Identification of Phytogenic Compounds with Antioxidant Action That Protect Porcine Intestinal Epithelial Cells from Hydrogen Peroxide Induced Oxidative Damage

Oxidative stress contributes to intestinal dysfunction. Plant extracts can have antioxidant action; however, the specific phytogenic active ingredients and their potential mechanisms are not well known. We screened 845 phytogenic compounds using a porcine epithelial cell (IPEC-J2) oxidative stress model to identify oxidative-stress-alleviating compounds. Calycosin and deoxyshikonin were evaluated for their ability to alleviate H2O2-induced oxidative stress by measuring their effects on malondialdehyde (MDA) accumulation, reactive oxygen species (ROS) generation, apoptosis, mitochondrial membrane potential (MMP), and antioxidant defense. Nrf2 pathway activation and the effect of Nrf2 knockdown on the antioxidative effects of hit compounds were investigated. Calycosin protected IPEC-J2 cells against H2O2-induced oxidative damage, likely by improving the cellular redox state and upregulating antioxidant defense via the Nrf2-Keap1 pathway. Deoxyshikonin alleviated the H2O2-induced decrease in cell viability, ROS production, and MMP reduction, but had no significant effect on MDA accumulation and apoptosis. Nrf2 knockdown did not weaken the effect of deoxyshikonin in improving cell viability, but it weakened its effect in suppressing ROS production. These results indicate that the mechanisms of action of natural compounds differ. The newly identified phytogenic compounds can be developed as novel antioxidant agents to alleviate intestinal oxidative stress in animals.


Introduction
Oxidative stress refers to a redox imbalance caused by excessive free radical production in the body. It is an important contributor to animal diseases and reduces growth and production performance. When reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulate to excessively high levels, they cause irreversible damage to cell lipids, proteins, and DNA [1], thus affecting animal physiological function and production performance. In livestock and poultry production, numerous factors, including changes in the environment, physiological stages, and exogenous pathogenic toxins, can cause oxidative stress, thus destroying the redox balance in the body. Oxidative stress negatively affects animal health. Excess ROS in the body can damage the mucosal barrier of tissues and organs, resulting in functional damage and ultimately, disease [2,3]. Further, oxidative stress reduces production performance, reproductive performance, and animal product quality, thus having serious economic effects. In certain circumstances, such as at high temperatures, during weaning, and during pregnancy, supplementing exogenous antioxidants can effectively alleviate oxidative stress in animals, reduce oxidative damage, and improve health and production performance. With the development of the animal plus 24 h. Then, the cells were treated with H 2 O 2 at 0, 200, 400, 600, 800, 1000, 1200, or 1400 µM for 4 h. After the treatments, a cell counting kit (CCK)-8 (Dojindo, Kumamoto, Japan) was used according to the manufacturer's instructions to detect cell viability. The absorbance at 450 nm was measured using a Multiskan FC instrument (Thermo Fisher Scientific, Waltham, MA, USA). Culture supernatants were collected and centrifuged at 1500 rpm for 10 min. Lactate dehydrogenase (LDH) levels in the supernatants were determined using an LDH assay kit (11644793001; Roche, Mannheim, Germany) and an automatic biochemical analyzer (Beckman Instruments, Brea, CA, USA).

Drug Toxicity Assay
Drug toxicity was evaluated before HTS. IPEC-J2 cells were seeded in 200 µL of medium in 96-well plates at 8000 cells/well and incubated overnight. The cells were treated with individual library compounds at 10 µM for 24 h. Cell viability was assessed using the CCK-8 assay to assess toxicity. The absorbance at 450 nm was measured using the Multiskan FC instrument.

HTS Assay
The HTS assay was conducted as previously described [15]. Briefly, IPEC-J2 cells were seeded in 96-well plates at 8000 cells/well. After overnight culture, the cells were stimulated with individual library compounds at 10 µM for 24 h. After incubation, the medium was discarded, and the cells were gently washed with warm phosphate-buffered saline (PBS). Then, H 2 O 2 was added to each well at 1000 µM and the plate was incubated for an additional 4 h. Following H 2 O 2 treatment, 10 µL of CCK-8 solution was added to each well. After 3 h of incubation, cell viability was determined by measuring the absorbance at 450 nm. For each compound, the cell viability after compound pretreatment plus H 2 O 2 treatment was normalized to that after compound treatment alone. A compound for which cell viability that was higher than 70%, implying that cell viability was improved by 15-20% compared with that after H 2 O 2 treatment, was considered a hit.
To assess the robustness of the HTS assay, the Z -factor [17] was calculated. Cells in a 96-well plate were treated, and not treated, with 1000 µM H 2 O 2 for 4 h and then subjected to the CCK-8 assay. The Z -factor was calculated as follows: where σ p and σ n are the standard deviations and µ p and µ n the means of positive and negative controls, respectively. Z ≥ 0.5 indicates that the assay can be used for HTS. The critical value was calculated to confirm the separation of the H 2 O 2 -induced cell damage model and untreated controls.

Secondary Screening and Validation of the Hit Compounds
Compounds for which the cell viability in the HTS assay was above 70% were further assayed at five concentrations (2.5, 5, 10, 20, and 40 µM) using the IPEC-J2 oxidative stress model in 96-well plates. Cells were grown overnight and then exposed to the hit compounds for 24 h. Then, cell viability was determined using the CCK-8 assay. Nontreated cells served as a control. The assay was performed three times.

Determination of Malondialdehyde (MDA) Accumulation and ROS Generation
IPEC-J2 cells were seeded in 6-well tissue culture plates at 2.5 × 10 5 cells/well and incubated overnight. Then, the cells were pretreated with calycosin or deoxyshikonin at 2.5 or 5 µM for 24 h. After the incubation, the cells were exposed, and not exposed, to 1000 µM of H 2 O 2 for 4 h. Nontreated cells served as a control. After the treatments, the cells were gently washed with PBS twice and lysed using RIPA lysis buffer (containing PMSF) (Solarbio, Beijing, China) for 10 min. After centrifugation at 10,000× g, 4 • C for Antioxidants 2022, 11, 2134 4 of 17 10 min, the supernatants were collected. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Madison, WI, USA). MDA concentrations were determined using a Porcine MDA ELISA kit (B162437; BIM Biosciences, San Francisco, CA, USA) according to the manufacturer's instructions.
To assess ROS production, treated cells were digested with trypsin, washed twice with buffer solution, and incubated with ROS reagent (MAK143; Sigma-Aldrich, Madrid, Spain) for 30 min. ROS production was detected using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using the CellQuest software (BD Biosciences). The experiments were performed in triplicate.

Detection of Apoptosis and the Mitochondrial Membrane Potential (MMP)
After the treatments, apoptosis was detected by flow cytometry using an annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) assay kit (Solarbio). The cells were collected and resuspended in binding buffer. The cells were stained with 5 µL of annexin V/PE at room temperature in the dark for 5 min. Then, 10 µL of 20 µg/mL 7-AAD and 400 µL of PBS were added and apoptotic cells were immediately detected using the BD FACSCalibur flow cytometer and analyzed using the CellQuest software.
The MMP was evaluated using MitoScreen (JC-1) (551302; BD Biosciences, Franklin Lakes, NJ, USA), which uses the membrane-permeable dye JC-1 to detect mitochondrial depolarization in cells. After the treatments, the cells were digested with trypsin and washed twice with binding buffer. Then, JC-1 was added for 30 min after which the cells were suspended in binding buffer. MMP levels were detected by using the FACSCalibur flow cytometer and analyzed using the CellQuest software. MMP is reflected by the proportion of JC-1 aggregates and monomers. The experiments were performed in triplicate.

Determination of Antioxidant Activities
Total antioxidant capacity (T-AOC), catalase (CAT) activity, and heme oxygenase (HO-1) activity in cell lysates were measured using commercial kits to determine the antioxidant capacity of IPEC-J2 cells after the different treatments. T-AOC and CAT kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and HO-1 ELISA kits were purchased from Enzo Life Sciences (Raamsdonksveer, The Netherlands). All assays were carried out following the manufacturer's instructions. After the treatments, the cells were gently washed with PBS twice and then lysed using RIPA Lysis Buffer R2220 (containing PMSF) (Solarbio). Protein concentrations were determined as described method above. The proteins were stored at −20 • C until analysis. The experiments were performed in triplicate.

Total RNA Isolation and Quantitative Reverse Transcription (RT-q) PCR
After the treatments, cells were lysed using RNAzol RT (Molecular Research Center, Cincinnati, OH, USA) and total RNA was isolated by alcohol precipitation. RNA concentrations and quality were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The RNA was transcribed into first-strand cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. qPCRs were run using iTaq Universal SYBR Green Supermix (Bio-Rad) in a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Target genes were amplified using porcine gene primers (Table S1) with 40 cycles of denaturation at 95 • C for 30 s, annealing at 60 • C for 30 s, and extension at 72 • C for 20 s. Target mRNA levels were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase gene, whose expression level was not altered by any of the compounds applied. The 2 −∆∆Ct method was used to calculate relative fold changes in gene expression [18].

Western Blot Analysis
After the treatments, the cells were gently washed with PBS twice and total proteins were extracted and quantified as described above. Equal amounts of proteins were Antioxidants 2022, 11, 2134 5 of 17 separated by sodium dodecyl sulfate gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. After blocking with a PBST buffer containing 5% skim-milk at room temperature for 1 h, the blots were incubated with the indicated primary antibodies at 4 • C overnight. After three washes with tris-buffered saline, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. The antibodies used in this study are listed in Table S2. Immunoreactive bands were detected using an enhanced chemiluminescence kit (ECL-Plus; Thermo Fisher Scientific) and imaged using a Bio-Rad gel detection system.

Statistical Analysis
Data were processed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). All data are presented as mean ± standard error of the mean (SEM). Means of two groups were compared using an unpaired two-tailed Student's t-test in SPSS (version 20.0, SPSS, Chicago, IL, USA). p < 0.05 was considered significant.

Establishment of an Oxidative Stress Model for HTS
Oxidative agents markedly decrease cell viability. H 2 O 2 is a commonly used strong oxidant. We developed an oxidative stress model for HTS of plant compounds by treating porcine IPEC-J2 cells with H 2 O 2 at 0, 200, 400, 600, 800, 1000, 1200, or 1400 µM for 4 h and then assessed cell viability using the CCK-8 assay. As shown in Figure 1A, H 2 O 2 concentration-dependently decreased the viability of IPEC-J2 cells. As 1000 µM H 2 O 2 reduced cell viability to approximately 50% after 4 h, we used this concentration and treatment time in subsequent experiments. To further clarify the effect of H 2 O 2 in inducing oxidative stress, we evaluated the effect of different concentrations of H 2 O 2 on cell LDH leakage ( Figure 1B). In agreement with the above findings, H 2 O 2 concentration-dependently increased LDH leakage in IPEC-J2 cells, which indicated that H 2 O 2 induces substantial cell membrane damage in these cells.

Variability and Robustness of the Model
To assess the quality of the oxidative stress model for use in cell-based HTS, cells were treated, and not treated, with 1000 µM H 2 O 2 for 4 h. Cell viability was determined in 30 H 2 O 2 -treated wells and 30 nontreated wells and the data (optical density values) were used to analyze the variability between wells and the robustness of the oxidative stress cell model by calculating the Z' factor. As shown in Figure 2, after treatment of IPEC-J2 cells with 1000 µM H 2 O 2 for 4 h, the Z' factor was 0.64, which is considered robust [18]. The critical values were 1.85% for the H 2 O 2 -free group and 1.48% for the H 2 O 2 -treated group. These results indicated that the H 2 O 2 -treated model group and the nontreated control group were well separated, and the assay could be used for HTS.

Variability and Robustness of the Model
To assess the quality of the oxidative stress model for use in cell-based HTS were treated, and not treated, with 1000 μM H2O2 for 4 h. Cell viability was determ in 30 H2O2-treated wells and 30 nontreated wells and the data (optical density va were used to analyze the variability between wells and the robustness of the oxid stress cell model by calculating the Z' factor. As shown in Figure 2, after treatme IPEC-J2 cells with 1000 μM H2O2 for 4 h, the Z' factor was 0.64, which is considered r [18]. The critical values were 1.85% for the H2O2-free group and 1.48% for the H2O2-tr group. These results indicated that the H2O2-treated model group and the nontreated trol group were well separated, and the assay could be used for HTS.

Variability and Robustness of the Model
To assess the quality of the oxidative stress model for use in cell-based HT were treated, and not treated, with 1000 μM H2O2 for 4 h. Cell viability was deter in 30 H2O2-treated wells and 30 nontreated wells and the data (optical density v were used to analyze the variability between wells and the robustness of the ox stress cell model by calculating the Z' factor. As shown in Figure 2, after treatm IPEC-J2 cells with 1000 μM H2O2 for 4 h, the Z' factor was 0.64, which is considered [18]. The critical values were 1.85% for the H2O2-free group and 1.48% for the H2O2-t group. These results indicated that the H2O2-treated model group and the nontreate trol group were well separated, and the assay could be used for HTS.

Identification of Active Monomers in a Chinese Herbal Compound Library
The established cell viability-based 96-well-plate HTS assay was used to identify compounds with antioxidant activity in a Chinese herbal compound library. Before the HTS, we screened out toxic compounds (inducing a >90% reduction in cell viability) using a drug toxicity assay. In total, 718 plant compounds were screened for their ability to alleviate the H 2 O 2 -induced reduction in cell viability. Compounds for which the cell viability was above 70% after H 2 O 2 treatment were considered potential active compounds. The top 22 hits from this primary screening were selected for further validation ( Figure 3A). compounds with antioxidant activity in a Chinese herbal compound library. Before the HTS, we screened out toxic compounds (inducing a >90% reduction in cell viability) using a drug toxicity assay. In total, 718 plant compounds were screened for their ability to alleviate the H2O2-induced reduction in cell viability. Compounds for which the cell viability was above 70% after H2O2 treatment were considered potential active compounds. The top 22 hits from this primary screening were selected for further validation ( Figure 3A).

Secondary Screening of the Hit Compounds
A dose-response-based secondary HTS was conducted to validate the antioxidant activity of the 22 hits. Most compounds alleviated H 2 O 2 -induced oxidative damage at at least one of five concentrations examined (2.5, 5, 10, 20, and 40 µM) ( Figure 3B). Different compounds had different optimal concentrations. Detailed information on these 22 compounds is provided in Table 1. Calycosin and deoxyshikonin were selected for further analysis of their antioxidant effect and their potential mechanisms. These two compounds have been shown to induce host defense peptides and to have immunomodulatory effects in porcine cells [19,20]. Further, they were effective at relatively low concentrations in the present study.

Characterization of the Antioxidant Activities of Calycosin and Deoxyshikonin
To further investigate the antioxidant activity of the two hit compounds, the lipid peroxidation product MDA, an indicator of oxidative damage, was first measured. The results demonstrated that H 2 O 2 treatment significantly increased the MDA level in IPEC-J2 cells (p < 0.05; Figure 4A) compared with the control treatment. Calycosin and deoxyshikonin pretreatments decreased the MDA level compared with the H 2 O 2 treatment alone; however, the effect of deoxyshikonin was not significant. Next, we evaluated intracellular ROS production. As shown in Figure 4B,C, ROS accumulation was significantly increased in IPEC-J2 cells after exposure to H 2 O 2 , whereas pretreatments with calycosin and deoxyshikonin significantly suppressed the ROS burst induced by H 2 O 2 . These results suggested that calycosin and deoxyshikonin have the potential to prevent cell lipid peroxidation and free radical accumulation, thus attenuating intestinal epithelial cell damage induced by H 2 O 2 .
Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 18 alone; however, the effect of deoxyshikonin was not significant. Next, we evaluated intracellular ROS production. As shown in Figure 4B,C, ROS accumulation was significantly increased in IPEC-J2 cells after exposure to H2O2, whereas pretreatments with calycosin and deoxyshikonin significantly suppressed the ROS burst induced by H2O2. These results suggested that calycosin and deoxyshikonin have the potential to prevent cell lipid peroxidation and free radical accumulation, thus attenuating intestinal epithelial cell damage induced by H2O2.

Effects of Calycosin and Deoxyshikonin on the MMP
The MMP is affected by H2O2-induced ROS release. Therefore, we investigated the potential regulatory effects of calycosin and deoxyshikonin on the MMP during the process of relieving oxidative stress. H2O2-induced oxidative stress significantly decreased the MMP level in IPEC-J2 cells ( Figure 5A,B). After calycosin and deoxyshikonin pretreat-

Effects of Calycosin and Deoxyshikonin on the MMP
The MMP is affected by H 2 O 2 -induced ROS release. Therefore, we investigated the potential regulatory effects of calycosin and deoxyshikonin on the MMP during the process of relieving oxidative stress. H 2 O 2 -induced oxidative stress significantly decreased the MMP level in IPEC-J2 cells ( Figure 5A,B). After calycosin and deoxyshikonin pretreatments, the MMP level was significantly increased compared with that after H 2 O 2 treatment alone, demonstrating the positive effects of calycosin and deoxyshikonin on the mitochondrial redox state in H 2 O 2 -damaged cells.

Effects of Calycosin and Deoxyshikonin on the Apoptosis
Excess ROS induce apoptosis. Flow cytometry results demonstrated that H2O2 trea ment significantly increased apoptosis. Calycosin and deoxyshikonin treatment and cal cosin pretreatment significantly alleviated H2O2-induced apoptosis, whereas deox shikonin pretreatment had no effect ( Figure 6A,B). To better understand the effects calycosin and deoxyshikonin on apoptosis, the expression levels of the apoptosis-assoc ated proteins Bcl-2, Bax, caspase 3, and cleaved caspase 3 in calycosin-or deoxyshikoni pretreated H2O2-challenged IPEC-J2 cells were determined by western blotting (Figu 6C,D). H2O2 treatment lowered the expression of the antiapoptotic factor Bcl-2. Calycos pretreatment significantly rescued Bcl-2 expression compared with H2O2 treatmen whereas deoxyshikonin pretreatment had no significant effect on Bcl-2 expression. H2O treatment enhanced the expression of the proapoptotic protein Bax. Calycosin and deo yshikonin pretreatments significantly suppressed the increase in Bax expression. Finall H2O2 treatment significantly increased the levels of caspase 3 and cleaved caspase whereas calycosin and pretreatment significantly suppressed these increases and deox shikonin pretreatment slightly, albeit not significantly, lowered the increase of cleave caspase 3.

Effects of Calycosin and Deoxyshikonin on the Apoptosis
Excess ROS induce apoptosis. Flow cytometry results demonstrated that H 2 O 2 treatment significantly increased apoptosis. Calycosin and deoxyshikonin treatment and calycosin pretreatment significantly alleviated H 2 O 2 -induced apoptosis, whereas deoxyshikonin pretreatment had no effect ( Figure 6A,B). To better understand the effects of calycosin and deoxyshikonin on apoptosis, the expression levels of the apoptosis-associated proteins Bcl-2, Bax, caspase 3, and cleaved caspase 3 in calycosin-or deoxyshikoninpretreated H 2 O 2 -challenged IPEC-J2 cells were determined by western blotting (Figure 6C,D). H 2 O 2 treatment lowered the expression of the antiapoptotic factor Bcl-2. Calycosin pretreatment significantly rescued Bcl-2 expression compared with H 2 O 2 treatment, whereas deoxyshikonin pretreatment had no significant effect on Bcl-2 expression. H 2 O 2 treatment enhanced the expression of the proapoptotic protein Bax. Calycosin and deoxyshikonin pretreatments significantly suppressed the increase in Bax expression. Finally, H 2 O 2 treatment significantly increased the levels of caspase 3 and cleaved caspase 3, whereas calycosin and pretreatment significantly suppressed these increases and deoxyshikonin pretreatment slightly, albeit not significantly, lowered the increase of cleaved caspase 3.

Effects of Calycosin and Deoxyshikonin on the Antioxidant Defense System
The effects of calycosin and deoxyshikonin on the cellular antioxidant defense system were examined to further investigate the mechanisms of these two compounds in alleviating oxidative stress. As shown in Figure 7A, the mRNA levels of HO-1, NQO1, and GPX1 were significantly elevated, indicating antioxidant defense system activation, in IPEC-J2 cells treated with H2O2. Compared with the levels after H2O2 treatment alone, calycosin and deoxyshikonin pretreatments alleviated HO-1 and NQO1 mRNA expression induced by H2O2, whereas they significantly increased CAT expression. The pretreatments had no significant effect on GPX1 expression compared with H2O2 treatment alone. Calycosin treatment alone significantly upregulated all genes detected except SOD1, whereas deoxyshikonin treatment alone significantly increased the expression of HO-1, NQO1, and CAT.
The T-AOC was significantly increased in calycosin-treated cells, and pretreatments with calycosin and deoxyshikonin significantly attenuated the H2O2-induced decrease in T-AOC ( Figure 7B). Next, HO-1 and CAT activities were evaluated ( Figure 7C,D). In line with the gene expression results, after pretreatments with the two compounds, HO-1 activity was alleviated when compared with that after H2O2 treatment alone, whereas CAT activity was increased, although the effect of deoxyshikonin treatment was not significant.

Effects of Calycosin and Deoxyshikonin on the Antioxidant Defense System
The effects of calycosin and deoxyshikonin on the cellular antioxidant defense system were examined to further investigate the mechanisms of these two compounds in alleviating oxidative stress. As shown in Figure 7A, the mRNA levels of HO-1, NQO1, and GPX1 were significantly elevated, indicating antioxidant defense system activation, in IPEC-J2 cells treated with H 2 O 2 . Compared with the levels after H 2 O 2 treatment alone, calycosin and deoxyshikonin pretreatments alleviated HO-1 and NQO1 mRNA expression induced by H 2 O 2 , whereas they significantly increased CAT expression. The pretreatments had no significant effect on GPX1 expression compared with H 2 O 2 treatment alone. Calycosin treatment alone significantly upregulated all genes detected except SOD1, whereas deoxyshikonin treatment alone significantly increased the expression of HO-1, NQO1, and CAT.
The T-AOC was significantly increased in calycosin-treated cells, and pretreatments with calycosin and deoxyshikonin significantly attenuated the H 2 O 2 -induced decrease in T-AOC ( Figure 7B). Next, HO-1 and CAT activities were evaluated ( Figure 7C,D). In line with the gene expression results, after pretreatments with the two compounds, HO-1 activity was alleviated when compared with that after H 2 O 2 treatment alone, whereas CAT activity was increased, although the effect of deoxyshikonin treatment was not significant.
These results suggested that the effects of calycosin and deoxyshikonin may attributed to the activation of the endogenous antioxidant defense system. These results suggested that the effects of calycosin and deoxyshikonin may attributed to the activation of the endogenous antioxidant defense system.

Activation of the Nrf2 Signaling Pathway
To further explore the antioxidant mechanism of calycosin and deoxyshikonin, we evaluated (p-)Nrf2 expression in IPEC-J2 cells treated with the two compounds and H2O2. As shown in Figure 8A,B, H2O2 treatment significantly lowered p-Nrf2 expression, whereas calycosin pretreatment significantly reversed this reduction. However, pretreatment with deoxyshikonin had no significant effect on p-Nrf2 expression when compared with H2O2 treatment alone. Calycosin or deoxyshikonin treatment alone had no significant effect on p-Nrf2 expression compared to the control treatment.

Activation of the Nrf2 Signaling Pathway
To further explore the antioxidant mechanism of calycosin and deoxyshikonin, we evaluated (p-)Nrf2 expression in IPEC-J2 cells treated with the two compounds and H 2 O 2 . As shown in Figure 8A,B, H 2 O 2 treatment significantly lowered p-Nrf2 expression, whereas calycosin pretreatment significantly reversed this reduction. However, pretreatment with deoxyshikonin had no significant effect on p-Nrf2 expression when compared with H 2 O 2 treatment alone. Calycosin or deoxyshikonin treatment alone had no significant effect on p-Nrf2 expression compared to the control treatment.

Effects of Calycosin and Deoxyshikonin on IPEC-J2 Cells Treated with Nrf2 siRNA
To clarify the role of the transcription factor Nrf2 in oxidative stress alleviation by the two compounds, we established Nrf2-knockdown IPEC-J2 cells and evaluated cell viability, MDA production, ROS accumulation, and apoptosis after the treatments. Nrf2 siRNA-transfected IPEC-J2 cells showed significantly lower cell viability than negative control cells when challenged with H 2 O 2 ( Figure 9A). The antioxidant effect of calycosin was blocked by Nrf2 knockdown; knockdown cells showed significantly reduced viability when compared with negative control siRNA-transfected cells after pretreatment with calycosin and subsequent H 2 O 2 treatment. The effect of deoxyshikonin pretreatment on cell viability was not significantly affected by Nrf2 knockdown. Furthermore, Nrf2 knockdown weakened the effects of calycosin in preventing MDA production and ROS generation in response to H 2 O 2 challenge ( Figure 9B-D). The effect of deoxyshikonin on ROS generation was also weakened after Nrf2 knockdown ( Figure 9C,D). Nrf2 knockdown abolished the effect of calycosin in alleviating apoptosis, whereas deoxyshikonin pretreatment still had no effect on apoptosis after Nrf2 knockdown ( Figure 9E,F). evaluated (p-)Nrf2 expression in IPEC-J2 cells treated with the two compounds and H2O2. As shown in Figure 8A,B, H2O2 treatment significantly lowered p-Nrf2 expression, whereas calycosin pretreatment significantly reversed this reduction. However, pretreatment with deoxyshikonin had no significant effect on p-Nrf2 expression when compared with H2O2 treatment alone. Calycosin or deoxyshikonin treatment alone had no significant effect on p-Nrf2 expression compared to the control treatment.

Effects of Calycosin and Deoxyshikonin on IPEC-J2 Cells Treated with Nrf2 siRNA
To clarify the role of the transcription factor Nrf2 in oxidative stress alleviation by the two compounds, we established Nrf2-knockdown IPEC-J2 cells and evaluated cell viability, MDA production, ROS accumulation, and apoptosis after the treatments. Nrf2 siRNA-transfected IPEC-J2 cells showed significantly lower cell viability than negative control cells when challenged with H2O2 ( Figure 9A). The antioxidant effect of calycosin was blocked by Nrf2 knockdown; knockdown cells showed significantly reduced viability when compared with negative control siRNA-transfected cells after pretreatment with calycosin and subsequent H2O2 treatment. The effect of deoxyshikonin pretreatment on cell viability was not significantly affected by Nrf2 knockdown. Furthermore, Nrf2 knockdown weakened the effects of calycosin in preventing MDA production and ROS generation in response to H2O2 challenge ( Figure 9B-D). The effect of deoxyshikonin on ROS generation was also weakened after Nrf2 knockdown ( Figure 9C,D). Nrf2 knockdown abolished the effect of calycosin in alleviating apoptosis, whereas deoxyshikonin pretreatment still had no effect on apoptosis after Nrf2 knockdown ( Figure 9E,F).

Discussion
We used a fast, in vitro HTS assay using IPEC-J2 cells to identify plant-derived compounds that have antioxidant capacity to provide a reference for later validation in animals and application in livestock production. In vitro assays have obvious advantages, including a low cost, short operation time, and the absence of ethical concerns [21][22][23]. Although differences exist between in vivo and in vitro cell responses to oxidative stress [24], in vitro assays allow for a preliminary selection.
The screening of 845 natural products led to the identification of 22 compounds that maintained cell viability above 70% after H 2 O 2 treatment. These compounds were confirmed to alleviate the H 2 O 2 -induced reduction in cell viability and attenuated oxidative damage in a concentration-dependent manner. Phenols and flavonoids were dominant, accounting for approximately half of the total compounds identified. Studies have demonstrated that phenols and flavonoids have strong antioxidant potential [25,26]. Among the top 10 identified compounds, bisabolangelone, morroniside, calycosin, sinapine, forsythoside A, and baicalin have been reported to show antioxidant activity and mitigate oxidative damage caused by various diseases, mainly in humans, both in vitro and in vivo [27][28][29][30][31]. These compounds may exhibit anti-inflammatory, antibacterial, and other comprehensive effects along with their antioxidant activity.
Two hit compounds, calycosin and deoxyshikonin, were further validated as they were among the most potent compounds. Calycosin, originally extracted from the roots of Astragalus membranaceus, is a typical phytoestrogen with a wide range of pharmacological activities, including anticancer, anti-inflammatory, antioxidant, antiosteoporosis, neuroprotective, hepatoprotective, cardioprotective, antidiabetic, and proangiogenic and vasoprotective activities [32]. Wang et al. [33] evaluated the ABTS radical-scavenging ability and ferric ion-reducing antioxidant power of calycosin and confirmed its antioxidant activity. The oxidative-stress-alleviating effect of calycosin has been investigated using different cell lines and mouse models [34][35][36]. However, this study is the first to report the antioxidant effect of calycosin in porcine cells. Pretreatment with calycosin remarkably reduced ROS accumulation in IPEC-J2 cells stimulated with H 2 O 2 . Cellular ROS accumulation has been linked to several gastrointestinal tract disorders [37]. MDA is the main product of ROS-induced membrane lipid peroxidation, and oxidative damage induces intracellular MDA accumulation. Calycosin pretreatment significantly inhibited MDA production induced by H 2 O 2 . Previous studies have clearly demonstrated the inhibitory effects of pretreatment with calycosin on ROS and MDA accumulation in oxidative-stress-damaged cells or hosts, thus alleviating oxidative-stress-induced impairments [34,36,38,39].
Deoxyshikonin, extracted from Lithosperraum erythrorhizon, is a promising drug candidate for the treatment of wounds and cancers [40,41]. Few studies have directly demonstrated the antioxidant activity of deoxyshikonin. Park et al. [40] demonstrated that Lithospermi Radix extract had concentration-dependent free radical-scavenging activity. In the present study, deoxyshikonin pretreatment suppressed ROS accumulation and the decrease in cell viability, but not MDA levels, in IPEC-J2 cells treated with H 2 O 2 , which indicates its potential protective effect on oxidative-stress-induced damage in porcine epithelial cells. Shikonin, a deoxyshikonin analog, has been shown to inhibit H 2 O 2 -induced increases in ROS and MDA levels in human HT29 cells, indicating that it exhibits antioxidant activity and has potential for the treatment of oxidative-damage-associated diseases [42]. These and our findings warrant further exploration of the antioxidant potential of deoxyshikonin in humans and animals.
H 2 O 2 markedly reduces cell viability by disrupting mitochondrial function and inducing apoptosis. H 2 O 2 treatment decreases the MMP, thus disrupting mitochondrial function [34]. In H 2 O 2 -treated cells, proapoptotic gene and protein expression are upregulated and antiapoptotic gene and protein expression downregulated, resulting in apoptosis [43,44]. Accordingly, we found that H 2 O 2 disrupted the MMP and induced apoptosis and apoptosis-related protein expression in IPEC-J2 cells. Calycosin pretreatment alleviated the reduction in MMP caused by H 2 O 2 and prevented H 2 O 2 -induced apoptosis. Previous studies have suggested that calycosin increases the expression of Bcl-2 while reducing the expression of Bax and Bad, thus inhibiting apoptosis, in endothelial cells [45,46]. Other studies have reported that calycosin induces apoptosis, mainly in cancer cells. Calycosin triggered apoptosis of osteosarcoma 143B cells and MCF-7 cells via mitochondrial-dependent intrinsic apoptotic pathways [47,48]. In our study, calycosin alleviated H 2 O 2 -induced oxidative damage and apoptosis in porcine epithelial cells by ameliorating mitochondrial dysfunction and inhibiting mitochondrial-pathway-mediated apoptosis. Its mechanism of action requires further study. Deoxyshikonin pretreatment alleviated the disruptive effect of H 2 O 2 on the MMP; however, it did not influence mitochondrial-mediated apoptosis. Likely, it may act on mitochondrial-mediated pyroptosis or other signaling pathways to alleviate H 2 O 2induced cell viability reduction, which requires further study. While shikonin has been demonstrated to attenuate H 2 O 2 -induced oxidative injury in HT29 human intestinal epithelial cells via antioxidant activities and the inhibition of mitochondrial-pathway-mediated apoptosis [42], the structural differences of the analogs may explain their differences in their activity and mechanism.
Nrf2 plays a key role in regulating oxidative-stress-derived damage [49]. In nonstressed conditions, Keap1, the main negative regulator of Nrf2, binds to Nrf2 to promote the degradation of Nrf2 in the cytoplasm [50]. Under oxidative stress and injury, Keap1 activity declines and Nrf2 is stabilized and translocates into the nucleus and activates the transcription of genes with antioxidant response elements. Numerous phytochemicals and natural bioactive agents activate the Nrf2-Keap1 pathway to increase the transcription of cytoprotective genes to protect cells against oxidative damage and combat certain diseases [51][52][53]. In the present study, the Nrf2-Keap1 pathway was significantly enhanced by calycosin pretreatment compared with H 2 O 2 treatment. Meanwhile, the expression and activity of several antioxidant enzymes were upregulated, likely via the activated Nrf2 pathway. Calycosin has shown similar effects in other cell lines [29,54], suggesting that Nrf2 transcriptional activity is involved in the increased antioxidant enzyme expression and the cytoprotective effect of calycosin against H 2 O 2 -mediated oxidative injury. Deoxyshikonin pretreatment had no significant effect on p-Nrf2 expression following H 2 O 2 challenge. Although several antioxidant enzymes were altered by deoxyshikonin treatment, we cannot conclude that the antioxidative effects of deoxyshikonin are mainly mediated by Nrf2-Keap1 pathway activation. It is highly likely that other signaling pathways are involved in its regulatory effects. We have previously found that deoxyshikonin strongly activated the MAPK signaling pathway and enhanced the gene expression of several antioxidant-related enzymes in IPEC-J2 cells based on RNA-sequencing obtained gene expression profiles after deoxyshikonin treatment [21], which may indicate that the MAPK pathway and other pathways may mediate the antioxidant and cytoprotective effects of deoxyshikonin.
To confirm the involvement of the Nrf2 signaling pathway, we evaluated MDA accumulation, ROS production, and apoptosis after Nrf2 knockdown in IPEC-J2 cells. The cytoprotective effect of calycosin on H 2 O 2 -induced cell damage was abolished under Nrf2 deficiency. These results confirmed that the Nrf2 pathway is crucial for the cytoprotective effect of calycosin against H 2 O 2 -induced oxidative stress in porcine epithelial cells. Lu et al. [29] obtained similar results in brain astrocytes; they found that calycosin protected against H 2 O 2 -induced astrocyte oxidative injury via the AKT-Nrf2-HO-1 signaling pathway, and siNrf2 application dramatically abolished these effects. In rheumatoid arthritis synovial fibroblasts, calycosin significantly potentiated Nrf2 translocation by inducing p62 accumulation and Keap1 degradation to activate HO-1 and NQO1, thus suppressing IL-6 and IL-33 secretion and COX-2 mRNA and protein expression [54]. The anti-inflammatory effect of calycosin has been demonstrated in various in vivo and in vitro studies [35,55]. Whether calycosin exerts its cytoprotective effect in porcine epithelial cells via both antioxidant and anti-inflammatory actions is worthy of further investigation. Nrf2 knockdown suppressed the effect of deoxyshikonin in reducing ROS accumulation but had no significant effect on the effects of deoxyshikonin on MDA and apoptosis levels, which further indicated that Nrf2 may not be the main signaling pathway, but is partially involved in the regulatory effect of deoxyshikonin on H 2 O 2 -mediated porcine epithelial cell damage. Shikonin attenuated oxidative stress in spiral ganglion neuron cells and spiral ganglion Schwann cells via Nrf2 pathway activation, thus improving neurological hearing damage in mice [56]. Despite their structural similarity, the analogs may have different mechanisms. More robust, in-depth studies are required to unravel the precise antioxidant mechanism of deoxyshikonin.

Conclusions
We successfully identified 22 natural products that alleviated H 2 O 2 -induced oxidative damage in IPEC-J2 cells using an HTS assay. Among them, calycosin and deoxyshikonin exhibited superior antioxidant capacity. Pretreatment with calycosin or deoxyshikonin alleviated H 2 O 2 -induced oxidative cell damage by regulating the redox state of cells and enhancing the antioxidant defense system. Nrf2-Keap1 signaling may be involved or partially involved in the protective mechanisms of the respective compounds. These oxidative-stress-alleviating natural compounds have the potential to be developed as novel therapeutic or protective agents for use in pigs and possibly, other livestock susceptible to oxidative stress. However, further research is required to fully understand the modes of action of the compounds. This was the first study to demonstrate the antioxidant effects of calycosin and deoxyshikonin in livestock animal cells. Further studies of the compounds in appropriate animal models will be needed to investigate their usability as antioxidants in livestock production.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11112134/s1, Table S1: sequences of the primers used in this study; Table S2: antibodies used in this study.