All-trans retinoic acid (ATRA, ≥98% HPLC) and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Shanghai, China). Rabbit anti-ZO-1(61–7300) was purchased from Invitrogen (CA, USA). Rabbit anti-Occludin (ab31721) was purchased from Abcam (Cambridge, UK). Mouse anti-IκBα (4814), mouse anti-NF-κB p65 (6956) and rabbit anti-p-NF-κB p65 (3033) were obtained from Cell signaling Technology (Beverly, USA). Mouse anti-p-IκBα (sc-8404), mouse anti-β-actin antibody (sc-47778) and goat anti-rabbit/mouse IgG -HRP secondary antibody (sc-2030 and sc-2031) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). The NF-κB inhibitor (BAY11-7082) was purchased from Selleck (Selleckchem, Huston USA).

IPEC-J2 cells and ST cells were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient Mixture (DMEM/F12, Gibco, Shanghai, China) or Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco, Shanghai, China) and 1% streptomycin and penicillin (Gibco, Shanghai, China), and maintained at 37°C with 5% CO₂ atmosphere incubator. The medium was changed every 2 days to avoid nutrient depletion. TGEV (SC-T strain) was provided by Prof. Zhiwen Xu, College of Veterinary Medicine, Sichuan Agricultural University. As described previously (24), the TGEV used for IPEC-J2 cells infection was harvested from ST cells and virus titers were determined by 50% tissue culture infective doses (TCID50). For TGEV infection, 70–80% confluent IPEC-J2 cells were inoculated with TGEV [multiplicity of infection (MOI) = 1] at 37°C for 1 h. After TGEV infection, the cells were washed 3 times with PBS and added fresh growth medium. Subsequently, cells and supernatants were harvested at the indicated time points.

The siRNA targeting porcine TLR-3, TLR-7, RIG-I, MDA5 and negative control siRNA (siNC) were synthesized by Sangon Biotech Co., Ltd (Shanghai, China), and the target sequences were listed in Table 1. Cells were transfected with siRNA at indicated concentrations using lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After transfection, the cells were used for subsequent analysis or treatment.

Cell viability was determined using a commercial CCK-8 kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, IPEC-J2 cells were cultured in 96-well plates at a density of 1×10⁴ cells/well and treated with various concentrations of ATRA (0, 1, 10, 20, 40, 60, 80, 100 and 200 μM) at 37°C for 48 h. Then, 10 ul CCK-8 assay solution was added into each well for 2 h at 37°C. Finally, the optical density (OD) of each well at 450 nm was measured using a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA).

To identify cell membrane damage, the activity of lactate dehydrogenase (LDH) in IPEC-J2 cells culture medium was detected by LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions.

The concentrations of IL-1β, IL-6, IL-8 and TNF-α in IPEC-J2 cells culture medium were determined by using the commercially ELISA kits (Jiangsu Meimian Biotechnology Co., Ltd., Jiangsu, China) according to the manufacturer’s instructions.

Total RNA was extracted from IPEC-J2 cells using TRIzol reagent (TaKaRa Biotechnology Co, Ltd, Dalian, China) following the manufacturer’s instructions. The integrity of RNA was checked by electrophoresis on a 1.5% agarose gel. Complementary DNA (cDNA) was synthesized from 1µg of total RNA using the PrimeScripte RT reagent kit (Takara) according to the manufacturer’s instructions. Then, the synthesized cDNA was diluted (1:4) and real-time quantitative PCR amplification was performed using SYBR Premix Ex TaqTM kits (TaKaRa) on the CFX96 Real-Time PCR Detection System (Bio-Rad). The primers were synthesized by TaKaRa Biotechnology Co, Ltd. (Dalian, China), which were listed in Table 2. The thermal cycling parameters were as follows: pre-denaturation at 95°C for 30s, then 40 cycles at 95°C for 5 s, 60°C for 34 s and extension at 72°C for 60 s. Each reaction was completed with a melting curve analysis to ensure the specificity of the reaction. The relative mRNA levels of target genes were analyzed using the 2^−ΔΔCt method and using β-actin as the reference gene (25). All reactions were performed in triplicate.

Proteins from IPEC-J2 cell samples were acquired using RIPA lysis buffer containing PMSF and phosphatase inhibitor. After incubating at 4°C for 30 min, samples were homogenized and centrifuged at 12,000 g for 15 min at 4°C, and the supernatants were collected. The protein concentrations in the supernatants were determined by a BCA protein assay kit (Thermo Scientific, MA, USA). Then, equal amounts of protein (25 μg) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Ltd., Tullagreen, Ireland). The membranes were blocked using 5% nonfat dry milk in 1× TBST for 1 h at room temperature followed by an overnight incubation at 4°C with indicated primary antibodies. Following three flushes with 1× TBST, the membranes were incubated with the corresponding HRP- conjugated secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized with ECL chemiluminescence kit (Beyotime Biotechnology, Shanghai, China) by the ChemiDoc™ XRS Imager System (Bio-Rad). The protein band densities were quantified using Image Lab software (Bio-Rad).

IPEC-J2 cells were cultured in a 12-well cell culture plate and infected with TGEV at MOI of 1 for 1 h. Afterwards, the cells were washed with PBS and cultured with 80 μM ATRA for 36 h. After cell treatment, IPEC-J2 cells were fixed with cold 4% paraformaldehyde for 15 min and permeated with 0.05% Triton X-100 for 15 min at room temperature. Then, the IPEC-J2 cells were incubated with primary anti-NF-κB p65 mouse antibodies overnight at 4°C (1:100 dilution, Cell signaling Technology, USA). The cells were then washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Abcam, Shanghai, China) for 2 h at room temperature in the dark. After washing with PBS, the cells were mounted using Vectashield Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Shanghai, China). Images were captured using a fluorescence microscope (Leica DMI 4000 B).

All data were presented as the means ± standard error of mean (SEM). Statistical differences were analyzed by the unpaired two-tailed Student’s t-test and/or one-way analysis of variance (ANOVA) using SPSS 22.0 statistics software (Chicago, IL, USA). P < 0.05 was considered statistically significant.

To investigate the cytotoxic effects of ATRA on IPEC-J2 cells, IPEC-J2 cells were treated with various concentrations of ATRA (0, 1, 10, 20, 40, 60, 80, 100 and 200 μM) for 48 h. As shown in Figure 1, ATRA concentrations ≤100 μM did not significantly affect cell viability (P > 0.05), but a marked decrease in cell viability was observed at ATRA concentration with 200 μM compared with the control group (P < 0.001). Therefore, an ATRA concentration range of 0~80 μM was used in the subsequent experiments.

To assess whether ATRA attenuated the inflammatory response induced by TGEV, the mRNA levels of the inflammatory cytokines were analyzed by real‐time PCR. The results showed that TGEV infection significantly up‐regulated the mRNA levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 (P < 0.001), whereas ATRA treatment significantly inhibited the up‐regulation of the gene expressions of inflammatory cytokines in a dose-dependent manner (P < 0.05). The highest concentration ATRA (80 μM) was the most effective in decreasing the mRNA expressions of inflammatory cytokines induced by TGEV (P < 0.001) (Figure 2). To further determine the effects of ATRA on the TGEV‐induced inflammatory response, we detected the concentrations of inflammatory cytokines in IPEC-J2 cells culture medium. Compared with the control group, ATRA treatment significantly decreased the concentrations of IL-1β and IL-6 in IPEC-J2 cells culture medium (P < 0.05). Compared with the control group, TGEV infection significantly increased the concentrations of IL-1β, IL-6, IL-8, TNF-α and IL-10 in IPEC-J2 cells culture medium (P < 0.01). However, the increase in the concentrations of inflammatory cytokines(IL-1β, IL-6, IL-8, TNF-α and IL-10)was not detected in IPEC-J2 cells culture medium treated with UV-inactivated TGEV (P > 0.05). Furthermore, ATRA treatment significantly inhibited the elevation of IL-1β, IL-6, TNF-α and IL-10 concentrations induced by TGEV (P < 0.05) (Figure 3). These results indicated that TGEV infection could induce the inflammatory response in IPEC-J2 Cells, and this ability required TGEV replication; ATRA treatment could attenuate the inflammatory response induced by TGEV.

LDH is an important marker for assessing cell permeability. To identify whether ATRA attenuated cell epithelial barrier integrity damage induced by TGEV, LDH activity in IPEC-J2 cell culture medium was detected. As shown in Figure 4A, compared with the control group, TGEV infection significantly increased the LDH activity in IPEC-J2 cells culture medium, whereas ATRA treatment significantly decreased LDH released from the TGEV-infected IPEC-J2 cells (P < 0.001). In addition, compared with the control group, ATRA treatment significantly increased Occludin protein level (P < 0.001). TGEV infection significantly reduced the protein levels of ZO-1 and Occludin compared with the control group (P < 0.001). However, ATRA treatment significantly reversed this reduction of ZO-1 and Occludin protein levels induced by TGEV (Pwfi 2 < 0.001) (Figures 4B–D).

Compared with the control group, TGEV infection significantly upregulated the phosphorylation levels of IκBα and NF-κB p65 in IPEC-J2 cells (P < 0.001), whereas UV-inactivated TGEV treatment did not induce the upregulation of NF-κB p65 phosphorylation level in IPEC-J2 cells (P > 0.05). Furthermore, ATRA treatment significantly prevented the upregulation of IκBα and NF-κB p65 phosphorylation levels induced by TGEV (P < 0.01) (Figures 5A–E). Further immunofluorescence analysis found that TGEV infection induced the translocation of NF-κB p65 from cytoplasm to nucleus in IPEC-J2 cells, and ATRA treatment inhibited this process (Figure 5F). These results indicated that TGEV infection could activate the NF-κB signaling pathway, and this ability required TGEV replication; ATRA treatment could prevent the NF-κB signaling pathway activation induced by TGEV.

To further investigate whether ATRA attenuated TGEV-induced inflammatory response via suppressing the NF‐κB signaling pathway, the effect of ATRA was examined by using by NF-κB inhibitors (BAY11-7082). As shown in Figure 6, we found that TGEV infection significantly increased the phosphorylation level of NF-κB p65 in IPEC-J2 cells compared with the control group, whereas BAY11-7082 treatment significantly inhibited the enhancing of NF-κB p65 phosphorylation level induced by TGEV (P < 0.001). Furthermore, we found that TGEV infection significantly upregulated the mRNA levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 in IPEC-J2 cells and increased the concentrations of IL-1β, IL-6, IL-8, TNF-α and IL-10 in IPEC-J2 cells culture medium compared with the control group (P < 0.05). However, BAY11-7082 treatment significantly inhibited the upregulation of IL-6, IL-8, TNF-α and IL-10 mRNA levels in IPEC-J2 cells and the enhancing of IL-1β, IL-6 and TNF-α concentrations in IPEC-J2 cells culture medium induced by TGEV (P < 0.05).

As shown in Figure 7, compared with the control group, ATRA treatment significantly increased the mRNA levels of TLR3, TLR4, TRIF and TRAF6 (P < 0.05). TGEV infection significantly upregulated the mRNA levels of TLR2, TLR3, TLR4, TLR7, MyD88, TRIF and TRAF6, and downregulated TLR9 mRNA level compared with the control group (P < 0.05). However, ATRA treatment significantly suppressed the upregulation of TLR3, TLR7, TRIF and TRAF6 mRNA levels and the downregulation of TLR9 mRNA level induced by TGEV (P < 0.01). Furthermore, TGEV infection significantly increased the protein levels of TLR3 and TLR7 compared with the control group, whereas ATRA treatment significantly inhibited the enhancing of TLR3 and TLR7 protein levels induced by TGEV (P < 0.01). As shown in Figure 8, the mRNA abundance of RIG-Ⅰ, MDA5 and MAVS was significantly upregulated in TGEV group compared with the control group, and ATRA treatment significantly inhibited the upregulation of RIG-Ⅰ, MDA5 and MAVS mRNA abundance induced by TGEV (P < 0.001). Furthermore, TGEV infection significantly increased the protein levels of RIG-I and MDA5 compared with the control group, whereas ATRA treatment significantly inhibited the enhancing of RIG-I and MDA5 protein levels induced by TGEV (P < 0.01).

To further confirm whether ATRA attenuated TGEV-induced inflammatory response via suppressing the TLRs/RLRs/NF‐κB signaling pathway, we used siRNAs targeting specific receptors to interfere with the signaling pathway. The knockdown efficiency of each siRNA was demonstrated by transfection and real‐time PCR assays (Figure 9A). The cells were transfected with specific siRNAs followed by infection with TGEV, then incubation with or without ATRA. As shown in Figures 9B, C, siRNAs targeting RIG-I and MDA5 but not TLR3 and TLR7 significantly reduced the phosphorylation level of NF-κB p65 in TGEV-infected cells (P < 0.01). Next, we further determine the effects of siRNAs on TGEV-induced inflammatory response. As shown in Figures 9D–H, knockdown of RIG-I and MDA5 significantly prevented the upregulation of IL-1β, IL-6 and TNF-α mRNA abundance and IL-1β, IL-8 and TNF-α concentrations induced by TGEV (P < 0.05); knockdown of RIG-I also significantly inhibited the enhancing of IL-10 concentration induced by TGEV (P < 0.01); knockdown of MDA5 also significantly inhibited the enhancing of IL-8 mRNA abundance and IL-6 concentration induced by TGEV (P < 0.01); knockdown of TLR3 significantly inhibited the upregulation of IL-6 mRNA abundance induced by TGEV (P < 0.01). However, knockdown of TLR7 had no significant effect on the levels of inflammatory factors in TGEV-infected cells (P > 0.05).