Intestinal epithelial cells (IECs) including the colorectal adenocarcinoma-derived enterocytes (HT-29, HCT-8, LS174T, DLD-1, and SW480) and the well-established non-tumorigenic normal enterocytes from the porcine gut (IPEC-1) were purchased from the American Type Culture Collection (Manassas, VA, USA) and Leibniz-Institute DSMZ (Brauschweig, Germany), respectively. In this study, HT-29 cells were used as a representative model of pathogenesis in the IECs because they show differentiated and polarized features in RPMI 1640-based media (32). In addition, HCT-8 or IPEC-1 cell-based barrier was assessed due to its rapid formation of epithelial monolayer among the colorectal adenocarcinoma-derived enterocytes. In particular, IPEC-1 cells have been extensively used as the non-tumorigenic enterocyte model of DON exposure (17, 33, 34). The colorectal adenocarcinoma-derived enterocytes were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from Welgene, Daegu, South Korea) in a 5% CO₂ humidified incubator at 37°C. Since RPMI 1640 media in this study do not contain HEPES buffer, 25 mM HEPES (LPS Solution, Daejeon, South Korea) was additionally administered in LS174T and SW480 cell culture for optimal buffering capacity. Other colon adenocarcinoma-derived cells optimally grow in HEPES-free RPMI-based media. IPEC-1 cells were maintained in DMEM/F12 (Welgene, Daegu, South Korea) supplemented with 10% (v/v) heat-inactivated FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin in a 5% CO₂ humidified incubator at 37°C. The viable cells determined by staining with trypan blue (Sigma-Aldrich, St. Louis, MO, USA) were counted using a hemocytometer. Mycotoxin-free adlay was kindly provided by Rural Development Administration, Korea. SB203580 and U0126 were purchased from Calbiochem (Merck Millipore, Billerica, MA, USA) and Enzo Life Science (Plymouth, PA, USA), respectively. All other chemicals were purchased from Sigma-Aldrich.

The ethyl acetate fraction of adlay bran was extracted because this fraction includes high levels of anti-inflammatory components such as chlorogenic acid, vanillic acid, caffeic acid, 4-hydroxyacetophenone, p-coumaric acid, syringaldehyde, ferulic acid, and 6-methoxy-2-benzoxazolinine as previously reported (29, 35). Briefly, dried adlay bran was extracted three times with 95% ethanol (200 ml each time) using a stirring apparatus in a brown bottle. The combined extracts were then passed through No. 1 Whatman filter paper, after which the filtrate was evaporated under vacuum at 38°C to obtain a residue, which was subsequently suspended in water and then successively partitioned with hexane (1:1) and ethyl acetate (1:1). This produced an ethyl acetate-soluble fraction, which was evaporated again and dissolved in DMSO.

Interleukin-8 (IL-8) transcriptional activity was measured using IL-8 promoter-luciferase reporter construct (36–38). IL-8 promoter-luciferase construct containing a part of the human IL-8 promoter ranging from nucleotides −416 to +44 was kindly provided from Dr. Kung, Hsing-Jien (University of California at Davis). The 3′ untranslated region (UTR) of the human IL-8 gene (+402/+1611) was cloned into the pGL3 control vector at the XbaI site. After polymerase chain reaction (PCR) of the promoter region with Pfu turbo DNA polymerase (Stratagene, La Jolla, CA, USA), the fragment was cloned into the TA vector (Invitrogen), sequenced, and subcloned into the pGL3 control vector. An antisense (AS) human antigen R (HuR) gene construct was kindly provided from Dr. Gorospe, Myriam (NIH, Baltimore, USA) and reconstituted into pcDNA3.1-Hyg vector system (Invitrogen). The final product was designated pcDNA3.1-AsHuR-Hyg (HuR-AS). CMV-driven small interference RNA (siRNA) expression vector was constructed by inserting the hairpin siRNA template into a pSilencer 4.1-CMV-neovector (Ambion Inc., Austin, TX, USA). The scrambled control vector and early growth response gene 1 (Egr1) siRNA insert-containing vector were denoted as pSilencer and pSiEgr1, respectively. Insert Egr1 siRNA (Dharmacon, Lafayette, CO, USA) targeted the sequence AAGTTACTACCTC TTATCCAT.

HT-29 cells (6 × 10³) were seeded in a 96-well culture plate in 6 replicates, cultured for 48 h, and then treated with the vehicle or each combination of chemicals for indicated times. At the end of treatment, 50 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (1 mg/ml) was added to each well (100 µl media), and the plates were incubated at 37°C for 4 h to measure its reduced metabolite (formazan crystal) via mitochondrial action (39). The formazan product was then dissolved in 150 µl DMSO at 37°C for 30 min, and the absorbance at 540 nm was measured with a microplate reader.

HT-29 cells (3 × 10⁵), HCT-8 cells (3 × 10⁵), DLD-1 cells (3 × 10⁵), LS174T cells (3 × 10⁵), SW480 cell (3 × 10⁵), and IPEC-1 (2 × 10⁵) were seeded, cultured for 48 h, and then treated with the vehicle (DMSO) or each dose of DON with or without adlay extract. Cellular RNA was extracted using RiboEX (GeneAll Biotech, Seoul, South Korea), and the subsequent procedure was performed based on the methods reported in our previous study (38). The mRNA was transcribed into cDNA by Prime RT premix (Genet Bio, Nonsan, South Korea). cDNA amplification was conducted with N-Taq DNA polymerase (Enzynomics, Seoul, South Korea) in a MyCycle thermal cycler (BioRad) using the following parameters: initial denaturation at 95°C for 2 min, followed by varying numbers of cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and elongation at 72°C for 30 s. An aliquot of each PCR product was subjected to 1.2% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining. Sequences of PCR primers for amplifying each gene were as follows: human GAPDH (5′-TCA ACG GAT TTG GTCGTA TT-3′ and 5′-CTGTGG TCA TGA GTC CTT CC-3′), human Egr1 (5′-AGC ACC TGA CCG CAG AGT CT-3′and 5′-AGA TGG TGC TGA GGA CGA GG-3′), human IL-8 (5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′5′-TCT CAG CCC TCT TCA AAA ACT TCT C-3′), porcine IL-8 (5′-ACT TCC AAA CTG GCT GTT GC-3′ and 5′-TGC TGT TGT TGT TGC TTC TCA-3′), and porcine GAPDH (5′-CAC GAC CAT GGA GAA GGC-3′ and 5′-GAA GCA GGG ATG ATG TTC TGG-3′). Real time PCR was conducted using an iCycler Thermal cycler (BioRad) with the following parameters: initial denaturation at 95°C for 15 min followed by cycles of denaturation at 95°C for 20 s, annealing at 59°C for 30 s, and elongation at 72°C for 30 s. Each sample was assessed in triplicate, after which the relative quantification of gene expression was performed by the comparative threshold cycle (C_t) method. GAPDH was used as the endogenous control, and each independent experiment was repeated three times.

The IL-8 concentrations in cell culture supernatants were determined using a commercially available ELISA kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s directions. HT-29 cells were seeded at 1 × 10⁵ cells/well of a 24-well plate (n = 6) and cultured for 48 h. After treatment with the vehicle (DMSO) or each combination of chemicals for 24 h, cell culture medium was collected, and cell debris was removed by centrifugation. Briefly, the capture antibody was coated onto the wells of ELISA plates overnight at 4°C. After washing with Tween 20-containing PBS and blocking with PBS supplemented with 10% (v/v) FBS overnight at 4°C, the plates were incubated with serial dilutions of IL-8 samples and standards. After incubation with the detection antibody and the tetramethylbenzidine substrate, the absorbance was measured at 450 nm using an ELISA reader, and the assay detection limit was 3.1 pg/ml of IL-8 (38). Results are representative of two independent experiments.

HT-29 cells (5 × 10⁵), DLD-1 cells (5 × 10⁵), LS174T cells (5 × 10⁵), SW480 cell (5 × 10⁵), and IPEC-1 (4 × 10⁵) were seeded, cultured for 48 h, and then treated with the vehicle (DMSO) or each combination of chemicals for 0.5, 1, or 2 h. Treated cells were washed with ice-cold phosphate buffer, lysed in boiling lysis buffer [1% (w/v) SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, PH 7.4] and sonicated for 5 s. The proteins in these lysates were then quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein (30 µg) were separated by SDS-PAGE gel in a BioRad gel mini electrophoresis system (BioRad, Hercules, CA, USA). The proteins were transferred onto a PVDF membrane (Pall Corporation, New York, NY, USA) and then blocked for 1 h with 5% skim milk in tris-buffered saline plus 0.1% Tween (TBST), after which they were incubated with the desired primary antibody overnight at 4°C. After washing three times with TBST, the blots were incubated with horseradish-conjugated secondary antibody for 2 h and then washed with TBST three times (40). Antibody binding was detected with an ECL substrate (ELPIS Biotech, Daejon, South Korea). The following antibodies were used for Western blot: rabbit polyclonal anti-Actin, mouse monoclonal anti-β-tubulin, rabbit polyclonal anti-p-p38, mouse monoclonal anti-p-Erk, rabbit polyclonal anti-Egr1, mouse monoclonal anti-hnRNP, mouse monoclonal anti-HuR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal p-(Ser) protein kinase C (PKC) Substrate Antibody, and rabbit anti-p-EGF Receptor (Y1068) (Cell Signaling Technology, Beverly, MA, USA).

HT-29 cells (7 × 10⁵) were seeded, cultured for 48 h, and then treated with the vehicle (DMSO) or each combination of DON and adlay for 1 h. Cells were harvested from culture plates by scraping in ice-cold PBS and centrifuged at 200 × g once for 3 min. The cell pellet after centrifugation was then resuspended in a lysis buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.5 mM PMSF, 0.1% Nonidet P-40, and protease inhibitor mixture (Sigma-Aldrich), incubated for 10 min on ice, and centrifuged at 900 × g once for 15 min. Next, the supernatant (cytosolic fraction) was collected, and the remaining pellet was resuspended in a buffer containing 20 mM HEPES, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 25% glycerol, and protease inhibitor mixture (Roche). After 10 min of incubation on ice, samples were centrifuged at 13,800 × g once for 30 min, and the supernatants (nuclear proteins) were collected, aliquots, and stored at −80°C before analysis. Next, rabbit polyclonal anti-p-PKC and mouse monoclonal anti-HuR were added to the cell cytosolic fraction lysate supernatant and rotated overnight at 4°C. Protein G-Sepharose (30 µl; Santa Cruz Biotechnology) in antibody-cell lysate were incubated by rotating at 4°C for 3 h. The antibody cytosolic fraction extracts were then washed three times with IP lysis buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1 mM EDTA, 400 mM Na₃VO₄, and 2.5 mM phenylmethanesulfonyl fluoride), after which 6× SDS sample buffer (60% glycerol, 300 mM Tris pH 6.8, 12 mM EDTA, 12% SDS, 864 mM 2-ME, and 0.05% bromophenol blue) was added. Finally, immunoprecipitates were collected by centrifugation and subjected to SDS-PAGE. The nuclear (hnRNP) and cytoplasmic (β-actin) markers verified the identity and purity of the fractions (38).

HT-29 cells (2.5 × 10⁵) were seeded, cultured for 48 h, and then transfected with a shRNA vector using OmicsFect (Omics Biotechnology Co., Taipei City, Taiwan), jetRPIME (polyplus transfection), or Lipofector-EXTReagent (Aptabio Therapeutics Inc., Gyeonggi-do, Korea) according to the manufacturer’s protocol. For transfection of the luciferase reporter gene, a mixture of 1 µg of luciferase reporter plasmids and 0.1 µg of Renilla luciferase pRL-null vector (Promega, Madison, WI, USA) per 1 µl of Carrigene (Kinovate, Seoul, Korea) was applied to wells of a 12-well culture plate. Transfected cells were exposed to vehicle (DMSO) or each combination of chemicals for 12 h and lysed for luciferase assay. All transfection efficiency was maintained at around 50–60%, which was confirmed with pMX-enhanced green fluorescent protein vector. Cells were washed with cold PBS and then lysed with passive lysis buffer (Promega, Madison, WI, USA). Cell lysates were centrifuged, and the supernatant was collected for luciferase activity, which was measured with a dual-mode luminometer (Model TD-20/20, Turner Designs Co., Sunnyvale, CA, USA) after briefly mixing 10 µl supernatant and 50 µl firefly luciferase assay substrate solution, followed by 50 µl stopping Renilla luciferase assay solution (Promega, Madison, WI, USA). The firefly luciferase activity was normalized against Renilla luciferase activity using the following formula: firefly luciferase activity/Renilla luciferase activity (41). This experiment was conducted in triplicate, and all of the results are representative of three independent experiments.

HT-29 cells (1 × 10⁵) or HCT-8 cells (1 × 10⁵) were cultured in glass-bottom culture dishes (SPL Life Science, Pocheon, Korea) and cultured for 48 h. Following treatment with the vehicle (DMSO) or each combination of chemicals for 1 or 24 h, cells were fixed with 4% paraformaldehyde (Biosesang, Sungnam, Korea) and permeabilized with 0.2% Tween 20 and 0.3% BSA in PBS for 10 min. After 2 h of blocking with 3% BSA in PBS, cells were incubated with primary antibody in 3% BSA PBS for 2 h at room temperature. Subsequently, the cells were washed with PBS and incubated with goat anti-mouse IgG (H + L) Dylight 488 conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) or Texas Red (SurModics, Eden Prairie, MN, USA) for 2 h at room temperature. The cells were repeatedly washed in PBS and stained with 100 ng/ml DAPI in PBS for 10 min (41). Next, confocal images were obtained using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). The images were acquired and processed with FV10-ASW software (Olympus, Tokyo, Japan), and the intensity of signals from individual cells was measured using the ImageJ 1.45 software. This experiment was conducted in triplicate, and all of the results are representative of three independent experiments.

Cells (HCT-8 or IPEC-1) were seeded at a density of 4 × 10⁵ cells per well and grown into monolayers in 24-well transwell filters with 0.4-µm pores (Becton-Dickinson Labware, Franklin Lakes, NJ, USA). For the cell differentiation, cell culture media containing 100 nM dexamethasone was added and exchanged every 2 days until complete differentiation (42). On 10th day of the differentiation process, cells were treated with the vehicle (DMSO) or each combination of chemicals. TEER was measured every 12 h with an EVOM2 epithelial voltohmmeter (World Precision Instruments, Sarasota, FL, USA). Experimental TEER values were expressed as Ω cm². This experiment was conducted in triplicate, and all of the results are representative of three independent experiments.

Fluorescein isothiocyanate (FITC)–dextran was used as a fluorescent probe to study cell permeability. Briefly, cells were seeded at a density of 4 × 10⁵ cells per well and grown into monolayers in 24-well transwell filters with 0.4 µm pores, and differentiated cells in the transwell inserts were treated with the vehicle (DMSO) or each combination of chemicals for 48 h. Next, FITC–dextran (Sigma-Aldrich Chemical Company) dissolved in cell culture media was added to the apical compartment at a final concentration of 2.2 mg/ml. After 1 h of incubation, the intensity of fluorescence emission in the outer part of the well was measured using a Victor 3 fluorometer (Perkin Elmer, Waltham, MA, USA), with excitation and emission wavelengths set to 490 and 535 nm, respectively (33). This experiment was conducted in triplicate, and all of the results are representative of three independent experiments.

For two-sided wound healing assays, cells (HCT-8 or IPEC-1) were seeded to a final density of 5 × 10⁵ cell/ml in an Ibidi Culture-Insert 2 well (Ibidi, Martinsried, Germany) according to the manufacturer’s directions. After cell attachment, the Ibidi Culture-Insert was gently removed creating a gap of ~500 μm. Cells were gently washed with PBS, and the dish was filled with serum-free medium containing the vehicle (DMSO) or each combination of chemicals. Images of the cells migrating into the wound were captured by an inverted microscope (×10; n = 3). The migration was measured by calculating mean of distances of the migrating cells from both wound edges using JR screen ruler. For the IPEC-1 cells, the relative area of migrating cells in the wound space was measured using the ImageJ 1.45 software since the migrating edge was not linear. For scratch-induced wound healing assays, cells were seeded in 12-well culture plates and grown to a monolayer. Because of irregular gap after scratching, one-sided migration was quantified. After scratching, cells were incubated with serum-free medium. Images of the cells migrating into the wound were captured by an inverted microscope (×10; n = 3–6). The average wound length was measured by calculating mean of distances of the migrating cells from one side of wound edges using JR screen ruler. This experiment was conducted in triplicate, and all of the results are representative of three independent experiments.

Statistical analysis was performed using the GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA). A Student’s t-test was used for comparative analysis of the two groups of data. To compare multiple groups, data were subjected to ANOVA, and pairwise comparisons were made by the Student–Newman–Keuls method.

Mucosal epithelium senses external toxic insults and transmits danger signals to activate a broad range of defensive inflammatory responses, including chemokine production. Adlay-treated HT-29 cells showed attenuated cellular induction of IL-8 in response to DON in a dose-dependent manner (Figure 1A). DON-triggered IL-8 release was thus reduced by adlay treatment (Figure 1B). Suppression of IL-8 mRNA was also demonstrated in enterocytes treated with lower levels of adlay extract (Figure 1C). In the early treatment (1–2 h) for the gene expression analysis, the cell growth was not significantly suppressed by DON or adlay (Figure S1A in Supplementary Material). However, extended exposure (24 h) to higher doses (>500 µg/ml) of adlay extract reduced the cell growth in presence or absence of DON (Figure S1B in Supplementary Material). Therefore, all biochemical readouts were divided by the total levels of cell number, protein, or RNA. Moreover, the prolonged impacts were assessed at the lower level of adlay (50 µg/ml; Figures 4–7). Consistent with responses in HT-29 cells, DON-induced IL-8 was also significantly reduced by adlay in other colonic epithelial cell lines, such as HCT-8, LS174T, DLD-1, and SW480 (Figure 1D). In addition to these colorectal adenocarcinoma-derived enterocytes, the similar responses in IL-8 expression were confirmed in the well-established non-tumorigenic normal enterocytes (IPEC-1; Figure 1D).

As a pivotal transcriptional regulator in DON-induced IL-8 production, Egr1 and MAPKs, which induce DON-induced Egr1 expression in IECs (43), were assessed in this study. As expected, DON treatment activated MAPK signals along with Egr1 induction in HT-29 cells (Figure 2A). Among these DON-elevated signaling mediators, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and p38 signals were positively associated with IL-8 mRNA expression in IECs (Figure 2B). In particular, ERK1/2 as a crucial mediator to regulate various physiological functions is activated by receptor tyrosine kinase-associated pathways such as epidermal growth factor receptor (EGFR) (43–45). In the present model, phosphorylation of EGFR and ERK1/2 were downregulated by adlay treatment; however, the JNK1/2 and p38 signals were not notably involved in this regulation (Figure 2C). Moreover, blocking of the EGFR-linked signal significantly downregulated DON-induced IL-8 transcriptional activation (Figure 2D). In terms of signaling pathways, inhibition of EGFR and ERK1/2 signals suppressed the DON-induced expression of Egr1 (Figure 2E), which is a key transcriptional modulator of pro-inflammatory IL-8 expression in IECs as mentioned in Section “Introduction” (Figure S2 in Supplementary Material). Furthermore, DON-induced Egr1 transcription was significantly attenuated in adlay-treated enterocytes (Figure 2F). Suppression of Egr1 levels by adlay treatment was consistently observed in other IECs, including DLD-1, SW480, and IPEC-1 cells (Figure 2G). Taken together, adlay-suppressed IL-8 expression was caused by signaling interference with Egr1 in EGFR- and ERK1/2-dependent manners in IECs.

Although DON-induced IL-8 transcription was suppressed by adlay exposure (Figure 3A), its mRNA stability was elevated by treatment with adlay in HT-29 cells (Figure 3B). Moreover, we constructed a luciferase reporter system containing 3′UTRs of IL-8, which have 10 AU-rich elements (AREs). DON-induced luciferase activity was also enhanced by adlay treatment in IECs (Figure 3C), indicating the elevated stability of DON-induced IL-8 mRNA by adlay. Although most RNA-binding proteins destabilize targeted mRNA, a few of these proteins, including HuR, bind to 3′UTRs with AREs and stabilize the transcripts of genes, including those encoding growth factors or pro-inflammatory cytokines (36, 46). Consistent with the promoting effects of adlay on IL-8 mRNA stability, adlay exposure enhanced the cytoplasmic translocation of HuR protein triggered by DON in enterocytes (Figure 3D). These patterns were also confirmed by visualizing nuclear localization of HuR protein in the cells by confocal microscopy (Figure 3E). In terms of the signaling pathway, PKC was evaluated for its involvement in HuR translocation to the cytoplasm (47, 48). Moreover, adlay-enhanced cytoplasmic translocation of HuR protein was significantly suppressed by PKC inhibition (Figure 3E). In addition, samples were analyzed to determine whether PKC is involved in regulation of HuR protein in adlay-exposed IECs. Adlay extract enhanced DON-induced HuR protein translocation to the cytoplasm via PKC-mediated phosphorylation in IECs (Figure 3F). Taken together, adlay-enhanced PKC-mediated cytoplasmic translocation of the HuR protein, although adlay treatment decreased DON-induced IL-8 production at the transcriptional level.

In addition to epithelial chemokine production, we assessed the effects of adlay on intestinal barrier integrity by measuring the TEER in vitro. The DON-disrupted intestinal barrier was partly restored by adlay extract treatment; however, HuR suppression and PKC inhibition attenuated the protective actions of adlay against barrier disruption (Figures 4A,B). Another readout of the intestinal permeability by quantifying the paracellular passage of 4 kDa FITC–dextran indicated a similar pattern of adlay-induced protection against DON-induced epithelial disruption in HuR- and PKC-dependent ways (Figures 4C,D). We next investigated how adlay restored DON-disrupted intestinal barrier integrity through enhanced translocation of the HuR protein.

To explore epithelial barrier restitution by cellular migration, we assessed wound healing of migrating enterocytes exposed to DON and adlay. DON exposure retarded the migration activities in response to physical wounds in enterocytes (Figure 5). In contrast, treatment with adlay significantly restored the epithelial restitution in DON-insulted cells, which was attenuated by inhibition of HuR expression and PKC activity in IECs (Figures 5A,B), indicating that DON-disrupted intestinal barrier integrity was rescued by adlay extract exposure via PKC- and HuR protein-mediated enhancement of migration of insulted IECs. The PKC- and HuR-linked signals were also positively involved in the scratch-induced wound healing process in the epithelial monolayer (Figures S3A,B in Supplementary Material, respectively).

Migrating HCT-8 enterocytes were closely observed after staining actin polymerization. DON treatment notably suppressed the staining of F-actin formation with lamellipodia and filopodia networks (Figure 6A) but was retarded by adlay exposure. In contrast, adlay-induced resistance to cytoskeletal rearrangement in the gut epithelial cells was notably mediated in PKC- and HuR-dependent manners (Figure 6A). Among the critical signaling mediators of lamellipodia or filopodia formation, RhoA GTPase was quantified in the present enterocyte exposure model. In agreement with the changes in F-actin formation, DON treatment suppressed the RhoA levels, which was counteracted by the adlay treatment. Adlay-induced restoration of the actin cytoskeleton network was dependent on PKC activation and HuR protein (Figure 6B). Functionally, inhibition of RhoA GTPase activities significantly suppressed adlay-restored enterocyte migration under the insult of enteropathogenic DON (Figure 6C). Assessment in the scratch-induced wound healing consistently confirms the positive involvement of RhoA signals in restoring of epithelial migration in the presence of DON and adlay (Figure S3C in Supplementary Material). Ultimately, inhibition of RhoA GTPase activities significantly suppressed adlay-restored epithelial barrier under the insult of DON in HCT-8 cells (Figure 6D). Finally, mechanistic implication was also tested using the non-tumorigenic enterocyte IPEC-1. Inhibition of RhoA GTPase activities significantly suppressed adlay-restored enterocyte migration in the presence of DON (Figure 7A). Furthermore, RhoA signal was positively involved in maintaining the adlay-restored epithelial barrier under the stress of DON in IPEC-1-based monolayer (Figure 7B). Taken together, RhoA GTPase-linked signal promotes cell migration via cytoskeletal rearrangement, which contributes to the epithelial restitution in the insulted gut barrier. Therefore, it can be concluded that adlay counteracts DON-induced migratory inhibition by restoring cytoskeletal rearrangement and RhoA expression in the gut epithelial cells in HuR and PKC-dependent pathways (Figure 7C).