Conceptualization.\u003cstrong\u003e\u0026nbsp;Gaiqun HUANG\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand \u003cstrong\u003eGang LIU\u003c/strong\u003e: Resources, Investigation. \u003cstrong\u003eKwang Sik LEE\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand \u003cstrong\u003eByung Rae JIN\u003c/strong\u003e: Supervision, Formal analysis. \u003cstrong\u003eZhongzheng GUI\u003c/strong\u003e: Writing\u0026ndash;review \u0026amp; editing, Supervision, Project administration.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by National Natural Science Foundation of China (No.32302687), Graduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (No.\u0026nbsp;KYCX24_4145), Jiangsu Provincial Agriculture Science and Technology Independent Innovation Fund (CX (24) 3084).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBell, L., Lamport, D. 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The SKN-1/Nrf2 transcription factor can protect against oxidative stress and increase lifespan in \u003cem\u003eC. elegans\u003c/em\u003e by distinct mechanisms. \u003cem\u003eAging Cell \u003c/em\u003e\u003cstrong\u003e16(5)\u003c/strong\u003e, 1191-1194 (2017). \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Nrf2 binding energy with MFAE predicted by molecular docking.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNrf2 binding energy\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eKcal\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNrf2 binding energy\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eKcal\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003eCyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-14.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eCyanidin-3-\u003cem\u003eO\u003c/em\u003e-sophoroside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-12.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003eCyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-15.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePelargonidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-16.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003ePelargonidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-7.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDelphinidin-3-\u003cem\u003eO\u003c/em\u003e-sophoroside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-13.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003ePelargonidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-14.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDelphinidin-3-\u003cem\u003eO\u003c/em\u003e-rhamnoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-16.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003eCyanidin-3-gentiobioside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-12.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eMalvidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-10.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003eCyanidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n 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\u003cp\u003e-14.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePeonidin-3-\u003cem\u003eO\u003c/em\u003e-sophoroside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-10.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003eCyanidin-3-\u003cem\u003eO\u003c/em\u003e-(6-\u003cem\u003eO\u003c/em\u003e-malonyl-beta-D-glucoside)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-16.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eProcyanidin A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-10.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003ePeonidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-14.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePetunidin-3-\u003cem\u003eO\u003c/em\u003e-arabinoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-15.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 169px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eNaringenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e-9.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anthocyanin, Oxidative stress, Anti-aging, Molecular docking, Nrf2/SKN-1 pathway, LO2 cells, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-7024360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7024360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Anthocyanin has been demonstrated that potential health-promoting and anti-aging through antioxidant activities, although the underlying mechanisms remain unclear. In this study, we identified cyanin-3-O-glucoside (61.7%) and cyanin-3-O-rutinoside (25.1%) as the primary components of mulberry fruit anthocyanin extracts (MFAE) using LC-MS/MS. Two experimental models including a hydrogen peroxide-induced hepatocyte oxidative damage and an age-synchronized Caenorhabditis elegans were established. These models were co-cultured with MFAE for varying time periods followed by comprehensive analyses of subcellular structures and comparisons of oxidative stress and senescence-related parameters across treatment groups. The expression levels of Nrf2/SKN-1 pathway-related genes and proteins predicted by molecular docking were investigated. The results demonstrate that MFAE treatment significantly alleviated H2O2-induced cellular oxidative damage, extended lifespan in C. elegans, and improved multiple health indices. Furthermore, MFAE activated the evolutionarily conserved Nrf2/SKN-1 pathway while modulating the expression of various upstream and downstream genes (gst-4, gcs-1, sod-3) and proteins (HO-1, P62, Keap1, BAX). These findings indicate that MFAE exerts anti-aging effects via antioxidant-driven activation of the Nrf2/SKN-1 pathway.","manuscriptTitle":"Oxidative stress relief to longevity: mulberry-derived anthocyanins enhanced antioxidant defense via Nrf2/SKN-1 activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 15:44:36","doi":"10.21203/rs.3.rs-7024360/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99b76d82-9db7-406b-a79e-94911f55dc92","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52696795,"name":"Biological sciences/Biochemistry"},{"id":52696796,"name":"Biological sciences/Cell biology"},{"id":52696797,"name":"Biological sciences/Molecular biology"},{"id":52696798,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-09-30T05:24:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 15:44:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7024360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7024360","identity":"rs-7024360","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}