Piceatannol, a natural stilbene, improves weaning piglets' growth performance through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota

Piceatannol, a natural stilbene, improves weaning piglets' growth performance through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota | 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 Research Article Piceatannol, a natural stilbene, improves weaning piglets' growth performance through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota Longlong Zhu, Yuyan Che, Meixia Chen, Long Cai, Qiujue Wu, Tao Feng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7540228/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Background Piglets are highly susceptible to oxidative stress, which can reduce growth performance and cause intestinal damage. Piceatannol (PIC), a natural bioactive substance enriched in Chinese rhubarb ( Rheum officinale ) and certain dark purple fruits, shows excellent antioxidant properties in our previous cell-based high-throughput screening. However, its effect on piglet growth performance and antioxidant capacity as well as underling mechanism has not been thoroughly investigated. Methods One hundred weaned pigs (28 d of age, 8.71 ± 0.20 kg) were randomly assigned to 4 treatments with 5 replicates of 5 pigs per replicate. The experimental diets consisted of: 1) basal diet, 2) basal diet + 100 mg/kg PIC, 3) basal diet + 200 mg/kg PIC, and 4) basal diet + 300 mg/kg PIC. On d 15 and 35, one pig from each replicate was selected for sampling. The growth performance was monitored during a 35-day trial. In addition, H 2 O 2 -challenged IPEC-J2 cells served as an in vitro model to investigate the antioxidant mechanisms of PIC. IPEC-J2 cells were treated with 1,000 µM H 2 O 2 in the presence or absence of 10 µM PIC. Results Dietary PIC at 200 mg/kg significantly enhanced growth performance, as evidenced by increased average daily gain and feed conversion rate ( P < 0.05). PIC supplementation markedly improved systemic antioxidant capacity, with elevated serum total antioxidant capacity, catalase activity, and glutathione levels, along with reduced malondialdehyde content ( P < 0.05; P < 0.01). Notably, PIC modulated the gut microbiota composition, increasing the amounts of beneficial genera (e.g., Blautia and Faecalibacterium ), and these microbial shifts significantly correlated with improved antioxidant indices. In vitro , PIC pretreatment effectively protected IPEC-J2 cells against H 2 O 2 -induced oxidative damage by reducing reactive oxygen species generation and lipid peroxidation ( P < 0.01). Mechanistically, PIC exerts its antioxidant effects through Nrf2 pathway activation, upregulating endogenous antioxidant enzymes ( P < 0.05; P < 0.01) while simultaneously inhibiting apoptosis via the regulation of the Bcl-2/Bax ratio and caspase-3 cleavage ( P < 0.01). Conclusions PIC improved the growth performance and health status of weaned piglets through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota, offering a potential new natural antioxidants for mitigating weaning stress in piglets. Piceatannol Weaning piglets Growth performance Intestinal flora Antioxidant Nrf2 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In the pig industry, oxidative stress often leads to impaired growth performance and various diseases, resulting in significant economic losses [ 1 ]. Young animals, including piglets, have immature antioxidant defense systems, rendering them particularly susceptible to free radical accumulation, lipid peroxidation propagation, and consequent oxidative stress [ 2 ]. In intensive pig production, piglets are highly susceptible to oxidative stress caused by multiple stressors (e.g., environmental, nutritional, and psychological) [ 3 ], leading to suppressed growth performance and health status. Previous studies have demonstrated that early weaned piglets exhibit obvious oxidative stress, as evidenced by significantly elevated reactive oxygen species (ROS) and malondialdehyde (MDA) in the jejunum and liver, hydroxyl radicals in the colon, and hydrogen peroxide (H 2 O 2 ) in the jejunum, colon, and liver [ 4 , 5 ]. Continuous oxidative stress in piglets can damage cell function and organs and reduce production performance. Moreover, oxidative stress is an early event in the onset and development of various diseases and is therefore considered an important factor in early-weaning syndrome in piglets. In this context, the use of antioxidant feed additives has been proposed as an effective nutritional strategy to improve piglet health and growth performance. Natural plant compounds and traditional Chinese medicine extracts have been widely investigated for their antioxidant properties [ 6 ]. Compounds such as carvacrol, thymol, quercetin, catechins, curcumin, oregano extract, and cinnamaldehyde have been demonstrated to function as effective antioxidants in livestock. These substances enhance the body’s antioxidant capacity and alleviate oxidative stress, thereby improving animal health and production performance. We previously established a cell-based high-throughput screening assay to identify potential antioxidants from a traditional Chinese herbal compound library comprising 845 active monomers [ 7 ]. Piceatannol (PIC) was among the top 22 hits in a primary screening. In a dose–response-based secondary screening, 10 µM PIC pretreatment maintained viability in H 2 O 2 -induced cells at 81.7%, compared to approximately 50% in non-pretreated cells [ 7 ]. PIC, a stilbene bioactive compound primarily derived from grapes, rhubarb, and sugarcane, has attracted considerable research interest due to its strong antioxidant properties [ 8 , 9 ]. Nevertheless, literature on the effect of PIC supplementation on piglet growth performance and antioxidant capacity is limited. Therefore, in the present study, we evaluated the effects of dietary PIC supplementation on growth performance and antioxidant capacity in weaning piglet and explored the underlying mechanisms of its antioxidant effects using a porcine intestinal epithelial cell model. We expect that our results may deepen the understanding of PIC’s antioxidant effects and mechanisms in piglets, thus providing a theoretical foundation for the development of effective antioxidants to alleviate oxidative stress in animals. Materials and methods Animal ethics statement The animal experiments were conducted in compliance with the ethical guidelines approved by the Laboratory Animal Ethical Commission of the Institute of Animal Husbandry and Veterinary Medicine (IAHVM), Beijing Academy of Agriculture and Forestry Sciences (BAAFS; Beijing, China) (approval No. IAHVM11-2304-21). Animal experimental design and management A total of 100 piglets (Duroc × Landrace; 28 days old; 8.71 ± 1.36 kg) were selected and randomly assigned to four treatment groups. Each treatment group included five replicates, with five piglets per replicate. Piglets in the control group were administered a basal diet, whereas piglets in the treatment groups received the basal diet supplemented with 100 (low dose), 200 (medium dose), or 300 (high dose) mg/kg PIC (Great Forest Biomedical, Hangzhou, China; purity > 99%), respectively. The detailed composition of the basal diet, formulated according to the NRC (2012) and the Nutrient Requirements of Swine (2020) [ 10 , 11 ], is presented in Table 1 . The doses of PIC used in this experiment were based on previous reports on structural analogs. Ad libitum feeding and watering was maintained for all piglets during the entire experimental period. Body weight and feed intake were recorded for all piglets throughout the experimental period to determine growth performance parameters, including average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion rate. Table 1 Ingredients and nutrient composition of basal diets (as-fed basis, %). Items Days 0 to 15 Days 15 to 35 Ingredient Corn 37.50 68.00 Extruded corn 11.00 Soybean meal 8.00 13.00 Extruded soybean 12.50 9.00 Wheat flour 18.50 Fish meal 5.10 3.50 Whey powder 4.00 3.70 Limestone 0.95 0.48 Dicalcium phosphate 0.11 0.70 Salt 0.30 0.30 L-Lysine HCl 0.58 0.32 DL-Methionine 0.13 L-Threonine 0.30 L-Tryptophan 0.03 Vitamin and mineral Premix 1 1.00 1.00 Total 100.00 100.00 Nutrient levels 2 Digestible energy, MJ/kg 14.98 14.68 Crude protein 18.52 17.13 Calcium 0.80 0.70 Phosphorus 0.70 0.58 Lysine 1.40 1.17 Methionine 0.46 0.31 Threonine 0.84 0.55 Tryptophan 0.25 0.18 1 Each kg of ration provides: VA 9800 IU; VB 1 4.5 mg; VB 2 11 mg; VB 6 6.5 mg; VB 12 0.1 mg; VD 3 2700 IU; VE 50 IU; VK 3 4.1 mg; Folic acid 1.9 mg; Nicotinic acid 48 mg; D-pantothenate acid 25 mg, Biotin 0.3 mg. Cu 64 mg; Fe 100 mg; Mn 41 mg; Zn 100 mg; Se 0.3 mg; I 0.5 mg. 2 Digestible energy is calculated according to NRC (2012), other nutrient levels are analysed according to PRC National Standard. Serum sample collection and biochemical analysis Following an overnight fast on days 15 and 35, blood samples were obtained from the anterior vena cava of each piglet. Serum was obtained by centrifugation at 4,000 × g for 10 minutes at 4°C. Serum levels of total antioxidant capacity (T-AOC), glutathione (GSH), and MDA, along with activities of total superoxide dismutase (T-SOD) and catalase (CAT), were determined using commercial kits per manufacturer instructions. T-AOC, CAT, and GSH kits were obtained from Jiancheng Bioengineering Institute (Nanjing, China), T-SOD and MDA kits from Beyotime Biotechnology (Shanghai, China). For data normalization, total protein concentrations were quantified using a bicinchoninic acid (BCA) assay (Huaxing bio, Beijing, China). 16S microbiome sequencing and bioinformatics analysis Total microbial genomic DNA was isolated from fecal samples using the E.Z.N.A. Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s protocol. The V3-V4 hypervariable regions of bacterial 16S rRNA genes were amplified with barcoded primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR amplicons were size-fractionated by 2% agarose gel electrophoresis, purified using a PCR Clean-Up Kit (YuHua, Shanghai, China), and quantified with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) employing the dsDNA HS Assay Kit. Equimolar amounts of purified amplicons were pooled for constructing sequencing libraries, which were paired-end sequenced (2 × 250 bp) on an Illumina NextSeq 2000 platform (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology (Shanghai, China) following standard protocols. Following demultiplexing, raw sequences were processed through quality filtering using fastp (version 0.19.6) and merged with FLASH (v1.2.11). The resulting high-quality sequences were then subjected to denoising via the DADA2 plugin within the QIIME 2 pipeline (using default parameters), which provides single-nucleotide resolution by modeling sample-specific error profiles to generate amplicon sequence variants (ASVs). Taxonomic classification of ASVs was performed using the naive Bayes consensus taxonomy classifier implemented in QIIME 2, with reference to the SILVA 16S rRNA database. Metagenomic functions were predicted based on ASV representative sequences, using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States). Cell culture IPEC-J2 porcine intestinal epithelial cells were generously provided by Dr. Glenn Zhang (Oklahoma State University, Stillwater, OK, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (Gibco/Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 µg/mL streptomycin, 100 U/mL penicillin, and 1% ITS supplement (5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium; ScienCell, San Diego, CA, USA) at 37°C in a humidified 5% CO 2 atmosphere. Routine subculturing was performed every 2–3 days. Oxidative stress model establishment and PIC treatment An in-vitro oxidative stress model was established by treating IPEC-J2 cells with H 2 O 2 . Cells were seeded in 96-well plates (Costar/Corning, Corning, NY, USA) at 8,000 cells/well (100 µL complete medium/well) and cultured for 40 h. Then, the cells were exposed to H 2 O 2 (0–1,400 µM in 100 µM increments) for 4 h. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a Multiskan FC instrument (Thermo Fisher Scientific). Given that 4-h treatment with 1,000 µM H 2 O 2 reduced cell viability by ∼50%, these parameter settings were employed in subsequent experiments. Cells were pretreated with PIC (0, 2.5, 5, 10, 20, or 40 µM) for 24 h and then exposed to 1,000 µM H 2 O 2 for 4 h prior to the CCK-8 assay. Non-treated cells served as controls. Pretreatment with 10 µM PIC conferred the strongest protective effect against H 2 O 2 -induced damage in terms of cell viability. Morphological changes in IPEC-J2 cells were examined using an optical microscope (Olympus, Tokyo, Japan; 100× magnification), and images were captured using a cellSens Entry system (Olympus). Culture supernatants were centrifuged (1,500 rpm, 10 min), and lactate dehydrogenase (LDH) activity was measured using a Cytotoxicity Detection assay kit (C0016, Beyotime Biotechnology) on an Infinite 200 PRO microplate reader (Tecan Trading AG, Switzerland). Determination of MDA production, ROS accumulation, and antioxidant activities IPEC-J2 cells were seeded in 6-well plates (2.5 × 10 5 cells/well) and cultured overnight. Cells were pretreated or not with 10 µM PIC for 24 h and then exposed or not to 1,000 µM H 2 O 2 for 4 h. Post-treatment, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (containing phenylmethylsulfonyl fluoride; Solarbio, Beijing, China) for 15 min. The lysates were centrifuged (12,000 × g , 4°C, 15 min), and the supernatants were collected for analysis. Proteins were quantified using a BCA assay kit. The concentration of MDA was determined using a Lipid Peroxidation MDA Assay Kit (S0131S; Beyotime Biotechnology) following the manufacturer’s instructions. Supernatants after incubation were collected to determine the 8-hydroxy-2′-deoxyguanine (8-OHDG) levels (Jiangsu Meibiao Biotechnology, Jiangsu, China). For ROS detection, cells were trypsinized, washed twice, stained with dichlorodihydrofluorescein diacetate probe (E004-1-1; Jiancheng Bioengineering Institute) at 37°C for 1 h, and analyzed using an Infinite 200 PRO microplate reader (Tecan Trading AG). T-AOC, T-SOD, and CAT activities and GSH levels in cell lysates were measured using the above-mentioned commercial kits. Quantitative reverse transcription (RT-qPCR) analysis Following cell treatments, total RNA was isolated using RNAzol (Molecular Research Center, Cincinnati, OH, USA) and alcohol precipitation. RNA concentration and quality were measured using a DS-11spectrophotometer (DeNovix, Wilmington, DE, USA). First-strand cDNA was synthesized from 1 µg RNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. qPCRs were run using iTaq Universal SYBR Green Supermix (Bio-Rad) on a QuantStudio 3 system (Thermo Fisher Scientific) with porcine-specific primers (Table S1 ). The thermal cycling protocol consisted of 40 cycles of 95°C for 30 s (denaturation), 60°C for 30 s (annealing), and 72°C for 20 s (extension). Target gene expression levels were normalized to GAPDH expression and calculated using the 2 –ΔΔCt method [ 12 ]. Western blot analysis Following cell treatments, proteins were extracted with RIPA lysis buffer, and their concentrations were quantified by BCA assay followed by normalization. Samples were denatured in 5× SDS loading buffer (Beyotime Biotechnology) at 95°C for 5 min and resolved by 10%–12% SDS polyacrylamide gel electrophoresis. Following electrophoretic separation, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% (w/v) skim milk in Tris-buffered saline containing Tween-20 for 3 h at room temperature, followed by overnight incubation with primary antibodies at 4°C overnight. After washing, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies for 1 h. All antibodies used in this study are listed in Table S2 . Immunoreactive bands were visualized using an ECL-Plus chemiluminescence substrate (Beyotime Biotechnology) and imaged with a ChemiDoc Imaging System (Bio-Rad Laboratories). Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). Establishment of Nrf2 -knockdown IPEC-J2 cells Porcine Nrf2 siRNA (sense strand: 5′-GCCCAUUGAUCUCUCUGAUTT-3′; antisense strand: 5′-AUCAGAGAGAUCAAUGGGCTT-3′), synthesized at GenePharma (Shanghai, China), was transfected into IPEC-J2 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Successfully transfected cells were pretreated or not with 10 µM PIC for 24 h and then exposed to 1,000 µM H 2 O 2 for 4 h. Non- siRNA-treated cells served as controls. Cell viability, MDA concentration, and ROS accumulation were measured as described above. Statistical analysis In-vivo and in-vitro experimental data were processed using GraphPad Prism v6 (GraphPad Software, San Diego, CA, USA). All data are presented as mean and standard error of the mean (SEM) and statistically analyzed using the mixed linear model in SPSS (v20.0, SPSS, Chicago, IL, USA) as follows: Y ijk = µ + T i + P j + S k + e ijk , where Y ijk is the dependent variable; µ is the overall mean; T i is the fixed treatment effect, P j is the period effect; S k is the steer effect; e ijk is random error. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test in SPSS was used to identify significant differences among the groups in piglet experiment. Linear and quadratic effects of dietary PIC supplementation doses were evaluated using regression analysis. An unpaired two-tailed Student’s t -test was used to compare group means in porcine cell experiments. Statistical significance was set to P < 0.05. Gut microbiota bioinformatics analysis was conducted using the Majorbio Cloud platform ( https://cloud.majorbio.com ). Microbial community similarity among samples was evaluated through principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity matrices using the Vegan package (v2.5-3). Treatment effects on community composition were statistically analyzed by PERMANOVA, which quantified the percentage of variation explained and its statistical significance. Linear discriminant analysis effect size (LEfSe) analysis was conducted to identify taxa with significantly different abundances (from phylum to genus level) of bacteria among groups based on linear discriminant analysis score > 2.5 and P 0.6 and false discovery rate-adjusted P < 0.01. Results PIC improves growth performance Dietary PIC supplementation improved weight gain in piglets (Table 2 ). On day 35 of the trial, the average weight in each treatment group was higher than that in the control group, with a significant difference observed in the medium-dose PIC group ( P < 0.05). PIC supplementation increased the ADG during the 0–15-day and 0–35-day periods, with a significant effect in the medium-dose group ( P < 0.05). During the 0–15-day period, the addition of medium and high doses of PIC significantly improved the ADFI of piglets ( P < 0.05). The addition of PIC reduced the feed conversion efficiency during piglet rearing, and the feed-to-gain ratio was significantly lower in the medium-dose group compared with the control group ( P < 0.05) over the entire 0–35-day period. Furthermore, with increasing PIC dose, the final weight of piglets on day 35 and the 0–35-day ADG showed linear and quadratic responses ( P < 0.05). The final weight on day 15, 0–15-day ADG, and ADFI exhibited a linear increase ( P < 0.05), whereas the 0–35-day feed-to-gain showed a linear decrease ( P < 0.05). Table 2 Effects of piceatannol (PIC) on growth performance in weaning piglets. Items Piceatannol, mg/kg SEM P -value 0 100 200 300 ANOVA Linear Quadratic BW, kg Day 0 8.72 8.70 8.69 8.70 0.133 1.000 0.965 0.965 Day 15 15.0 15.3 15.9 15.7 0.157 0.163 0.047 0.437 Day 35 28.0 b 29.2 ab 29.9 a 29.4 ab 0.241 0.015 0.010 0.035 ADG, g Day 0 to 15 418 b 440 ab 479 a 469 ab 8.796 0.039 0.011 0.285 Day 15 to 35 649 693 703 682 11.51 0.397 0.296 0.178 Day 0 to 35 550 b 584 ab 607 a 591 ab 7.135 0.020 0.012 0.042 ADFI, g Day 0 to 15 717 b 751 ab 799 a 791 a 11.05 0.016 0.003 0.254 Day 15 to 35 1199 1212 1222 1196 13.94 0.919 0.995 0.522 Day 0 to 35 993 1014 1041 1022 9.886 0.412 0.211 0.326 F/G Day 0 to 15 1.74 1.71 1.67 1.69 0.029 0.874 0.519 0.653 Day 15 to 35 1.86 1.75 1.74 1.75 0.024 0.264 0.127 0.217 Day 0 to 35 1.81 a 1.74 ab 1.72 b 1.73 ab 0.012 0.028 0.015 0.053 BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion rate. Mean and total SEM are listed in separate columns. Different superscript letters within a row indicate significant differences ( P < 0.05). PIC improves serum antioxidant status The effects of PIC on piglet antioxidant indicators are shown in Table 3 . In comparison to the control group, the low-dose PIC group had significantly enhanced serum T-AOC on day 15 and CAT activity on day 35 ( P < 0.05). The medium-dose PIC group showed more comprehensive effects, with significantly increased T-AOC and reduced MDA content on days 15 and 35, along with elevated CAT activity on day 35 ( P < 0.05). The high-dose PIC group exhibited distinct regulatory effects, with significantly reduced MDA contents on day 15 and increased GSH levels on day 35 ( P 0.05). Dose–response analysis revealed that day 15 T-AOC levels responded to PIC supplementation in both linear and quadratic patterns ( P = 0.078; P < 0.01). Linear relationships were observed for GSH levels on days 35, as well as day-15 MDA content ( P < 0.01). Quadratic responses were identified for day-35 T-AOC levels, CAT activity, and MDA content ( P < 0.05, P < 0.01), indicating complex dose-dependent effects of PIC on the antioxidant system during different developmental stages. Table 3 Effects of piceatannol (PIC) on serum antioxidant capacity in weaning piglets. Items Piceatannol, mg/kg SEM P -value 0 100 200 300 ANOVA Linear Quadratic Day 15 T-SOD, U/g protein 0.90 0.97 0.94 0.79 0.031 0.166 0.184 0.067 T-AOC, mmol/g protein 0.77 b 0.91 a 0.92 a 0.87 ab 0.018 0.006 0.023 0.004 CAT, U/mL 1.65 1.94 2.52 2.19 0.178 0.390 0.190 0.394 GSH, µmol/g protein 10.32 9.19 10.50 9.30 0.247 0.121 0.400 0.934 MDA, µmol/mg protein 3.63 a 3.28 ab 2.33 b 2.52 b 0.174 0.010 0.003 0.332 Day 35 T-SOD, U/g protein 0.85 1.17 0.89 0.82 0.074 0.321 0.545 0.199 T-AOC, mmol/g protein 0.19 b 0.22 ab 0.23 a 0.20 ab 0.006 0.007 0.408 0.001 CAT, U/mL 1.50 b 2.15 a 2.12 a 1.80 ab 0.083 0.006 0.139 0.001 GSH, µmol/g protein 12.41 b 13.35 ab 13.62 ab 13.92 a 0.190 0.015 0.003 0.308 MDA, µmol/mg protein 7.68 a 6.16 ab 4.54 b 6.68 ab 0.427 0.046 0.162 0.021 T-SOD, total superoxide dismutase; T-AOC, total antioxidant capacity; CAT, catalase; GSH, glutathione; MDA, malondialdehyde. Mean and total SEM are listed in separate columns. Different superscript letters within a row indicate significant differences ( P < 0.05). Dietary PIC supplementation alters gut microbial diversity and structure in weanling piglets PIC supplementation significantly altered β-diversity in the gut microbiota of piglets as indicated by PCoA results ( P < 0.01) (Fig. 1 A). On day 15 post-weaning, the fecal microbiota composition was dominated by four major phyla: Firmicutes, Bacteroidota, Actinobacteriota, and Spirochaetota, which collectively accounted for > 99% of the total relative abundance (Fig. 1 B). The dominant bacterial genera included norank_o_Clostridia_UCG-014 , norank_f_Muribaculaceae , norank_o_RF39 , Clostridium_sensu_stricto_1 , norank_f_Eubacterium_coprostanoligenes_group , and Prevotella (Fig. 1 C). LEfSe analysis revealed significant microbial enrichments across groups (Fig. 1 D). Genera such as Prevotellaceae_NK3B31_group , UCG-002 , and UCG-005 were significantly enriched in the low-dose PIC group. Catenisphaera , Prevotella , and Blautia , were significantly enriched specifically in the medium-dose PIC group, whereas the high-dose PIC group was significantly enriched in Bacillus , Subdoligranulum , and Asteroleplasma . Dietary PIC supplementation attenuates oxidative stress in association with gut microbiota alterations in weaning piglets The correlation heatmap based on correlation coefficients in Fig. 1 E depicts the relationships between microbial species in piglet feces and serum antioxidant indices. T-AOC exhibited positive correlations with norank_o__Clostridia_UCG-014 and Faecalibacterium , and negative correlations with NK4A214_group and norank_o__RF39 ( P < 0.05). Serum T-SOD activity was significantly positively correlated with UCG-002 , and negatively with Terrisporobacter ( P < 0.05). Serum CAT activity exhibited a significant negative correlation with norank_o__RF39 ( P < 0.05). Serum MDA content was significantly positively correlated with Collinsella , NK4A214_group , Prevotellaceae_NK3B31_group ( P < 0.05), but negatively with Blautia and Faecalibacterium ( P < 0.05). KEGG functional prediction analysis of microbial communities using PICRUSt2 annotated a total of 1,770 enzymes. Figure 1 F presents a heatmap of the top 20 most significantly enriched enzyme categories. Dietary supplementation with 200 mg/kg PIC significantly increased the abundances of phosphomethylpyrimidine synthase [EC 4.1.99.17], methionine synthase [EC 2.1.1.13], and adenosylcobinamide kinase [EC 2.7.1.156] in the piglet gut microbiota, whereas it significantly reduced the abundances of phosphoenolpyruvate carboxylase [EC 4.1.1.31], adenosine deaminase [EC 3.5.4.4], and oligopeptidase B [EC 3.4.21.83]. PIC exerts a cytoprotective in IPEC-J2 cells Cell viability was the highest in the (non-treated) control group, indicating normal cellular conditions. Exposure to H 2 O 2 decreased cell viability in a concentration-dependent manner, indicating strong oxidative stress-induced cytotoxicity (Fig. 2 A). PIC pretreatment improved viability compared to H 2 O 2 alone, particularly at PIC concentrations of 10–20 µM, suggesting a protective role of PIC against oxidative damage (Fig. 2 B). Morphological analysis revealed that cells in the control group showed normal cell morphology, whereas H 2 O 2 -treated IPEC-J2 cells exhibited enlarged intercellular gaps and disrupted membrane integrity (Fig. 2 C). PIC pretreatment significantly attenuated these morphological alterations and effectively preserved cellular integrity. LDH activity is a marker of cell membrane integrity. LDH levels were the lowest in the control group, consistent with minimal cellular damage (Fig. 2 D). Cells in the H 2 O 2 -treated group displayed significantly elevated LDH activity ( P < 0.01), indicating cellular membrane disruption. Cells in the PIC + H 2 O 2 group showed reduced LDH release compared with those treated with H 2 O 2 alone ( P < 0.05), indicating a cytoprotective effect of PIC. Characterization of the antioxidant activities of PIC Figure 3 illustrates the production of oxidative stress products, including MDA, 8-OHDG, and ROS, in IPEC-J2 cells in the different treatment groups. The control group exhibited the lowest MDA content. The H 2 O 2 -treated group displayed a significant increase in MDA levels, suggesting pronounced oxidative stress. Pretreatment with PIC significantly reduced the MDA content compared with H 2 O 2 treatment alone ( P < 0.01), implying that PIC effectively mitigates H 2 O 2 -induced lipid peroxidation. 8-OHDG is a major hallmark of DNA oxidative damage. PIC pretreatment significantly suppressed 8-OHDG production compared with H 2 O 2 treatment alone ( P < 0.05). ROS generation was measured based on fluorescence intensity. H 2 O 2 treatment enhanced ROS levels by 1.45-fold compared to those in the control group, indicating oxidative stress induction. ROS levels were maintained at control levels in the PIC alone group, and the PIC + H 2 O 2 group exhibited only partial ROS elevation compared to H 2 O 2 alone group. These findings suggest that PIC prevents spontaneous ROS formation and partially counteracts H 2 O 2 -induced oxidative stress. Effects of PIC on cell apoptosis Excess intracellular ROS trigger apoptosis. The effects of PIC on H 2 O 2 -induced apoptosis are shown in Fig. 4 . RT-qPCR analysis revealed that H 2 O 2 treatment markedly upregulated pro-apoptotic genes while downregulating anti-apoptotic genes. Notably, PIC alone significantly elevated B-cell lymphoma 2 ( BCL2 ) expression and decreased the expression of Caspase 3 ( CASP3 ) ( P < 0.05). PIC pretreatment significantly alleviated H 2 O 2 -induced apoptosis as demonstrated by decreased Bcl-2-associated X protein ( BAX ) and CASP3 expression and increased BCL2 expression compared to the levels after H 2 O 2 treatment alone ( P < 0.05; P < 0.01). Consistent with the trends observed in the RT-qPCR results, western blotting revealed that H 2 O 2 treatment reduced Bcl-2 expression and enhanced Bax and Cleaved Caspase 3 expression, whereas PIC reversed these effects. Effects of PIC on the antioxidant defense system As shown in Fig. 5 A, PIC treatment alone significantly upregulated the mRNA levels of SOD1 , CAT , GPX1 , HO1 , and NQO1 , demonstrating its intrinsic antioxidant capacity. Compared with H 2 O 2 treatment alone, PIC pretreatment significantly attenuated H 2 O 2 -induced alterations in these antioxidant enzyme transcript levels. Furthermore, PIC pretreatment markedly elevated T-AOC levels compared to H 2 O 2 treatment alone (Fig. 5 B). Consistent with these gene expression profiles, PIC pretreatment alleviated T-SOD, GSH, and CAT activities compared to H 2 O 2 treatment alone, although the effect on CAT was not significant. These results suggest that PIC may confer protective effects by activating endogenous antioxidant defense systems. PIC antioxidant capacity involves Nrf2 signaling To explore the protective mechanisms of PIC further, we assessed whether Nrf2 activation contributed to the enhanced antioxidant capacity observed in PIC-pretreated IPEC-J2 cells (Fig. 6 ). Immunoblotting results revealed that PIC pretreatment promoted Nrf2 accumulation, indicating Nrf2 pathway activation. To further elucidate the role of Nrf2 in PIC-mediated attenuation of oxidative stress, we generated Nrf2 -knockdown IPEC-J2 cells and assessed treatment effects on cell viability, MDA generation, and ROS production. Nrf2 siRNA-transfected cells demonstrated significant downregulation of Nrf2 expression compared to negative control siRNA-treated cells, as confirmed by RT-qPCR and western blot analyses. Following PIC pretreatment and H 2 O 2 challenge, Nrf2 -knockdown IPEC-J2 cells exhibited significantly reduced viability compared to control siRNA-transfected cells ( P < 0.01). Nrf2 knockdown significantly weakened PIC’s preventing effect on H 2 O 2 -induced MDA and ROS accumulation ( P < 0.05; P < 0.01). These results suggest that the oxidation-protective effect of PIC was blocked by Nrf2 siRNA, indicating the involvement of Nrf2 signaling in the antioxidant function of PIC. Discussion Accumulating evidence has established that plant polyphenols such as curcumin, resveratrol, ferulic acid, ellagic acid, quercetin, chlorogenic acid, and catechin exhibit potent antioxidant properties and therapeutic potential against oxidative stress and its related pathologies [ 13 , 14 ]. Particularly resveratrol has attracted extensive attention owing to its beneficial effects on human and animal health [ 15 , 16 , 17 ]. As a resveratrol analog, PIC features two hydroxyl groups in ortho position of the resveratrol structure. PIC exhibits an 11-fold higher peroxyl radical-scavenging activity than its parent compound, resveratrol [ 18 ]. In C2C12 myotubes, PIC upregulated the expression of HO1 and SOD1 , thereby reducing H 2 O 2 -induced intracellular ROS accumulation [ 19 ]. In mice, PIC supplementation mitigated lipopolysaccharide-induced oxidative stress in liver tissues [ 20 ]. In piglets, dietary PIC supplementation mitigated hepatic injury, likely via the modulation of redox homeostasis, maintenance of mitochondrial function, and prevention of excessive apoptosis [ 21 ]. However, information on the supplementation effects of PIC in weaning piglets and its potential mechanism remained insufficient. The growth performance of piglets is intrinsically associated with their health status, with oxidative stress typically leading to impaired growth performance [ 1 , 22 ]. Many multifunctional natural compounds exert health-promoting effects, improving growth and production performance, in livestock animals [ 23 , 24 ]. Jia et al. [ 21 ] studied the effects of PIC administration on weaning piglet performance under short-term acute stress induced by diquat. They found no significant differences during the first week post-weaning, but demonstrated that PIC restored the body weight reduction induced by diquat challenge. However, this study did not evaluate the effects of PIC on weaned piglet growth performance throughout the nursery period. In the present investigation, the administration of medium and high doses of PIC significantly improved the ADG during the early weaning phase and throughout the nursery period, and high-dose PIC significantly improved the feed conversion efficiency. While we did not use an in-vivo oxidative stress challenge model, our study clearly demonstrated the growth-promoting effects of PIC in weaning piglets. Further studies are needed to confirm its beneficial effects, particularly under oxidative stress conditions, in piglets. Weaning induces multiple stress pathways in piglets, disrupting intestinal redox homeostasis and leading to oxidative stress [ 25 ]. Early weaning compromises intestinal antioxidant capacity in piglets, as evidenced by elevated MDA and ROS levels coupled with significantly reduced antioxidant enzyme activities in the jejunum [ 13 ]. PIC has been demonstrated to stimulate the antioxidant system and enhance antioxidant enzyme capacity, thereby mitigating oxidative stress damage in various cell lines and hosts. Treatment with PIC conferred protection against oxidative stress in skeletal muscle cells through upregulation of antioxidant enzymes, including HO-1 and SOD1 [ 19 ]. PIC administration improved hepatic SOD activity and reduced lipid peroxidation in lipopolysaccharide-induced endotoxemic mice [ 20 ]. In the study in piglets by Jia et al. [ 21 ], dietary PIC administration improved hepatic SOD and GPX activities and decreased the amount of MDA after diquat challenge. In porcine intestinal epithelial cells, PIC pretreatment significantly attenuated deoxynivalenol-induced oxidative damage through coordinated upregulation of protein expression of phase II detoxification enzymes (NQO1 and GCLM) and transcriptional activation of antioxidant genes ( CAT , SOD1 , PRX3 , and GSTA4 ) [ 26 ]. In the present study, PIC supplementation improved piglet serum T-AOC and CAT activity and decreased the amount of MDA, indicating its antioxidant potential in piglets. In vitro , it enhanced antioxidative gene expression and enzyme activity. To our knowledge, this represents the first experimental demonstration of the antioxidant efficacy of PIC in piglets through feeding trials and intestinal epithelial cell studies. Our results provide novel insights supporting the application of PIC as a functional additive in pig production. Early weaning reduces gut microbial diversity and induces microbiota dysbiosis, enhancing the susceptibility to diarrheal diseases and other health complications, in piglets [ 27 ]. Intestinal oxidative stress during the weaning stage triggers gut microbiota dysbiosis, thereby contributes to the pathogenesis of post-weaning diarrhea and enteric infections. The present study demonstrated that dietary PIC supplementation alters the gut microbiota composition, which may contribute to the maintenance of intestinal homeostasis. Blautia is a functional genus with probiotic characteristics [ 28 ]. In weaning piglets supplemented with a composite probiotic, Blautia abundance was significantly negatively correlated with MDA contents, and positively with GSH activity [ 29 ]. In line herewith, significant negative correlation between Blautia abundance and MDA levels was observed in our study. Increased Faecalibacterium abundance has been found to benefit intestinal antioxidant capacity and gut barrier integrity [ 30 ]. Consistent herewith, we found a significant positive correlation between Faecalibacterium abundance and T-AOC, and negative correlation with MDA levels, in weaning piglets. Blautia and Faecalibacterium were the dominant bacteria in the medium-dose supplementation group in the present study, which indicates that PIC-induced elevation of these bacteria contributes to enhanced systemic antioxidant capacity. In drinking water supplemented with acidifier, Xu et al. [ 31 ] found a significant positive correlation between Subdoligranulum abundance and serum antioxidant capacity. In the present study, Subdoligranulum was the dominant bacterium in the high-dose supplementation group. We found a negative correlation between Subdoligranulum relative abundance and serum MDA concentrations and positive associations with T-AOC and CAT activity (although statistical significance was not reached), implying that increased Subdoligranulum abundance may promote antioxidant defenses. Norank_o_RF39 has been proposed as a potentially harmful bacterium and may be associated with reduced antioxidant enzyme activity [ 32 ]. In line herewith, we found negative correlations between norank_o_RF39 and T-AOC and CAT activity, supporting its potential negative impact on host antioxidant function. Collectively, our results indicate that PIC positively modulates the intestinal microbial community, including enhancing the abundance of bacteria with antioxidant activity, thus enhancing the antioxidant capacity and growth rate of piglets. Phosphomethylpyrimidine synthase plays an essential role in the biosynthesis of thiamine (vitamin B 1 ) [ 33 ], which is pivotal in sulfur metabolism and hence antioxidant capacity [ 34 ]. KEGG pathway enrichment analysis revealed that phosphomethylpyrimidine synthase [EC 4.1.99.17] was significantly enriched by PIC supplementation, suggesting the potential benefit of PIC in improving piglet antioxidant status. Cobalamin has been shown to promote porcine oocyte maturation while reducing ROS generation and elevating GSH levels [ 35 ]. Adenosylcobinamide kinase [EC 2.7.1.156], a key enzyme in cobalamin biosynthesis [ 36 ], was upregulated upon PIC supplementation and thus may have contributed to the enhanced antioxidant capacity in piglets. Finally, PIC supplementation significantly enriched methionine synthase [EC 2.1.1.13], which participates in methionine biosynthesis. In swine, dietary supplementation with 0.12% dl -methionine analogs significantly increased serum T-SOD activity and GSH content while improving growth performance [ 37 ]. PIC's oxidative stress-mitigating effects were comprehensively assessed using in-silico , in-vitro , and mouse models [ 38 ]. H 2 O 2 can directly enter cells and induce reversible oxidative stress; therefore, it is widely employed as an oxidative stress inducer in in vitro systems [ 39 ]. In IPEC-J2 cells, H 2 O 2 treatment stimulated MDA secretion and triggered an intracellular ROS burst. Pretreatment with PIC significantly suppressed both MDA production and H 2 O 2 -induced ROS accumulation. These findings align with previous findings that PIC pretreatment effectively inhibits ROS and MDA accumulation in oxidative stress-damaged cells and animal models, ameliorating oxidative stress-induced cellular damage [ 40 , 41 ]. Apoptosis is typically triggered when cells experience oxidative stress. Oxidative stress can trigger apoptosis through the coordinated upregulation of pro-apoptotic factors and downregulation of anti-apoptotic mediators at both transcriptional and translational levels [ 42 ]. PIC downregulated Bcl-XL and inhibited the activation of caspase-3 and caspase-8 in PC12 cells incubated with hydrogen peroxide or SIN-1, thus inhibiting apoptosis [ 43 ]. PIC supplementation significantly attenuated H 2 O 2 -induced apoptosis in ARPE-19 cells, as evidenced by a reduced Bax/Bcl-2 ratio and decreased cleavage of caspase-3 and PARP, potentially through the modulation of Akt phosphorylation status [ 40 ]. In our study, PIC alleviated H 2 O 2 -induced oxidative damage and apoptosis in porcine epithelial cells. However, the signaling pathways involved, and whether PIC affects mitochondrial or non-mitochondrial apoptotic pathways, require further investigation. Nrf2 is a crucial redox-sensitive transcription factor that serves as the primary regulator of certain antioxidant enzymes and detoxification genes, playing a pivotal role in maintaining cellular redox homeostasis [ 44 ]. Key antioxidant factors regulated by Nrf2 include NQO1, HO-1, SOD, CAT, and related enzymes [ 45 ]. PIC protected retinal pigment epithelium cells against H 2 O 2 -mediated damage via Nrf2 signaling and GCLc , SOD , and HO1 antioxidant gene transcription [ 40 ], indicating that PIC may exert its cytoprotective effects through Nrf2 pathway activation. Our data showed that PIC pretreatment potently activated Nrf2 signaling, as evidenced by significantly elevated Nrf2 phosphorylation levels as compared to those in control and H 2 O 2 treatments. This activation was accompanied by increased antioxidant enzyme expression, indicating that Nrf2 transcriptional activity mediates the cytoprotective effects of PIC against H 2 O 2 -induced oxidative damage. To validate Nrf2 pathway involvement, we assessed MDA production and ROS accumulation following Nrf2 knockdown in IPEC-J2 cells. Notably, the protective effects of PIC against H 2 O 2 -induced cellular damage were completely abolished under Nrf2-deficient conditions, confirming that Nrf2 signaling is essential for PIC-mediated cytoprotection against oxidative stress in porcine intestinal epithelial cells. PIC’s antioxidant effects may also involve other signaling pathways, such as PI3K/Akt or STAT3, or organelle functional regulation [ 40 , 46 ]. More comprehensive investigations are warranted to fully elucidate the precise molecular mechanisms underlying the antioxidant properties of PIC on porcine cells. Conclusions This study provides the first experimental evidence for the beneficial effects of dietary PIC supplementation on weaning piglet growth performance, demonstrating that its mechanism may involve gut microbiota modulation and Nrf2-mediated antioxidant defense system activation. These findings highlight PIC acts as a potential antioxidant agent, thereby enhancing growth performance and health status in piglets. Further investigations are warranted to fully elucidate its mechanisms of action, its application in appropriate animal models, and its bioavailability and toxicity in animals to investigate its efficacy as a natural antioxidant in livestock production. Abbreviations ADFI Average daily feed intake ADG Average daily gain BAX Bcl-2-associated X protein BCL2 B-cell lymphoma 2 CASP3 Caspase 3 CAT Catalase GAPDH Glyceraldehyde-3-phosphate dehydrogenase GSH Glutathione HO1 Heme oxygenase-1 LDH Lactate dehydrogenase MDA Malondialdehyde NQO1 Quinone oxidoreductase-1 Nrf2 Nuclear factor erythroid 2-related factor 2 ROS Reactive oxygen species T-AOC Total antioxidant capacity T-SOD Total superoxide dismutase 8-OHDG 8-hydroxy-2′-deoxyguanine Declarations Author contributions Conceptualization and design, JW and TF; methodology, LLZ and YYC; formal analysis and writing-original draft preparation, LLZ; investigation and resources, MXC and LC; project administration and data curation, LLZ and QJW; funding acquisition, supervision, writing-review and editing, JW and TF. All authors read and approved the final manuscript. Funding This work was financially supported by the Innovation Capacity Building—Science and Technology Research Project of BAAFS (grant No. KJCX20251005), the National Key R&D Program of China (grant No. 2022YFD130040311), and the Special Program on Science and Technology Innovation Capacity Building of BAAFS (grant No. KJCX 20240339). Availability of data and materials The data generated and/or analyzed during the current study are available from the corresponding authors upon request. Ethics approval and consent to participate The animal experiments were conducted in compliance with the ethical guidelines approved by the Laboratory Animal Ethical Commission of the Institute of Animal Husbandry and Veterinary Medicine (IAHVM), Beijing Academy of Agriculture and Forestry Sciences (BAAFS; Beijing, China) (approval No. IAHVM11-2304-21). Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. References Hao Y, Xing MJ, Gu XH. 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Supplementary Files Fig4BBax.jpg Fig4BBcl2.jpg Fig4BCleavedCaspase3.jpg Fig4Bactin.jpg Fig6ANrf2.jpg Fig6Aactin.jpg Fig6ApNrf2.jpg Fig6CNrf2.jpg Fig6Cactin.jpg GraphicalAbstract.png SupplementaryTable1.docx SupplementaryTable2.docx Cite Share Download PDF Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Minor revision 14 Oct, 2025 Reviewers agreed at journal 29 Sep, 2025 Reviewers invited by journal 17 Sep, 2025 Editor assigned by journal 13 Sep, 2025 First submitted to journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":1713726,"visible":true,"origin":"","legend":"\u003cp\u003e16S rRNA microbiome sequencing in weaning piglets on PIC supplementation, and bioinformatics analysis. (A) Principal coordinate analysis (PCoA) plot generated based on an unweighted UniFrac matrix. (B) Phylum-level microbiota profiles of PIC-treated and non-treated control piglets. (C) Genus-level microbiota profiles of PIC-treated and control piglets. (D) Differences between bacterial taxa in PIC-treated and control piglets. The histogram shows differentially abundant bacterial species in the treatment and control groups ranked by LDA scores. (E) Spearman correlation heatmaps of the correlations between serum antioxidant indices and relative abundances of fecal bacterial genera. Only the top 20 bacterial genera are shown. Asterisks represent significant positive or negative correlations (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (F) Significant differences in PICRUSt2-predicted gene counts for enzymes between medium dose treatment (200 mg/kg PIC) and control groups. Only the top 20 enzymes in terms of significance are shown.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/684d163bcbf3e804dffabc47.png"},{"id":92270669,"identity":"c94083c3-4134-43d6-bf14-5c5dfe287a30","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5571230,"visible":true,"origin":"","legend":"\u003cp\u003ePIC ameliorates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage in IPEC-J2 cells. (A) Cell viability of IPEC-J2 cells after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at different concentrations. (B) Protective effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cell damage at different concentrations. (C) Morphological analysis of the protective effect of PIC against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced IPEC-J2 cell damage using microscopy. (D) Effect of PIC on lactate dehydrogenase (LDH) release. CON, cells not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PIC; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h; PIC, cells treated with 10 μM PIC for 24 h; PIC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells pretreated with 10 μM PIC for 24 h and then treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Data represent means ± SEMs. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/4c7c7447d77134ef5b1f066c.png"},{"id":92270670,"identity":"851adfac-4654-464c-bddc-ddb70a06752b","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2317618,"visible":true,"origin":"","legend":"\u003cp\u003ePIC attenuates redox disturbance in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced IPEC-J2 cells. (A) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced MDA production. (B) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced 8-OHDG production. (C) Representative images of dichlorodihydrofluorescein diacetate staining. (D) Quantitation of ROS levels based on dichlorodihydrofluorescein diacetate fluorescence in panel C. CON, cells not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PIC; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h; PIC, cells treated with 10 μM PIC for 24 h; PIC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells pretreated with 10 μM PIC for 24 h and then treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Data represent means ± SEMs. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/63e21809ad9bec163cf608ee.png"},{"id":92270671,"identity":"891d1cb7-c98d-4bdf-aa1c-f30a025ef9f1","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1055524,"visible":true,"origin":"","legend":"\u003cp\u003ePIC attenuates apoptosis in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced IPEC-J2 cells. (A) mRNA levels of apoptosis-associated genes as analyzed using RT-qPCR. (B) Representative western blot image of apoptosis-related proteins. (C) Quantitative results of protein levels in panel B. CON, cells not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PIC; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h; PIC, cells treated with 10 μM PIC for 24 h; PIC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells pretreated with 10 μM PIC for 24 h and then treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Data represent means ± SEMs. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/91b6facd539da28cd8e53105.png"},{"id":92271495,"identity":"a1f4fda3-9cec-4cea-a6c2-df864034735d","added_by":"auto","created_at":"2025-09-26 14:44:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":450812,"visible":true,"origin":"","legend":"\u003cp\u003ePIC improves antioxidant capacity in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced IPEC-J2 cells. (A) mRNA levels of \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eGPX1\u003c/em\u003e, \u003cem\u003eHO1\u003c/em\u003e, and \u003cem\u003eNQO1\u003c/em\u003e as measured using RT-qPCR. (B) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced total antioxidant capacity. (C) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced superoxide dismutase activity. (D) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced catalase activity. (E) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced reduced glutathione. CON, cells not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PIC; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h; PIC, cells treated with 10 μM PIC for 24 h; PIC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells pretreated with 10 μM PIC for 24 h and then treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Data represent means ± SEMs. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/22310393afea6c812f8bb538.png"},{"id":92271499,"identity":"6fd3c74d-b3eb-4abe-be47-73ccf6aa241a","added_by":"auto","created_at":"2025-09-26 14:44:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3021833,"visible":true,"origin":"","legend":"\u003cp\u003ePIC alleviates oxidative stress via the Nrf2 pathway in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced IPEC-J2 cells. (A) Western blots of Nrf2 and p-Nrf2 levels. (B)\u0026nbsp;Quantitation of Nrf2 and p-Nrf2 levels in panel A. (C) mRNA and protein levels of Nrf2 after Nrf2 silencing. (D) Cell viability, normalized to that of negative control siRNA-transfected cells. (E) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced MDA concentration\u003cem\u003e \u003c/em\u003ein \u003cem\u003eNrf2\u003c/em\u003e-knockdown cells. (F) Effect of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS production\u003cem\u003e \u003c/em\u003ein \u003cem\u003eNrf2\u003c/em\u003e-knockdown cells. (G) Quantitation of ROS levels in panel F. CON, cells not treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and PIC; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h; PIC, cells treated with 10 μM PIC for 24 h; PIC + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cells pretreated with 10 μM PIC for 24 h and then treated with 1,000 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Data represent means ± SEMs. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003cem\u003e \u003c/em\u003eIn \u003cem\u003eNrf2\u003c/em\u003e-knockdown cells\u003cstrong\u003e, \u003c/strong\u003e\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/08a006e15cbd3387dfaeccbd.png"},{"id":101692376,"identity":"1b634d07-ed3c-4aee-ae7f-44ae7df6f095","added_by":"auto","created_at":"2026-02-02 16:18:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14905101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/a6e0000e-645d-4c2f-b891-20d1c3f8299a.pdf"},{"id":92271497,"identity":"b80e701c-cb49-4abb-a7a5-ae64fa467789","added_by":"auto","created_at":"2025-09-26 14:44:36","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":860803,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4BBax.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/aed9634a74d58bb6ac253ac9.jpg"},{"id":92270687,"identity":"d55b01bf-d6e6-4957-913b-6e7cacd0102c","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":985290,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4BBcl2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/d91c4f9ad606398bb69c0960.jpg"},{"id":92270679,"identity":"a79a67e8-b005-4559-a31b-3852d264bdd3","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":1293799,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4BCleavedCaspase3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/1ac17b1350206fd2dcffdf2d.jpg"},{"id":92270699,"identity":"45536697-7fed-4acc-8960-73a8da45e21f","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":920757,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4Bactin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/4020f6c531983aed353d1db0.jpg"},{"id":92271504,"identity":"dccae7a6-6db7-4647-9ed2-28bb481baac5","added_by":"auto","created_at":"2025-09-26 14:44:37","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":943361,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6ANrf2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/c62ad19b81df98f43a6b047b.jpg"},{"id":92270680,"identity":"d5b2d959-1c89-4166-9816-0cf335645ae4","added_by":"auto","created_at":"2025-09-26 14:36:36","extension":"jpg","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":845982,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6Aactin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/af3b4fd23ee2b4f9778e6eed.jpg"},{"id":92271500,"identity":"6232fb37-fb0c-47ba-8579-6a7f9f459b14","added_by":"auto","created_at":"2025-09-26 14:44:37","extension":"jpg","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":871629,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6ApNrf2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/e4d8cda76b413a8e11942987.jpg"},{"id":92270711,"identity":"db5cc0bd-a24a-40cb-bc07-66970462304d","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"jpg","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":803227,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6CNrf2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/b6b97284032c32faf06a2b87.jpg"},{"id":92270709,"identity":"5c23de5e-d58e-4050-9c66-c74757eb5717","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"jpg","order_by":21,"title":"","display":"","copyAsset":false,"role":"supplement","size":822094,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6Cactin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/f4696990f9a8916fde13e1ab.jpg"},{"id":92270719,"identity":"dae2615d-24b6-4c01-88fd-6fc0bb735fc9","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"supplement","size":2336689,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/b5523e08072480595cd8fd63.png"},{"id":92270718,"identity":"80b979f9-a28e-4e20-b72d-e9d7d9e0f005","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"docx","order_by":23,"title":"","display":"","copyAsset":false,"role":"supplement","size":43404,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/fa4de0c0adf324eaeed7b54a.docx"},{"id":92270721,"identity":"ea49aa77-1550-4763-ae64-dd7a64b6d56b","added_by":"auto","created_at":"2025-09-26 14:36:37","extension":"docx","order_by":24,"title":"","display":"","copyAsset":false,"role":"supplement","size":39932,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7540228/v1/5a77149d813d34aa7c0d5320.docx"}],"financialInterests":"","formattedTitle":"Piceatannol, a natural stilbene, improves weaning piglets' growth performance through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the pig industry, oxidative stress often leads to impaired growth performance and various diseases, resulting in significant economic losses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Young animals, including piglets, have immature antioxidant defense systems, rendering them particularly susceptible to free radical accumulation, lipid peroxidation propagation, and consequent oxidative stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In intensive pig production, piglets are highly susceptible to oxidative stress caused by multiple stressors (e.g., environmental, nutritional, and psychological) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], leading to suppressed growth performance and health status. Previous studies have demonstrated that early weaned piglets exhibit obvious oxidative stress, as evidenced by significantly elevated reactive oxygen species (ROS) and malondialdehyde (MDA) in the jejunum and liver, hydroxyl radicals in the colon, and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in the jejunum, colon, and liver [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Continuous oxidative stress in piglets can damage cell function and organs and reduce production performance. Moreover, oxidative stress is an early event in the onset and development of various diseases and is therefore considered an important factor in early-weaning syndrome in piglets. In this context, the use of antioxidant feed additives has been proposed as an effective nutritional strategy to improve piglet health and growth performance.\u003c/p\u003e\u003cp\u003eNatural plant compounds and traditional Chinese medicine extracts have been widely investigated for their antioxidant properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Compounds such as carvacrol, thymol, quercetin, catechins, curcumin, oregano extract, and cinnamaldehyde have been demonstrated to function as effective antioxidants in livestock. These substances enhance the body\u0026rsquo;s antioxidant capacity and alleviate oxidative stress, thereby improving animal health and production performance. We previously established a cell-based high-throughput screening assay to identify potential antioxidants from a traditional Chinese herbal compound library comprising 845 active monomers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Piceatannol (PIC) was among the top 22 hits in a primary screening. In a dose\u0026ndash;response-based secondary screening, 10 \u0026micro;M PIC pretreatment maintained viability in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cells at 81.7%, compared to approximately 50% in non-pretreated cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. PIC, a stilbene bioactive compound primarily derived from grapes, rhubarb, and sugarcane, has attracted considerable research interest due to its strong antioxidant properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nevertheless, literature on the effect of PIC supplementation on piglet growth performance and antioxidant capacity is limited.\u003c/p\u003e\u003cp\u003eTherefore, in the present study, we evaluated the effects of dietary PIC supplementation on growth performance and antioxidant capacity in weaning piglet and explored the underlying mechanisms of its antioxidant effects using a porcine intestinal epithelial cell model. We expect that our results may deepen the understanding of PIC\u0026rsquo;s antioxidant effects and mechanisms in piglets, thus providing a theoretical foundation for the development of effective antioxidants to alleviate oxidative stress in animals.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimal ethics statement\u003c/h2\u003e\u003cp\u003e The animal experiments were conducted in compliance with the ethical guidelines approved by the Laboratory Animal Ethical Commission of the Institute of Animal Husbandry and Veterinary Medicine (IAHVM), Beijing Academy of Agriculture and Forestry Sciences (BAAFS; Beijing, China) (approval No. IAHVM11-2304-21).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal experimental design and management\u003c/h3\u003e\n\u003cp\u003eA total of 100 piglets (Duroc \u0026times; Landrace; 28 days old; 8.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 kg) were selected and randomly assigned to four treatment groups. Each treatment group included five replicates, with five piglets per replicate. Piglets in the control group were administered a basal diet, whereas piglets in the treatment groups received the basal diet supplemented with 100 (low dose), 200 (medium dose), or 300 (high dose) mg/kg PIC (Great Forest Biomedical, Hangzhou, China; purity\u0026thinsp;\u0026gt;\u0026thinsp;99%), respectively. The detailed composition of the basal diet, formulated according to the NRC (2012) and the Nutrient Requirements of Swine (2020) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The doses of PIC used in this experiment were based on previous reports on structural analogs. \u003cem\u003eAd libitum\u003c/em\u003e feeding and watering was maintained for all piglets during the entire experimental period. Body weight and feed intake were recorded for all piglets throughout the experimental period to determine growth performance parameters, including average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion rate.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIngredients and nutrient composition of basal diets (as-fed basis, %).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDays 0 to 15\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDays 15 to 35\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIngredient\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e37.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e68.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtruded corn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtruded soybean\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWheat flour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFish meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhey powder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLimestone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicalcium phosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalt\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Lysine HCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDL-Methionine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Threonine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Tryptophan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitamin and mineral Premix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNutrient levels\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDigestible energy, MJ/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhosphorus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLysine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethionine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThreonine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTryptophan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e Each kg of ration provides: VA 9800 IU; VB\u003csub\u003e1\u003c/sub\u003e 4.5 mg; VB\u003csub\u003e2\u003c/sub\u003e 11 mg; VB\u003csub\u003e6\u003c/sub\u003e 6.5 mg; VB\u003csub\u003e12\u003c/sub\u003e 0.1 mg; VD\u003csub\u003e3\u003c/sub\u003e 2700 IU; VE 50 IU; VK\u003csub\u003e3\u003c/sub\u003e 4.1 mg; Folic acid 1.9 mg; Nicotinic acid 48 mg; D-pantothenate acid 25 mg, Biotin 0.3 mg. Cu 64 mg; Fe 100 mg; Mn 41 mg; Zn 100 mg; Se 0.3 mg; I 0.5 mg.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e2\u003c/sup\u003e Digestible energy is calculated according to NRC (2012), other nutrient levels are analysed according to PRC National Standard.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eSerum sample collection and biochemical analysis\u003c/h3\u003e\n\u003cp\u003eFollowing an overnight fast on days 15 and 35, blood samples were obtained from the anterior vena cava of each piglet. Serum was obtained by centrifugation at 4,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. Serum levels of total antioxidant capacity (T-AOC), glutathione (GSH), and MDA, along with activities of total superoxide dismutase (T-SOD) and catalase (CAT), were determined using commercial kits per manufacturer instructions. T-AOC, CAT, and GSH kits were obtained from Jiancheng Bioengineering Institute (Nanjing, China), T-SOD and MDA kits from Beyotime Biotechnology (Shanghai, China). For data normalization, total protein concentrations were quantified using a bicinchoninic acid (BCA) assay (Huaxing bio, Beijing, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S microbiome sequencing and bioinformatics analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal microbial genomic DNA was isolated from fecal samples using the E.Z.N.A. Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer\u0026rsquo;s protocol. The V3-V4 hypervariable regions of bacterial 16S rRNA genes were amplified with barcoded primers 338F (5\u0026prime;-ACTCCTACGGGAGGCAGCAG-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;) in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR amplicons were size-fractionated by 2% agarose gel electrophoresis, purified using a PCR Clean-Up Kit (YuHua, Shanghai, China), and quantified with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) employing the dsDNA HS Assay Kit. Equimolar amounts of purified amplicons were pooled for constructing sequencing libraries, which were paired-end sequenced (2 \u0026times; 250 bp) on an Illumina NextSeq 2000 platform (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology (Shanghai, China) following standard protocols. Following demultiplexing, raw sequences were processed through quality filtering using fastp (version 0.19.6) and merged with FLASH (v1.2.11). The resulting high-quality sequences were then subjected to denoising via the DADA2 plugin within the QIIME 2 pipeline (using default parameters), which provides single-nucleotide resolution by modeling sample-specific error profiles to generate amplicon sequence variants (ASVs). Taxonomic classification of ASVs was performed using the naive Bayes consensus taxonomy classifier implemented in QIIME 2, with reference to the SILVA 16S rRNA database. Metagenomic functions were predicted based on ASV representative sequences, using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eIPEC-J2 porcine intestinal epithelial cells were generously provided by Dr. Glenn Zhang (Oklahoma State University, Stillwater, OK, USA). Cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/Nutrient Mixture F-12 (Gibco/Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 \u0026micro;g/mL streptomycin, 100 U/mL penicillin, and 1% ITS supplement (5 \u0026micro;g/mL insulin, 5 \u0026micro;g/mL transferrin, 5 ng/mL selenium; ScienCell, San Diego, CA, USA) at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Routine subculturing was performed every 2\u0026ndash;3 days.\u003c/p\u003e\n\u003ch3\u003eOxidative stress model establishment and PIC treatment\u003c/h3\u003e\n\u003cp\u003eAn \u003cem\u003ein-vitro\u003c/em\u003e oxidative stress model was established by treating IPEC-J2 cells with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cells were seeded in 96-well plates (Costar/Corning, Corning, NY, USA) at 8,000 cells/well (100 \u0026micro;L complete medium/well) and cultured for 40 h. Then, the cells were exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0\u0026ndash;1,400 \u0026micro;M in 100 \u0026micro;M increments) for 4 h. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 450 nm using a Multiskan FC instrument (Thermo Fisher Scientific).\u003c/p\u003e\u003cp\u003eGiven that 4-h treatment with 1,000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reduced cell viability by \u0026sim;50%, these parameter settings were employed in subsequent experiments. Cells were pretreated with PIC (0, 2.5, 5, 10, 20, or 40 \u0026micro;M) for 24 h and then exposed to 1,000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h prior to the CCK-8 assay. Non-treated cells served as controls. Pretreatment with 10 \u0026micro;M PIC conferred the strongest protective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced damage in terms of cell viability. Morphological changes in IPEC-J2 cells were examined using an optical microscope (Olympus, Tokyo, Japan; 100\u0026times; magnification), and images were captured using a cellSens Entry system (Olympus). Culture supernatants were centrifuged (1,500 rpm, 10 min), and lactate dehydrogenase (LDH) activity was measured using a Cytotoxicity Detection assay kit (C0016, Beyotime Biotechnology) on an Infinite 200 PRO microplate reader (Tecan Trading AG, Switzerland).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of MDA production, ROS accumulation, and antioxidant activities\u003c/h2\u003e\u003cp\u003eIPEC-J2 cells were seeded in 6-well plates (2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) and cultured overnight. Cells were pretreated or not with 10 \u0026micro;M PIC for 24 h and then exposed or not to 1,000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Post-treatment, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (containing phenylmethylsulfonyl fluoride; Solarbio, Beijing, China) for 15 min. The lysates were centrifuged (12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, 15 min), and the supernatants were collected for analysis. Proteins were quantified using a BCA assay kit.\u003c/p\u003e\u003cp\u003eThe concentration of MDA was determined using a Lipid Peroxidation MDA Assay Kit (S0131S; Beyotime Biotechnology) following the manufacturer\u0026rsquo;s instructions. Supernatants after incubation were collected to determine the 8-hydroxy-2\u0026prime;-deoxyguanine (8-OHDG) levels (Jiangsu Meibiao Biotechnology, Jiangsu, China). For ROS detection, cells were trypsinized, washed twice, stained with dichlorodihydrofluorescein diacetate probe (E004-1-1; Jiancheng Bioengineering Institute) at 37\u0026deg;C for 1 h, and analyzed using an Infinite 200 PRO microplate reader (Tecan Trading AG). T-AOC, T-SOD, and CAT activities and GSH levels in cell lysates were measured using the above-mentioned commercial kits.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative reverse transcription (RT-qPCR) analysis\u003c/h3\u003e\n\u003cp\u003eFollowing cell treatments, total RNA was isolated using RNAzol (Molecular Research Center, Cincinnati, OH, USA) and alcohol precipitation. RNA concentration and quality were measured using a DS-11spectrophotometer (DeNovix, Wilmington, DE, USA). First-strand cDNA was synthesized from 1 \u0026micro;g RNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer\u0026rsquo;s protocol. qPCRs were run using iTaq Universal SYBR Green Supermix (Bio-Rad) on a QuantStudio 3 system (Thermo Fisher Scientific) with porcine-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The thermal cycling protocol consisted of 40 cycles of 95\u0026deg;C for 30 s (denaturation), 60\u0026deg;C for 30 s (annealing), and 72\u0026deg;C for 20 s (extension). Target gene expression levels were normalized to \u003cem\u003eGAPDH\u003c/em\u003e expression and calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eFollowing cell treatments, proteins were extracted with RIPA lysis buffer, and their concentrations were quantified by BCA assay followed by normalization. Samples were denatured in 5\u0026times; SDS loading buffer (Beyotime Biotechnology) at 95\u0026deg;C for 5 min and resolved by 10%\u0026ndash;12% SDS polyacrylamide gel electrophoresis. Following electrophoretic separation, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% (w/v) skim milk in Tris-buffered saline containing Tween-20 for 3 h at room temperature, followed by overnight incubation with primary antibodies at 4\u0026deg;C overnight. After washing, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies for 1 h. All antibodies used in this study are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Immunoreactive bands were visualized using an ECL-Plus chemiluminescence substrate (Beyotime Biotechnology) and imaged with a ChemiDoc Imaging System (Bio-Rad Laboratories). Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstablishment of\u003c/b\u003e \u003cb\u003eNrf2\u003c/b\u003e\u003cb\u003e-knockdown IPEC-J2 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePorcine Nrf2 siRNA (sense strand: 5\u0026prime;-GCCCAUUGAUCUCUCUGAUTT-3\u0026prime;; antisense strand: 5\u0026prime;-AUCAGAGAGAUCAAUGGGCTT-3\u0026prime;), synthesized at GenePharma (Shanghai, China), was transfected into IPEC-J2 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;s protocol. Successfully transfected cells were pretreated or not with 10 \u0026micro;M PIC for 24 h and then exposed to 1,000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h. Non- siRNA-treated cells served as controls. Cell viability, MDA concentration, and ROS accumulation were measured as described above.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e experimental data were processed using GraphPad Prism v6 (GraphPad Software, San Diego, CA, USA). All data are presented as mean and standard error of the mean (SEM) and statistically analyzed using the mixed linear model in SPSS (v20.0, SPSS, Chicago, IL, USA) as follows:\u003c/p\u003e\u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003eijk\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003e\u0026micro;\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e + \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ej\u003c/em\u003e\u003c/sub\u003e + \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e + \u003cem\u003ee\u003c/em\u003e\u003csub\u003e\u003cem\u003eijk\u003c/em\u003e\u003c/sub\u003e,\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003eijk\u003c/em\u003e\u003c/sub\u003e is the dependent variable; \u003cem\u003e\u0026micro;\u003c/em\u003e is the overall mean; \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the fixed treatment effect, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ej\u003c/em\u003e\u003c/sub\u003e is the period effect; \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ek\u003c/em\u003e\u003c/sub\u003e is the steer effect; \u003cem\u003ee\u003c/em\u003e\u003csub\u003e\u003cem\u003eijk\u003c/em\u003e\u003c/sub\u003e is random error.\u003c/p\u003e\u003cp\u003eOne-way analysis of variance (ANOVA) followed by Duncan\u0026rsquo;s multiple range test in SPSS was used to identify significant differences among the groups in piglet experiment. Linear and quadratic effects of dietary PIC supplementation doses were evaluated using regression analysis. An unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to compare group means in porcine cell experiments. Statistical significance was set to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eGut microbiota bioinformatics analysis was conducted using the Majorbio Cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Microbial community similarity among samples was evaluated through principal coordinate analysis (PCoA) based on Bray\u0026ndash;Curtis dissimilarity matrices using the Vegan package (v2.5-3). Treatment effects on community composition were statistically analyzed by PERMANOVA, which quantified the percentage of variation explained and its statistical significance. Linear discriminant analysis effect size (LEfSe) analysis was conducted to identify taxa with significantly different abundances (from phylum to genus level) of bacteria among groups based on linear discriminant analysis score\u0026thinsp;\u0026gt;\u0026thinsp;2.5 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significant correlations between nodes were defined as Spearman\u0026rsquo;s |ρ| \u0026gt;0.6 and false discovery rate-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePIC improves growth performance\u003c/h2\u003e\u003cp\u003eDietary PIC supplementation improved weight gain in piglets (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On day 35 of the trial, the average weight in each treatment group was higher than that in the control group, with a significant difference observed in the medium-dose PIC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PIC supplementation increased the ADG during the 0\u0026ndash;15-day and 0\u0026ndash;35-day periods, with a significant effect in the medium-dose group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). During the 0\u0026ndash;15-day period, the addition of medium and high doses of PIC significantly improved the ADFI of piglets (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The addition of PIC reduced the feed conversion efficiency during piglet rearing, and the feed-to-gain ratio was significantly lower in the medium-dose group compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) over the entire 0\u0026ndash;35-day period. Furthermore, with increasing PIC dose, the final weight of piglets on day 35 and the 0\u0026ndash;35-day ADG showed linear and quadratic responses (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The final weight on day 15, 0\u0026ndash;15-day ADG, and ADFI exhibited a linear increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the 0\u0026ndash;35-day feed-to-gain showed a linear decrease (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of piceatannol (PIC) on growth performance in weaning piglets.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003ePiceatannol, mg/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eANOVA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLinear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBW, kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.965\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.437\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.2\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.241\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADG, g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e418\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e440\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e479\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e469\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.796\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.285\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e649\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e703\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e682\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.397\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.296\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.178\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e550\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e584\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e607\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e591\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.135\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.042\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADFI, g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e717\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e751\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e799\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e791\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.254\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1196\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e13.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.919\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.522\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1041\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.886\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.412\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.326\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF/G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.874\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.519\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.653\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.217\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0 to 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.74\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.73\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.028\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.053\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eBW, body weight; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion rate. Mean and total SEM are listed in separate columns. Different superscript letters within a row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePIC improves serum antioxidant status\u003c/h2\u003e\u003cp\u003eThe effects of PIC on piglet antioxidant indicators are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In comparison to the control group, the low-dose PIC group had significantly enhanced serum T-AOC on day 15 and CAT activity on day 35 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The medium-dose PIC group showed more comprehensive effects, with significantly increased T-AOC and reduced MDA content on days 15 and 35, along with elevated CAT activity on day 35 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The high-dose PIC group exhibited distinct regulatory effects, with significantly reduced MDA contents on day 15 and increased GSH levels on day 35 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PIC administration did not significantly affect T-SOD activity on day 15 or 35, nor CAT activity and GSH content on day 15 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Dose\u0026ndash;response analysis revealed that day 15 T-AOC levels responded to PIC supplementation in both linear and quadratic patterns (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.078;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Linear relationships were observed for GSH levels on days 35, as well as day-15 MDA content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Quadratic responses were identified for day-35 T-AOC levels, CAT activity, and MDA content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating complex dose-dependent effects of PIC on the antioxidant system during different developmental stages.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of piceatannol (PIC) on serum antioxidant capacity in weaning piglets.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003ePiceatannol, mg/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eANOVA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLinear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT-SOD, U/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.166\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.067\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT-AOC, mmol/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.87\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCAT, U/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.394\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGSH, \u0026micro;mol/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.934\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMDA, \u0026micro;mol/mg protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.332\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT-SOD, U/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.074\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.545\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.199\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT-AOC, mmol/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.22\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.408\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCAT, U/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.80\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.083\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGSH, \u0026micro;mol/g protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.41 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.35 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.62 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.308\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMDA, \u0026micro;mol/mg protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.16\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.68\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.046\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.021\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eT-SOD, total superoxide dismutase; T-AOC, total antioxidant capacity; CAT, catalase; GSH, glutathione; MDA, malondialdehyde. Mean and total SEM are listed in separate columns. Different superscript letters within a row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDietary PIC supplementation alters gut microbial diversity and structure in weanling piglets\u003c/h2\u003e\u003cp\u003ePIC supplementation significantly altered β-diversity in the gut microbiota of piglets as indicated by PCoA results (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On day 15 post-weaning, the fecal microbiota composition was dominated by four major phyla: Firmicutes, Bacteroidota, Actinobacteriota, and Spirochaetota, which collectively accounted for \u0026gt;\u0026thinsp;99% of the total relative abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The dominant bacterial genera included \u003cem\u003enorank_o_Clostridia_UCG-014\u003c/em\u003e, \u003cem\u003enorank_f_Muribaculaceae\u003c/em\u003e, \u003cem\u003enorank_o_RF39\u003c/em\u003e, \u003cem\u003eClostridium_sensu_stricto_1\u003c/em\u003e, \u003cem\u003enorank_f_Eubacterium_coprostanoligenes_group\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). LEfSe analysis revealed significant microbial enrichments across groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Genera such as \u003cem\u003ePrevotellaceae_NK3B31_group\u003c/em\u003e, \u003cem\u003eUCG-002\u003c/em\u003e, and \u003cem\u003eUCG-005\u003c/em\u003e were significantly enriched in the low-dose PIC group. \u003cem\u003eCatenisphaera\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eBlautia\u003c/em\u003e, were significantly enriched specifically in the medium-dose PIC group, whereas the high-dose PIC group was significantly enriched in \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eSubdoligranulum\u003c/em\u003e, and \u003cem\u003eAsteroleplasma\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDietary PIC supplementation attenuates oxidative stress in association with gut microbiota alterations in weaning piglets\u003c/h2\u003e\u003cp\u003eThe correlation heatmap based on correlation coefficients in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE depicts the relationships between microbial species in piglet feces and serum antioxidant indices. T-AOC exhibited positive correlations with \u003cem\u003enorank_o__Clostridia_UCG-014\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e, and negative correlations with \u003cem\u003eNK4A214_group\u003c/em\u003e and \u003cem\u003enorank_o__RF39\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum T-SOD activity was significantly positively correlated with \u003cem\u003eUCG-002\u003c/em\u003e, and negatively with \u003cem\u003eTerrisporobacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum CAT activity exhibited a significant negative correlation with \u003cem\u003enorank_o__RF39\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum MDA content was significantly positively correlated with \u003cem\u003eCollinsella\u003c/em\u003e, \u003cem\u003eNK4A214_group\u003c/em\u003e, \u003cem\u003ePrevotellaceae_NK3B31_group\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but negatively with \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eKEGG functional prediction analysis of microbial communities using PICRUSt2 annotated a total of 1,770 enzymes. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF presents a heatmap of the top 20 most significantly enriched enzyme categories. Dietary supplementation with 200 mg/kg PIC significantly increased the abundances of phosphomethylpyrimidine synthase [EC 4.1.99.17], methionine synthase [EC 2.1.1.13], and adenosylcobinamide kinase [EC 2.7.1.156] in the piglet gut microbiota, whereas it significantly reduced the abundances of phosphoenolpyruvate carboxylase [EC 4.1.1.31], adenosine deaminase [EC 3.5.4.4], and oligopeptidase B [EC 3.4.21.83].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePIC exerts a cytoprotective in IPEC-J2 cells\u003c/h2\u003e\u003cp\u003eCell viability was the highest in the (non-treated) control group, indicating normal cellular conditions. Exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decreased cell viability in a concentration-dependent manner, indicating strong oxidative stress-induced cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). PIC pretreatment improved viability compared to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone, particularly at PIC concentrations of 10\u0026ndash;20 \u0026micro;M, suggesting a protective role of PIC against oxidative damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Morphological analysis revealed that cells in the control group showed normal cell morphology, whereas H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated IPEC-J2 cells exhibited enlarged intercellular gaps and disrupted membrane integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). PIC pretreatment significantly attenuated these morphological alterations and effectively preserved cellular integrity. LDH activity is a marker of cell membrane integrity. LDH levels were the lowest in the control group, consistent with minimal cellular damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Cells in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group displayed significantly elevated LDH activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating cellular membrane disruption. Cells in the PIC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group showed reduced LDH release compared with those treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a cytoprotective effect of PIC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of the antioxidant activities of PIC\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the production of oxidative stress products, including MDA, 8-OHDG, and ROS, in IPEC-J2 cells in the different treatment groups. The control group exhibited the lowest MDA content. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group displayed a significant increase in MDA levels, suggesting pronounced oxidative stress. Pretreatment with PIC significantly reduced the MDA content compared with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), implying that PIC effectively mitigates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced lipid peroxidation. 8-OHDG is a major hallmark of DNA oxidative damage. PIC pretreatment significantly suppressed 8-OHDG production compared with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ROS generation was measured based on fluorescence intensity. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment enhanced ROS levels by 1.45-fold compared to those in the control group, indicating oxidative stress induction. ROS levels were maintained at control levels in the PIC alone group, and the PIC\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group exhibited only partial ROS elevation compared to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone group. These findings suggest that PIC prevents spontaneous ROS formation and partially counteracts H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eEffects of PIC on cell apoptosis\u003c/h2\u003e\u003cp\u003eExcess intracellular ROS trigger apoptosis. The effects of PIC on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. RT-qPCR analysis revealed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment markedly upregulated pro-apoptotic genes while downregulating anti-apoptotic genes. Notably, PIC alone significantly elevated B-cell lymphoma 2 (\u003cem\u003eBCL2\u003c/em\u003e) expression and decreased the expression of Caspase 3 (\u003cem\u003eCASP3\u003c/em\u003e) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PIC pretreatment significantly alleviated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis as demonstrated by decreased Bcl-2-associated X protein (\u003cem\u003eBAX\u003c/em\u003e) and \u003cem\u003eCASP3\u003c/em\u003e expression and increased \u003cem\u003eBCL2\u003c/em\u003e expression compared to the levels after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Consistent with the trends observed in the RT-qPCR results, western blotting revealed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment reduced Bcl-2 expression and enhanced Bax and Cleaved Caspase 3 expression, whereas PIC reversed these effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEffects of PIC on the antioxidant defense system\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, PIC treatment alone significantly upregulated the mRNA levels of \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eGPX1\u003c/em\u003e, \u003cem\u003eHO1\u003c/em\u003e, and \u003cem\u003eNQO1\u003c/em\u003e, demonstrating its intrinsic antioxidant capacity. Compared with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone, PIC pretreatment significantly attenuated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced alterations in these antioxidant enzyme transcript levels. Furthermore, PIC pretreatment markedly elevated T-AOC levels compared to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Consistent with these gene expression profiles, PIC pretreatment alleviated T-SOD, GSH, and CAT activities compared to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment alone, although the effect on CAT was not significant. These results suggest that PIC may confer protective effects by activating endogenous antioxidant defense systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePIC antioxidant capacity involves Nrf2 signaling\u003c/h2\u003e\u003cp\u003eTo explore the protective mechanisms of PIC further, we assessed whether Nrf2 activation contributed to the enhanced antioxidant capacity observed in PIC-pretreated IPEC-J2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Immunoblotting results revealed that PIC pretreatment promoted Nrf2 accumulation, indicating Nrf2 pathway activation. To further elucidate the role of Nrf2 in PIC-mediated attenuation of oxidative stress, we generated \u003cem\u003eNrf2\u003c/em\u003e-knockdown IPEC-J2 cells and assessed treatment effects on cell viability, MDA generation, and ROS production. Nrf2 siRNA-transfected cells demonstrated significant downregulation of Nrf2 expression compared to negative control siRNA-treated cells, as confirmed by RT-qPCR and western blot analyses. Following PIC pretreatment and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e challenge, \u003cem\u003eNrf2\u003c/em\u003e-knockdown IPEC-J2 cells exhibited significantly reduced viability compared to control siRNA-transfected cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). \u003cem\u003eNrf2\u003c/em\u003e knockdown significantly weakened PIC\u0026rsquo;s preventing effect on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced MDA and ROS accumulation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggest that the oxidation-protective effect of PIC was blocked by Nrf2 siRNA, indicating the involvement of Nrf2 signaling in the antioxidant function of PIC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccumulating evidence has established that plant polyphenols such as curcumin, resveratrol, ferulic acid, ellagic acid, quercetin, chlorogenic acid, and catechin exhibit potent antioxidant properties and therapeutic potential against oxidative stress and its related pathologies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Particularly resveratrol has attracted extensive attention owing to its beneficial effects on human and animal health [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As a resveratrol analog, PIC features two hydroxyl groups in ortho position of the resveratrol structure. PIC exhibits an 11-fold higher peroxyl radical-scavenging activity than its parent compound, resveratrol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In C2C12 myotubes, PIC upregulated the expression of \u003cem\u003eHO1\u003c/em\u003e and \u003cem\u003eSOD1\u003c/em\u003e, thereby reducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced intracellular ROS accumulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In mice, PIC supplementation mitigated lipopolysaccharide-induced oxidative stress in liver tissues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In piglets, dietary PIC supplementation mitigated hepatic injury, likely via the modulation of redox homeostasis, maintenance of mitochondrial function, and prevention of excessive apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, information on the supplementation effects of PIC in weaning piglets and its potential mechanism remained insufficient.\u003c/p\u003e\u003cp\u003eThe growth performance of piglets is intrinsically associated with their health status, with oxidative stress typically leading to impaired growth performance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Many multifunctional natural compounds exert health-promoting effects, improving growth and production performance, in livestock animals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Jia et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] studied the effects of PIC administration on weaning piglet performance under short-term acute stress induced by diquat. They found no significant differences during the first week post-weaning, but demonstrated that PIC restored the body weight reduction induced by diquat challenge. However, this study did not evaluate the effects of PIC on weaned piglet growth performance throughout the nursery period. In the present investigation, the administration of medium and high doses of PIC significantly improved the ADG during the early weaning phase and throughout the nursery period, and high-dose PIC significantly improved the feed conversion efficiency. While we did not use an \u003cem\u003ein-vivo\u003c/em\u003e oxidative stress challenge model, our study clearly demonstrated the growth-promoting effects of PIC in weaning piglets. Further studies are needed to confirm its beneficial effects, particularly under oxidative stress conditions, in piglets.\u003c/p\u003e\u003cp\u003eWeaning induces multiple stress pathways in piglets, disrupting intestinal redox homeostasis and leading to oxidative stress [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Early weaning compromises intestinal antioxidant capacity in piglets, as evidenced by elevated MDA and ROS levels coupled with significantly reduced antioxidant enzyme activities in the jejunum [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PIC has been demonstrated to stimulate the antioxidant system and enhance antioxidant enzyme capacity, thereby mitigating oxidative stress damage in various cell lines and hosts. Treatment with PIC conferred protection against oxidative stress in skeletal muscle cells through upregulation of antioxidant enzymes, including HO-1 and SOD1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. PIC administration improved hepatic SOD activity and reduced lipid peroxidation in lipopolysaccharide-induced endotoxemic mice [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the study in piglets by Jia et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], dietary PIC administration improved hepatic SOD and GPX activities and decreased the amount of MDA after diquat challenge. In porcine intestinal epithelial cells, PIC pretreatment significantly attenuated deoxynivalenol-induced oxidative damage through coordinated upregulation of protein expression of phase II detoxification enzymes (NQO1 and GCLM) and transcriptional activation of antioxidant genes (\u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003ePRX3\u003c/em\u003e, and \u003cem\u003eGSTA4\u003c/em\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, PIC supplementation improved piglet serum T-AOC and CAT activity and decreased the amount of MDA, indicating its antioxidant potential in piglets. \u003cem\u003eIn vitro\u003c/em\u003e, it enhanced antioxidative gene expression and enzyme activity. To our knowledge, this represents the first experimental demonstration of the antioxidant efficacy of PIC in piglets through feeding trials and intestinal epithelial cell studies. Our results provide novel insights supporting the application of PIC as a functional additive in pig production.\u003c/p\u003e\u003cp\u003eEarly weaning reduces gut microbial diversity and induces microbiota dysbiosis, enhancing the susceptibility to diarrheal diseases and other health complications, in piglets [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Intestinal oxidative stress during the weaning stage triggers gut microbiota dysbiosis, thereby contributes to the pathogenesis of post-weaning diarrhea and enteric infections. The present study demonstrated that dietary PIC supplementation alters the gut microbiota composition, which may contribute to the maintenance of intestinal homeostasis. \u003cem\u003eBlautia\u003c/em\u003e is a functional genus with probiotic characteristics [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In weaning piglets supplemented with a composite probiotic, \u003cem\u003eBlautia\u003c/em\u003e abundance was significantly negatively correlated with MDA contents, and positively with GSH activity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In line herewith, significant negative correlation between \u003cem\u003eBlautia\u003c/em\u003e abundance and MDA levels was observed in our study. Increased \u003cem\u003eFaecalibacterium\u003c/em\u003e abundance has been found to benefit intestinal antioxidant capacity and gut barrier integrity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistent herewith, we found a significant positive correlation between \u003cem\u003eFaecalibacterium\u003c/em\u003e abundance and T-AOC, and negative correlation with MDA levels, in weaning piglets. \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e were the dominant bacteria in the medium-dose supplementation group in the present study, which indicates that PIC-induced elevation of these bacteria contributes to enhanced systemic antioxidant capacity. In drinking water supplemented with acidifier, Xu et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] found a significant positive correlation between \u003cem\u003eSubdoligranulum\u003c/em\u003e abundance and serum antioxidant capacity. In the present study, \u003cem\u003eSubdoligranulum\u003c/em\u003e was the dominant bacterium in the high-dose supplementation group. We found a negative correlation between \u003cem\u003eSubdoligranulum\u003c/em\u003e relative abundance and serum MDA concentrations and positive associations with T-AOC and CAT activity (although statistical significance was not reached), implying that increased \u003cem\u003eSubdoligranulum\u003c/em\u003e abundance may promote antioxidant defenses. \u003cem\u003eNorank_o_RF39\u003c/em\u003e has been proposed as a potentially harmful bacterium and may be associated with reduced antioxidant enzyme activity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In line herewith, we found negative correlations between \u003cem\u003enorank_o_RF39\u003c/em\u003e and T-AOC and CAT activity, supporting its potential negative impact on host antioxidant function. Collectively, our results indicate that PIC positively modulates the intestinal microbial community, including enhancing the abundance of bacteria with antioxidant activity, thus enhancing the antioxidant capacity and growth rate of piglets.\u003c/p\u003e\u003cp\u003ePhosphomethylpyrimidine synthase plays an essential role in the biosynthesis of thiamine (vitamin B\u003csub\u003e1\u003c/sub\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which is pivotal in sulfur metabolism and hence antioxidant capacity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. KEGG pathway enrichment analysis revealed that phosphomethylpyrimidine synthase [EC 4.1.99.17] was significantly enriched by PIC supplementation, suggesting the potential benefit of PIC in improving piglet antioxidant status. Cobalamin has been shown to promote porcine oocyte maturation while reducing ROS generation and elevating GSH levels [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Adenosylcobinamide kinase [EC 2.7.1.156], a key enzyme in cobalamin biosynthesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], was upregulated upon PIC supplementation and thus may have contributed to the enhanced antioxidant capacity in piglets. Finally, PIC supplementation significantly enriched methionine synthase [EC 2.1.1.13], which participates in methionine biosynthesis. In swine, dietary supplementation with 0.12% \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003edl\u003c/span\u003e-methionine analogs significantly increased serum T-SOD activity and GSH content while improving growth performance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePIC's oxidative stress-mitigating effects were comprehensively assessed using \u003cem\u003ein-silico\u003c/em\u003e, \u003cem\u003ein-vitro\u003c/em\u003e, and mouse models [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can directly enter cells and induce reversible oxidative stress; therefore, it is widely employed as an oxidative stress inducer in \u003cem\u003ein vitro\u003c/em\u003e systems [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In IPEC-J2 cells, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment stimulated MDA secretion and triggered an intracellular ROS burst. Pretreatment with PIC significantly suppressed both MDA production and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS accumulation. These findings align with previous findings that PIC pretreatment effectively inhibits ROS and MDA accumulation in oxidative stress-damaged cells and animal models, ameliorating oxidative stress-induced cellular damage [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Apoptosis is typically triggered when cells experience oxidative stress. Oxidative stress can trigger apoptosis through the coordinated upregulation of pro-apoptotic factors and downregulation of anti-apoptotic mediators at both transcriptional and translational levels [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. PIC downregulated Bcl-XL and inhibited the activation of caspase-3 and caspase-8 in PC12 cells incubated with hydrogen peroxide or SIN-1, thus inhibiting apoptosis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. PIC supplementation significantly attenuated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis in ARPE-19 cells, as evidenced by a reduced Bax/Bcl-2 ratio and decreased cleavage of caspase-3 and PARP, potentially through the modulation of Akt phosphorylation status [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In our study, PIC alleviated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage and apoptosis in porcine epithelial cells. However, the signaling pathways involved, and whether PIC affects mitochondrial or non-mitochondrial apoptotic pathways, require further investigation.\u003c/p\u003e\u003cp\u003eNrf2 is a crucial redox-sensitive transcription factor that serves as the primary regulator of certain antioxidant enzymes and detoxification genes, playing a pivotal role in maintaining cellular redox homeostasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Key antioxidant factors regulated by Nrf2 include NQO1, HO-1, SOD, CAT, and related enzymes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. PIC protected retinal pigment epithelium cells against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated damage via Nrf2 signaling and \u003cem\u003eGCLc\u003c/em\u003e, \u003cem\u003eSOD\u003c/em\u003e, and \u003cem\u003eHO1\u003c/em\u003e antioxidant gene transcription [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], indicating that PIC may exert its cytoprotective effects through Nrf2 pathway activation. Our data showed that PIC pretreatment potently activated Nrf2 signaling, as evidenced by significantly elevated Nrf2 phosphorylation levels as compared to those in control and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatments. This activation was accompanied by increased antioxidant enzyme expression, indicating that Nrf2 transcriptional activity mediates the cytoprotective effects of PIC against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage. To validate Nrf2 pathway involvement, we assessed MDA production and ROS accumulation following \u003cem\u003eNrf2\u003c/em\u003e knockdown in IPEC-J2 cells. Notably, the protective effects of PIC against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cellular damage were completely abolished under Nrf2-deficient conditions, confirming that Nrf2 signaling is essential for PIC-mediated cytoprotection against oxidative stress in porcine intestinal epithelial cells. PIC\u0026rsquo;s antioxidant effects may also involve other signaling pathways, such as PI3K/Akt or STAT3, or organelle functional regulation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. More comprehensive investigations are warranted to fully elucidate the precise molecular mechanisms underlying the antioxidant properties of PIC on porcine cells.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides the first experimental evidence for the beneficial effects of dietary PIC supplementation on weaning piglet growth performance, demonstrating that its mechanism may involve gut microbiota modulation and Nrf2-mediated antioxidant defense system activation. These findings highlight PIC acts as a potential antioxidant agent, thereby enhancing growth performance and health status in piglets. Further investigations are warranted to fully elucidate its mechanisms of action, its application in appropriate animal models, and its bioavailability and toxicity in animals to investigate its efficacy as a natural antioxidant in livestock production.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"607\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eADFI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eAverage daily feed intake\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eADG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eAverage daily gain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eBAX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eBcl-2-associated X protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eBCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eB-cell lymphoma 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eCASP3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eCaspase 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eGAPDH\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eGlutathione\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eHO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eHeme oxygenase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eLDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eLactate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eNQO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eQuinone oxidoreductase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eNrf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eNuclear factor erythroid 2-related factor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eReactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eT-AOC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eTotal antioxidant capacity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003eT-SOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003eTotal superoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e8-OHDG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 483px;\"\u003e\n \u003cp\u003e8-hydroxy-2\u0026prime;-deoxyguanine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and design, JW and TF; methodology, LLZ and YYC; formal analysis and writing-original draft preparation, LLZ; investigation and resources, MXC and LC; project administration and data curation, LLZ and QJW; funding acquisition, supervision, writing-review and editing, JW and TF. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Innovation Capacity Building\u0026mdash;Science and Technology Research Project of BAAFS (grant No. KJCX20251005), the National Key R\u0026amp;D Program of China (grant No. 2022YFD130040311), and the Special Program on Science and Technology Innovation Capacity Building of BAAFS (grant No. KJCX 20240339).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and/or analyzed during the current study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were conducted in compliance with the ethical guidelines approved by the Laboratory Animal Ethical Commission of the Institute of Animal Husbandry and Veterinary Medicine (IAHVM), Beijing Academy of Agriculture and Forestry Sciences (BAAFS; Beijing, China) (approval No. IAHVM11-2304-21).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHao Y, Xing MJ, Gu XH. 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Nutrients. 2023;15(13):2973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu15132973\u003c/span\u003e\u003cspan address=\"10.3390/nu15132973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Piceatannol, Weaning piglets, Growth performance, Intestinal flora, Antioxidant, Nrf2 pathway","lastPublishedDoi":"10.21203/rs.3.rs-7540228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7540228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePiglets are highly susceptible to oxidative stress, which can reduce growth performance and cause intestinal damage. Piceatannol (PIC), a natural bioactive substance enriched in Chinese rhubarb (\u003cem\u003eRheum officinale\u003c/em\u003e) and certain dark purple fruits, shows excellent antioxidant properties in our previous cell-based high-throughput screening. However, its effect on piglet growth performance and antioxidant capacity as well as underling mechanism has not been thoroughly investigated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eOne hundred weaned pigs (28 d of age, 8.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 kg) were randomly assigned to 4 treatments with 5 replicates of 5 pigs per replicate. The experimental diets consisted of: 1) basal diet, 2) basal diet\u0026thinsp;+\u0026thinsp;100 mg/kg PIC, 3) basal diet\u0026thinsp;+\u0026thinsp;200 mg/kg PIC, and 4) basal diet\u0026thinsp;+\u0026thinsp;300 mg/kg PIC. On d 15 and 35, one pig from each replicate was selected for sampling. The growth performance was monitored during a 35-day trial. In addition, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-challenged IPEC-J2 cells served as an in vitro model to investigate the antioxidant mechanisms of PIC. IPEC-J2 cells were treated with 1,000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the presence or absence of 10 \u0026micro;M PIC.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDietary PIC at 200 mg/kg significantly enhanced growth performance, as evidenced by increased average daily gain and feed conversion rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PIC supplementation markedly improved systemic antioxidant capacity, with elevated serum total antioxidant capacity, catalase activity, and glutathione levels, along with reduced malondialdehyde content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, PIC modulated the gut microbiota composition, increasing the amounts of beneficial genera (e.g., \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e), and these microbial shifts significantly correlated with improved antioxidant indices. \u003cem\u003eIn vitro\u003c/em\u003e, PIC pretreatment effectively protected IPEC-J2 cells against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage by reducing reactive oxygen species generation and lipid peroxidation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Mechanistically, PIC exerts its antioxidant effects through Nrf2 pathway activation, upregulating endogenous antioxidant enzymes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) while simultaneously inhibiting apoptosis via the regulation of the Bcl-2/Bax ratio and caspase-3 cleavage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003ePIC improved the growth performance and health status of weaned piglets through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota, offering a potential new natural antioxidants for mitigating weaning stress in piglets.\u003c/p\u003e","manuscriptTitle":"Piceatannol, a natural stilbene, improves weaning piglets' growth performance through the regulation of Nrf2-mediated redox homeostasis and modulation of the related gut microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 14:36:31","doi":"10.21203/rs.3.rs-7540228/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2025-10-14T22:36:14+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-29T12:39:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T07:25:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-13T05:24:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2025-09-11T04:08:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77e8248a-304d-4d6c-ae45-fceb2d306704","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:17:53+00:00","versionOfRecord":{"articleIdentity":"rs-7540228","link":"https://doi.org/10.1186/s40104-025-01320-8","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2026-02-01 15:59:27","publishedOnDateReadable":"February 1st, 2026"},"versionCreatedAt":"2025-09-26 14:36:31","video":"","vorDoi":"10.1186/s40104-025-01320-8","vorDoiUrl":"https://doi.org/10.1186/s40104-025-01320-8","workflowStages":[]},"version":"v1","identity":"rs-7540228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7540228","identity":"rs-7540228","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}