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EPA and DHA confer protection against deoxynivalenol-induced endoplasmic reticulum stress and iron imbalance in IPEC-1 cells

Published online by Cambridge University Press:  14 September 2021

Jia Lin
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Feifei Huang
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Tianzeng Liang
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Qin Qin
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Qiao Xu
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Xingfa Huang
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Jing Zhang
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Kan Xiao
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Huiling Zhu
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
Jiangchao Zhao
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China Department of Animal Science, Division of Agriculture, University of Arkansas, Fayetteville, Arkansas 72701, USA
Yulan Liu*
Affiliation:
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
*
*Corresponding author: Yulan Liu, email yulanflower@126.com
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Abstract

This study assessed the molecular mechanism of EPA or DHA protection against intestinal porcine epithelial cell line 1 (IPEC-1) cell damage induced by deoxynivalenol (DON). The cells were divided into six groups, including the CON group, the EPA group, the DHA group, the DON group, the EPA + DON group and the DHA + DON group. RNA sequencing was used to investigate the potential mechanism, and qRT-PCR was employed to verify the expression of selected genes. Changes in ultrastructure were used to estimate pathological changes and endoplasmic reticulum (ER) injury in IPEC-1 cells. Transferrin receptor 1 (TFR1) was tested by ELISA. Fe2+ and malondialdehyde (MDA) contents were estimated by spectrophotometry, and reactive oxygen species (ROS) was assayed by fluorospectrophotometry. RNA sequencing analysis showed that EPA and DHA had a significant effect on the expression of genes involved in ER stress and iron balance during DON-induced cell injury. The results showed that DON increased ER damage, the content of MDA and ROS, the ratio of X-box binding protein 1s (XBP-1s)/X-box binding protein 1u (XBP-1u), the concentration of Fe2+ and the activity of TFR1. However, the results also showed that EPA and DHA decreased the ratio of XBP-1s/XBP-1u to relieve DON-induced ER damage of IPEC-1 cells. Moreover, EPA and DHA (especially DHA) reversed the factors related to iron balance. It can be concluded that EPA and DHA reversed IPEC-1 cell damage induced by DON. DHA has the potential to protect IPEC-1 cells from DON-induced iron imbalance by inhibiting ER stress.

Type
Research Article
Copyright
© The Author(s), 2021. Published by Cambridge University Press on behalf of The Nutrition Society

The intestinal tract is the largest digestive and absorptive organ in the animal body. Under physiological conditions, the intestinal tract forms a barrier, effectively protecting the body from various exogenous toxins. Disruptions of the intestinal barrier increase the probability of pathogen translocation to the intestinal lamina propria and systemic circulation, thereby influencing health(Reference Winer, Luck and Tsai1). Therefore, treatment and prevention of intestinal barrier dysfunction induced by various exogenous toxins are essential.

Fusarium mycotoxins are the largest group of mycotoxins, including deoxynivalenol (DON), nivalenol and T-2 toxin, which are the most dangerous to human and animal health(Reference Sobrova, Adam and Vasatkova2). In EU, DON remains one of the most prevalent mycotoxin (82 %, range 22·2–1037 mug/kg)(Reference Kemboi, Ochieng and Antonissen3). In recent years, increasing evidence suggests that dietary exposure to DON causes gastrointestinal illness, including induction and promotion of inflammatory reactions, disruption of the intestinal barrier and reduced nutrient absorption(Reference Lucke, Bohm and Zebeli4Reference Vignal, Djouina and Pichavant6). Feed contamination with DON has reached alarming magnitudes. It negatively affects the intestinal barrier function and increases intestinal protein availability, promoting the development of intestinal breakdown in broiler chickens(Reference Antonissen, Van Immerseel and Pasmans5,Reference Awad, Ruhnau and Hess7) . Application of DON concentrations at 2 μg/ml in polarised epithelial cells of porcine small intestinal origin (such as intestinal porcine epithelial cell line 1 (IPEC-1) and IPEC-J2) showed significant toxic effects, as indicated by a reduction in cell number, the disintegration of the tight junction protein ZO-1, increase of cell cycle phase G2/M and activation of caspase 3(Reference Kemboi, Ochieng and Antonissen3). The ability to reduce an excessive intestinal inflammatory response is crucial to attenuate the progression of intestinal tract injury. DON exposure induced endoplasmic reticulum (ER) stress, and impaired intestinal barrier function and microbiota(Reference Lin, Sun and Mu8). DON increased the expressions of genes associated with inflammation and apoptosis, such as IL-1β, cyclooxygenase-2, IL-6, TNF-α, caspase-3, caspase-8 and caspase-9, and decreased the cellular anti-oxidative status in IPEC-J2 cells(Reference Kang, Li and Dai9). Therefore, it is very important to find an effective therapeutic agent that protects the intestinal cell from DON-induced damage.

DON is well studied, but the cytoprotective agents that help alleviate DON-induced toxicity are less reported. n-3 PUFA, including EPA and DHA, exerts anti-inflammatory effects by inhibiting TLR4 and NOD signalling pathways(Reference Liu, Chen and Odle10,Reference Kim, Jung and Lee11) . Fish oil rich in n-3 PUFA reduced lipopolysaccharide-induced over-expression of pro-inflammatory genes (such as IL-1 receptor type 1 (IL1R1), IL-1 receptor accessory protein (IL1RAP), CCAAT/enhancer binding protein (C/EBP) beta (CEBPB) and C-reactive protein (CRP)). Additionally, fish oil increased lipopolysaccharide-induced expression of anti-inflammatory genes (such as IL-18 binding protein (IL-18BP), NF-kB inhibitor alpha (NFKBIA), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) and activating transcription factor 3 (ATF3))(Reference Zhang, Xu and Zhu12). EPA and DHA can alleviate DON-induced IL-6-dependent IgA nephropathy in mice(Reference Shi and Pestka13Reference Shi and Pestka15). A previous study showed that fish oil interfered with DON-induced immediate-early gene (including cytokines: IL-1α, IL-1β, and IL-6 and IL-11, chemokines: MCP-1, MCP-3, CINC-1 and MIP-2, etc.) expression and suppressed mitogen-activated protein kinase activation in mice(Reference Kinser, Li and Jia16). Recent research has shown that EPA and DHA may play a beneficial role in preventing intestinal cell injury and barrier function impairment by regulating the necroptosis signalling pathway(Reference Xiao, Liu and Qin17). A previous study showed that EPA and DHA protect goblet cells against ER stress induced by palmitic acid(Reference Escoula, Bellenger and Narce18). However, there is limited evidence on studying the exact protective mechanism of EPA and DHA against DON-induced changes in the intestinal cell.

In the present study, IPEC-1 cells, which are highly sensitive to DON, were used as an intestinal cell model(Reference Diesing, Nossol and Panther19). We aimed to identify novel EPA and DHA modulated gene expression using RNA sequencing during DON-induced damage in IPEC-1 cells.

Materials and methods

Cells and reagents

IPEC-1 cells, originally isolated from both the jejunum and ileum of a neonatal unsuckled piglet, were generously provided by Dr. Guoyao Wu’s Laboratory at Texas A&M University. Dulbecco’s Modified Eagle Medium-F12 was purchased from HyClone. Insulin-transferrin-selenium, penicillin/streptomycin, epidermal growth factor and trypsin/EDTA (0·25 %, 0·9 mM EDTA) were supplied by Gibco. Fetal bovine serum, EPA, DHA, DON and all other cell culture reagents were obtained from Sigma-Aldrich Ltd.

Cell culture

IPEC-1 cells were grown to confluence in 50 cm2 flasks supplemented with Dulbecco’s Modified Eagle Medium-F12 and 5 % fetal bovine serum (v/v), containing 1 % insulin-transferrin-selenium, 1 % penicillin/streptomycin, 5 ng/ml epidermal growth factor in a CO2 incubator with 5 % CO2. All assays were carried out within twenty passages to ensure uniformity of cell population and reproducibility.

Ultrastructure observation

After IPEC-1 cells were cultured to 70–80 % confluency, they were first treated with 12·5 μg/ml EPA or DHA for 24 h and then stimulated with PBS or 0·5 μg/ml DON for 48 h(Reference Xiao, Liu and Qin17). This dose of DON was chosen because 0·5 μg/ml DON can cause reduced cellular activity(Reference Xiao, Liu and Qin17). Previous research chose 6·25, 12·5 and 25 μg/ml as the dosage of EPA and DHA, and research has shown that 12·5 μg/ml EPA and DHA can alleviate the toxicity of 0·5 μg/ml DON(Reference Xiao, Liu and Qin17). Thus, supplementation with EPA and DHA (12·5 μg/ml) may serve as a proper dosage to intervene in DON-induced IPEC-1 toxicity. We also conducted a preliminary experiment that proved that the selected dosage of EPA, DHA and DON was appropriate.

The treatments of IPEC-1 cells are shown in online Supplementary Table S1. There were three biological replicates for each treatment. Transmission electron microscopy (FEI TECNAI) was used to evaluate the ultrastructural changes in IPEC-1 cells. Cell samples from each group were fixed with 2·5 % glutaraldehyde, fixed in 1 % osmium tetroxide, dehydrated in a graded series of ethanol solutions and then immersed in a mixture of acetone and epoxy resin. After cutting ultrathin sections (60–80 nm) and staining with uranyl acetate and lead citrate, images were captured by transmission electron microscopy.

RNA sequencing and data analysis

The total RNA was extracted from each sample as previously described(Reference Liu, Chen and Odle10). Then, according to the standardised process, cDNA library construction and RNA sequencing were performed by BGI Company. The NOISeq method was used to screen differentially expressed genes (DEG) between treatment groups(Reference Tarazona, Garcia-Alcalde and Dopazo20). We screened DEG according to the following default criteria: log2 (fold change) of 1 and diverge probability of 0·8. We used a corrected P value less than or equal to 0·05 to screen DEG.

Quantitative RT-PCR measurement

Total RNA extraction, quantification and cDNA transcription were carried out as previously described(Reference Liu, Chen and Odle10). The specific oligonucleotide primers are listed in online Supplementary Table S2. The primers used for qRT-PCR are previously described(Reference Liu, Chen and Odle10). The relative mRNA expression was analysed using the 2−ΔΔCt method and normalised to the housekeeping gene GAPDH.

Reactive oxygen species and malondialdehyde concentration measurement

Ten percentage cell homogenate was prepared by 0·01 g cell sample and 0·09 ml saline. ROS and malondialdehyde (MDA) were detected in 10 % of cell homogenates using the detection kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s instructions. The levels of ROS were assayed by fluorospectrophotometry, using the SpectraMax Gemini EM (Shanghai Molecular Devices Co., Ltd.). The levels of MDA were assayed by colorimetric method at 532 nm, using the 723PCS visible spectrophotometer (Shanghai Jingke Technology Instrument Co., Ltd.).

Transferrin receptor 1 concentration measurement

The cell culture was centrifuged at 1000 × g for 20 min to separate the supernatant, and the assay was performed according to the instructions of the porcine transferrin receptor 1 (TFR1) kit (ELISA kit, Lunchangshuo Biological Technology Co., LTD). Samples, standards and the horseradish peroxidase-labelled detection antibody were added to the coated microwells pre-coated with porcine TFR1 capture antibody to incubate and washed thoroughly. The colour of the sample was developed with the substrate tumor mutational burden, which was converted into blue under the catalysis of peroxidase, and finally into yellow under the action of acid. The colour intensity was positively correlated with TFR1 concentration in the sample. The absorbance (OD value) of the samples was measured with a microplate reader (Rayto RT-6100) at a wavelength of 450 nm, and the TFR1 concentration of samples was calculated by the instructions of the TFR1 kit.

Fe2+ concentration measurement

The Fe2+ content was detected by the ferrozine colorimetric method. In the acidic medium, Fe3+ was dissociated from the compound, and then reduced to Fe2+ by a reducing agent, and it formed a purple-red compound with ferrozine (such as haemoglobin azide, HiN3). The absorbance (OD value) of the samples was measured with a microplate reader (Rayto RT-6100) at a wavelength of 562 nm, and the Fe2+ concentration = 35·81 × (△Adetermination – △Ablank)÷(△Astandard – △Ablank) according to the instructions of the Fe detection kit (Lunchangshuo Biological Technology Co., LTD).

Statistical analyses

All data were analysed by ANOVA using the general linear model procedure of Statistical Analysis System (SAS Institute). The model included the effects of DON, n-3 PUFA and their interaction terms. Means and standard deviation were calculated as simple statistics and presented graphically using Graphpad Prism 7. When significant n-3 PUFA × DON interaction occurred, multiple comparison test was performed using Tukey multiple comparisons. Differences were considered significant at values of P ≤ 0·05.

Results

EPA and DHA alleviate deoxynivalenol-induced ultrastructural changes in IPEC-1 cells

The ultrastructural changes in EPA, DHA and/or DON treatment groups were investigated and shown in Fig. 1. The ultrastructures of the ER and mitochondria were normal in the CON, EPA and DHA groups. Compared with the CON group, DON challenge induced severe ER swelling and empty vacuoles (marked by red arrow). However, EPA and DHA treatment reduced DON-induced ultrastructural changes in IPEC-1 cells.

Fig. 1. Effects of EPA, DHA and/or DON on ultrastructural damage in IPEC-1 cells. Cells were preincubated with 12·5 μg/ml EPA or DHA for 24 h and then treated with PBS or 0·5 μg/ml DON for 48 h. Endoplasmic reticulum injury was significantly attenuated. Original magnifications 7800×. Scale bars = 500 nm. The red arrow refers to the swollen endoplasmic reticulum.

EPA and DHA alleviate deoxynivalenol-induced significant changes in gene expression in IPEC-1 cells

The volcano plot of DEG in each pairwise is shown in Fig. 2. As expected, there were substantially more DEG following DON challenge in CON, EPA and DHA-treated IPEC-1 cells. Following pairwise comparisons between groups, we detected 3372 DEG (1343 up-regulated and 2029 down-regulated genes) in IPEC-1 cells between the CON group v. DON group, 110 DEG (fifty-five up-regulated and fifty-five down-regulated genes) between the EPA group v. DON + EPA group and 469 DEG (237 up-regulated and 232 down-regulated genes) between the DHA group v. DON + DHA group, respectively (Fig. 3(a) and (b)). Moreover, a total of fifty-six DEG were overlapping in CON v. DON and DON v. DON + EPA (Fig. 3(c)). A total of 203 DEG were overlapping in CON v. DON and DON v. DON + DHA, in which forty-two DEG were also found in DON v. DON + EPA (Fig. 3(c) online Supplementary Table S3). Genes associated with the enriched Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways are listed in Fig. 3(d) and (e). As shown in Fig. 3(d), the specific DEG from CON v. DON, DON v. EPA + DON and DON v. DHA + DON were predominately associated with cellular processes, metabolic processes, single-organism processes and regulation of biological processes. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated the specific DEG from CON v. DON, DON v. EPA + DON and DON v. DHA + DON and was mainly involved in metabolic pathways. These data support that the n-3 PUFA supplement influenced the expression of metabolic pathway-related genes in IPEC-1 cells during DON-induced injury.

Fig. 2. Volcano graph of all expressed genes in pairwise comparison. X-axis and Y-axis present threshold value in log transform. Each dot is a differentially expressed gene (DEG). Dots in red mean significant DEG which passed the screening threshold, and black dots are non-significant DEG. The threshold was defined as: log2(fold change) ≥ 1 and diverge probability ≥ 0·8. The red dots pointing to up-regulated DEG (log2(fold change) ≥ 1, P ≤ 0·05), the blue dots pointing to up-regulated DEG (log2(fold change) ≤ −1, P ≤ 0·05), the grey dots pointing to no-DEG (log2(fold change) < 1, P > 0·05).

Fig. 3. Effects of EPA, DHA and/or DON on gene expression in IPEC-1 cells. (a) Hierarchical clustering of DEG. (b) Statistics of differentially expressed genes from CON v. DON, DON v. EPA + DON, DON v. DHA + DON pairwise. (c) Venn chart of overlapped DEG from CON v. DON, DON v. EPA + DON, DON v. DHA + DON pairwise. (d) Gene Ontology (GO) functional analysis of the specific differentially expressed genes (DEG). (e) KEGG pathway analysis of the specific differentially expressed genes (DEG). , up-regulated; , down-regulated.

EPA and DHA alleviate deoxynivalenol-induced significant changes in endoplasmic reticulum stress

Based on our RNA sequencing data and previous literature, some important ER stress-related genes are listed and summarised in Table 1. To verify the RNA sequencing data, we selected eleven genes for further qRT-PCR (Fig. 4). The ER stress process is strongly associated with the presence of misfolded protein aggregation. For this reason, we conducted the evaluation of the nuclear factor XBP-1 mRNA splicing. The levels of X-box binding protein 1s (XBP-1s) and X-box binding protein 1u (XBP-1u) mRNAs were tested using the semi-quantitative PCR assay, allowing the ratio of XBP-1s to XBP-1u (XBP-1s/u ratio) to be calculated as a measure of XBP-1 splicing. IPEC-1 cell receiving DON had a significantly higher ratio of XBP-1s to XBP-1u (XBP-1s/u ratio) than the CON group (P < 0·001, Fig. 4(a) and (b)). DHA significantly decreased the ratio of XBP1s/XBP1u in IPEC-1 cell compared with the DON group (P < 0·01, Fig. 4(a) and (b)). The TRAF1 mRNA level in the DON group was increased significantly compared with the CON group (P < 0·001, Fig. 4(c)). DHA significantly decreased the expression of TRAF1 mRNA level compared with the DON group (P < 0·01, Fig. 4(c)). DON challenge decreased the mRNA abundance of AP1G2, AP1M1, AP1S2 and AP1S3 in IPEC-1 cells, compared with the CON group (P < 0·001, Fig. 4(d)). DHA significantly increased the expression of GSS, eIF2α, AP1AR, AP1B1, AP1G2, AP1M1, AP1S1, AP1S2 and AP1S3 compared with the DON group (P < 0·01, Fig. 4(d)).

Table 1. Endoplasmic reticulum stress-related genes with evidence for differential regulation after EPA/DHA and/or DON treatment in IPEC-1

The red font refer to significant changes.

Fig. 4. Effects of EPA, DHA and/or DON on ER stress in IPEC-1 cells. (a) The mRNA expression of XBP-1s and XBP-1u. (b) Effects of EPA, DHA and/or DON on the ratio of XBP-1s/XBP-1u. (c) Effects of EPA, DHA and/or DON on the mRNA expression of TRAF1. (d) Effects of EPA, DHA and/or DON on the mRNA expression of ER stress-related factors. Values are mean values and standard deviations, n 5. abcdeMeans without a common letter difference, P < 0·05. , CON; , EPA; , DHA; , DON; , EPA + DON; , DHA + DON.

EPA and DHA alleviate deoxynivalenol-induced significant changes in iron balance regulation

Based on our RNA sequencing data and previous literature, some important Fe balance-related genes are listed and summarised in Table 2. The mRNA level responses of the variables (TFR1, SLC7A11 and PRNP) in the EPA group were higher than the CON group; the mRNA level responses of the variables (TFR1 and PRNP) in the DON group were lower than the CON group, whereas the mRNA level responses of the variables (TFR1, SLC7A11 and PRNP) in the DHA + DON group were higher than the DON group (P < 0·01, Fig. 5(a)).

Table 2. Fe equilibrium-related genes with evidence for differential regulation after EPA/DHA and/or DON treatment in IPEC-1

Fig. 5. Effects of EPA, DHA and/or DON on Fe balance in IPEC-1 cells. (a) The mRNA expression of TFR1, SLC7A11 and PRNP. (b) Effects of EPA, DHA and/or DON on TFR1 activity. (c) Effects of EPA, DHA and/or DON on Fe2+ concentration. (d) Effects of EPA, DHA and/or DON on MDA concentration. (e) Effects of EPA, DHA and/or DON on ROS concentration. Values are mean value and standard devaitions, n 5. abcdeMeans without a common letter difference, P < 0·05. , CON; , EPA; , DHA; , DON; , EPA + DON; , DHA + DON.

EPA and DHA alleviate deoxynivalenol-induced iron imbalance changes in IPEC-1 cells

The protein concentration of TFR1 in DON, EPA + DON and DHA + DON groups was increased significantly compared with the CON group (P < 0·001, Fig. 5(b)). DON challenge increased the protein abundance of TFR1, while TFR1 was decreased significant in the EPA + DON and DHA + DON groups, compared with the DON group (P < 0·001, Fig. 5(b)). The changes of Fe2+ content in IPEC-1 cells are presented in Fig. 5(c). The Fe2+ content was significantly increased in the DON group compared with the CON group, while it was significantly decreased in the EPA + DON and DHA + DON groups compared with the DON group (P < 0·001, Fig. 5(c)). The concentration of MDA and ROS in the DON group was significantly higher compared with the CON group (P < 0·001, Fig. 5(d) and (e)). The changes in MDA and ROS concentration in the EPA + DON and DHA + DON groups were significantly decreased compared with the DON group (P < 0·001, Fig. 5(d) and (e)).

Discussion

Investigators are very concerned about the adverse effects of environmental DON residues on livestock or humans worldwide. As extensively reviewed, pigs are highly susceptible to mycotoxins(Reference Bertero, Moretti and Spicer21). The intestinal epithelium is the first barrier against food contaminants and is highly sensitive to Fusarium toxins, especially DON. A previous study investigated the negative effects of DON on intestinal functions in piglets, including activation of inflammatory responses, damage of epithelial barrier and changes in the intestinal microbiome(Reference Jia, Liu and Zhao22). Several studies have shown that DON can decrease enterocyte viability and compromise immune function, leading to decrease in growth(Reference Chaytor, See and Hansen23Reference Khoshal, Novak and Martin25). The n-3 PUFA can be advantageous by favouring propagation of beneficial micro-organisms, enhancing gut barrier function and supporting intestinal health and immune function in suckling piglets(Reference Chen, Xu and Li26). DON challenge decreased cell viability and cell number and increased LDH activity, which were recovered by EPA and DHA(Reference Xiao, Liu and Qin17). This present study further investigated the molecular mechanism(s) of n-3 PUFA (including EPA and DHA) in IPEC-1 cells under the challenge of DON based on the research results of Xiao et al. (Reference Xiao, Liu and Qin17)

The ER plays an important role in the synthesis, folding and transport of proteins(Reference Barlowe and Miller27) and is very sensitive to changes in the intracellular environment that can regulate cell stress(Reference Kanemoto, Nitani and Murakami28). According to a previous study, DON induced an abundance of very short, dilated cisterns and vesicles in ER of hepatocytes of pigs(Reference Skiepko, Przybylska-Gornowicz and Gajecka29), which was confirmed in our study in DON-treated IPEC-1 cells. Moreover, we found that DON can induce ER swelling, leading to ER injury. Some mycotoxins, such as α-/β-zearalenol and DON, induce ER stress and unfolded protein reactions that can result in apoptosis(Reference Lin, Sun and Mu8,Reference Ben Salem, Boussabbeh and Prola30) . According to the RNA sequencing data, some important ER stress-related genes showed significant alterations after EPA, DHA and/or DON treatment. Thus, thirteen DEG (eIF2α, ATF4, IRE1, TRAF2, JNK, NFκB, XBP1, AP1AR, AP1B1, AP1M1, AP1S1, AP1S2 and AP1S3) revealed the effects of EPA/DHA and/or DON on IPEC-1 ER stress to confirm that EPA/DHA can regulate DON-increased IPEC-1 ER stress. To our knowledge, this study is the first to study the gene expression profiles of IPEC-1 cells in a trial of EPA/DHA supplementation during DON-induced ER injury in a cell model. And the results showed that EPA and DHA modulated DON-induced ER damage, leading to better maintenance of the ER health. XBP-1 is a marker gene of ER stress; the expression of XBP-1u protein is enhanced, acting as a feedback inhibitor for XBP-1s protein. Our result reveals that treatment with DON markedly induced the expression of ER stress mRNA levels (the increases of the ratio of XBP-1s/XBP-1u and the reduction of AP1G2, AP1M1, AP1S2 and AP1S3 gene level). Furthermore, the ER stress-related genes were significantly increased by EPA. However, EPA had a negligible effect on the regulation of down-regulation of ER stress-related genes induced by DON. Meanwhile, DHA supplementation attenuated DON-induced ER stress and the ratio of XBP-1s/XBP-1u. Recent studies have shown that EPA had no significant effect on MeHg-induced cell death, whereas DHA can promote it(Reference Takanezawa, Nakamura and Hamaguchi31). Our results show that only DHA efficiently down-regulated basal XBP-1s/XBP-1u ratio and TRAF1 gene expression; EPA and DHA (especially DHA) could protect IPEC-1 cells from ER stress caused by DON to some extent. Treatment with EPA markedly induced the expression of ER stress-related genes (GSS, eIF2α, AP1AR, AP1B1, AP1G1, AP1M1, AP1S1, AP1S2 and AP1S3) mRNA levels. A recent study revealed that DHA enhanced methylmercury-induced ER stress and cell death, while EPA potentially attenuated these effects in mouse embryonic fibroblasts(Reference Takanezawa, Nakamura and Hamaguchi31). Our results also revealed the differential impact of DHA and EPA on DON-induced cell injury; however, DHA attenuates DON-induced toxicity more so than EPA. An increase in ER stress and Fe balance genes induced by EPA may be the reason that EPA is not as effective as DHA to attenuate DON-induced IPEC-1 toxicity.

We observed some specific DEG (SLC7A11, TFR1, SLC40A1 and PRNP) from CON v. DON were involved in Fe homoeostasis regulation according to the RNA sequencing data. In addition to being closely related to erythropoiesis, Fe plays an essential role in intestinal health. Ferroptosis inducers (artesunate and erastin) can lead to autophagosome formation via the ER stress response(Reference Lee, Kalimuthu and Seok Park32). Ferroptosis is involved in diabetes mellitus and myocardial ischaemia/reperfusion (I/R) injury (IRI) that is related to ER stress, which aggravates cardiomyocyte injury(Reference Li, Li and Leng33). According to previous research, Fe accumulation in non-alcoholic fatty liver disease is mainly due to impaired Fe export from hepatocytes and Kupffer cells which might be the consequence of hepcidin induction by ER stress(Reference Belot, Gourbeyre and Palin34). Suitable oral doses and forms of Fe are mainly absorbed through the small intestine and then distributed to various organs and tissues(Reference Qi, Zhang and Guo35). Excessive Fe dosage usually catalyses the production of active oxygen by the Fenton reaction(Reference Knutson, Walter and Ames36); then, ROS breaks the intestinal tight junction barrier, leading to intestinal dysfunction(Reference Morgan, Tickle and Sokol37). DON can damage intestinal function, as indicated by lower transepithelial electrical resistance and higher permeability of fluorescein isothiocyanate-labelled dextran (FD4) flux as well as decreased proportions of tight junction proteins located in the plasma membrane(Reference Xiao, Liu and Qin17). DON-induced intestinal tight junction barrier damage may be related to Fe imbalance. In the present study, DON induced excess Fe transfer into cells, catalysing the formation of toxic hydroxyl radicals that cause intestinal damage. We also found that EPA markedly induced the expression of Fe balance-related genes (TFR1, SLC7A11 and PRNP) mRNA levels and had a limited effect on IPEC-1 Fe equilibrium regulation gene expression with DON treatment; however, DHA had significant effects during DON-induced Fe equilibrium-related genes disorders. An increase in Fe balance genes induced by EPA may be a reason that DHA was more effective than EPA on attenuating DON-induced IPEC-1 toxicity.

Xiao et al. (Reference Xiao, Liu and Qin17) found that EPA and DHA decreased DON-induced ROS overproduction and cell necrosis, as demonstrated by down-regulating protein expressions of necroptosis-related signals, including tumour necrosis factor receptor (TNFR1), receptor-interacting protein kinase 1 (RIP1), RIP3, phosphorylated mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase family 5 (PGAM5), dynamin-related protein 1 (Drp1) and high mobility group box-1 protein (HMGB1). This study proved that EPA and DHA could suppress DON-induced increased ROS and MDA. Peroxide accumulation is not only the cause of programmed cell necrosis(Reference Ding, Yu and Feng38) but also that of Fe imbalance-induced ferroptosis(Reference Liu, Guo and Yan39).

Newborn piglets suffering from Fe deficiency were susceptible to viral infection, and TFR1 was highly expressed in intestinal villi of that piglets(Reference Zhang, Cao and Yang40). Our study shows that DON increased protein expression of TFR1 and Fe2+ content. EPA and DHA reversed DON-induced TFR1 and Fe2+ protein content disorders. And the expression of Fe balance-related genes decreased by DON and reversed by only DHA, indicating that there might be other mechanisms which regulate Fe homoeostasis by EPA and need our further investigation. Fe can catalyse H2O2 to produce hydroxyl radicals and potent ROS(Reference Moya, Baker and Liu41). In our study, we found that Fe2+ concentration was higher than those of EPA or DHA-treated groups in DON-treated IPEC-1 cells. In other words, the Fe accumulation enhanced MDA induction. It has been reported that TFR1 plays a vital role in hepatic Fe metabolism(Reference Polonifi, Politou and Kalotychou42). DON induced MDA aggregation and Fe deposition. However, EPA and DHA treatment highly reversed all of these above changes. A recent study has shown that DHA significantly lowered both basal and TNF-α-stimulated pro-inflammatory adipokine chemerin production in 3T3-L1 and human adipocytes(Reference Sainz, Fernandez-Galilea and Costa43). EPA did not modify basal chemerin production, while it attenuated the induction of chemerin by TNF-α (Reference Sainz, Fernandez-Galilea and Costa43). According to our data, DHA had a greater regulatory effect on DON-induced Fe imbalance than EPA.

Conclusions

Our results illustrated that EPA and DHA treatment had a specific effect on DON-induced ER stress and Fe metabolic disorders in IPEC-1 cells (Fig. 6), and DHA had a greater therapeutic potential for the treatment of DON-induced IPEC-1 cell damage than that of EPA. The potential mechanism is closely associated with the activation of TFR1, resulting in the promotion of Fe metabolism and the inhibition of ER stress in IPEC-1 cells by regulating the expression of related genes (XBP-1s/XBP-1u, AP1G2, AP1M1, AP1S2 and AP1S3).

Fig. 6. The molecular mechanism of EPA and DHA protecting against DON-induced IPEC-1 cell damage.

Acknowledgements

The authors would like to thank Associate Prof. Milton Talukder of Patuakhali Science, Technology University, Bangladesh and Xiaochen Sun of the University of Queensland, Australia and Samantha Howe of the University of Arkansas, USA for review of this manuscript.

This study has received assistance from the Natural Science Foundation of Hubei Province (2019CFA015 and 2019CFB831), the National Natural Science Foundation of China (No.31772615) and the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (grant number DKXY2020008).

J. L. and K. X. formulated the research question and designed the study; F. H., T. L., Q. Q., Q. X., X. H. and J. Z. analysed the data; J. L. and F. H. wrote the first draft; H. Z., J. Z. and Y. L. interpreted the findings and revised the manuscript; J. L. and Y. L. have primary responsibility for final content. All authors read and approved the final manuscript.

The authors declare that there are no conflicts of interest.

Supplementary material

For supplementary materials referred to in this article, please visit https://doi.org/10.1017/S0007114521003688

Footnotes

These authors contributed equally to this work.

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Figure 0

Fig. 1. Effects of EPA, DHA and/or DON on ultrastructural damage in IPEC-1 cells. Cells were preincubated with 12·5 μg/ml EPA or DHA for 24 h and then treated with PBS or 0·5 μg/ml DON for 48 h. Endoplasmic reticulum injury was significantly attenuated. Original magnifications 7800×. Scale bars = 500 nm. The red arrow refers to the swollen endoplasmic reticulum.

Figure 1

Fig. 2. Volcano graph of all expressed genes in pairwise comparison. X-axis and Y-axis present threshold value in log transform. Each dot is a differentially expressed gene (DEG). Dots in red mean significant DEG which passed the screening threshold, and black dots are non-significant DEG. The threshold was defined as: log2(fold change) ≥ 1 and diverge probability ≥ 0·8. The red dots pointing to up-regulated DEG (log2(fold change) ≥ 1, P ≤ 0·05), the blue dots pointing to up-regulated DEG (log2(fold change) ≤ −1, P ≤ 0·05), the grey dots pointing to no-DEG (log2(fold change) < 1, P > 0·05).

Figure 2

Fig. 3. Effects of EPA, DHA and/or DON on gene expression in IPEC-1 cells. (a) Hierarchical clustering of DEG. (b) Statistics of differentially expressed genes from CON v. DON, DON v. EPA + DON, DON v. DHA + DON pairwise. (c) Venn chart of overlapped DEG from CON v. DON, DON v. EPA + DON, DON v. DHA + DON pairwise. (d) Gene Ontology (GO) functional analysis of the specific differentially expressed genes (DEG). (e) KEGG pathway analysis of the specific differentially expressed genes (DEG). , up-regulated; , down-regulated.

Figure 3

Table 1. Endoplasmic reticulum stress-related genes with evidence for differential regulation after EPA/DHA and/or DON treatment in IPEC-1

Figure 4

Fig. 4. Effects of EPA, DHA and/or DON on ER stress in IPEC-1 cells. (a) The mRNA expression of XBP-1s and XBP-1u. (b) Effects of EPA, DHA and/or DON on the ratio of XBP-1s/XBP-1u. (c) Effects of EPA, DHA and/or DON on the mRNA expression of TRAF1. (d) Effects of EPA, DHA and/or DON on the mRNA expression of ER stress-related factors. Values are mean values and standard deviations, n 5. abcdeMeans without a common letter difference, P < 0·05. , CON; , EPA; , DHA; , DON; , EPA + DON; , DHA + DON.

Figure 5

Table 2. Fe equilibrium-related genes with evidence for differential regulation after EPA/DHA and/or DON treatment in IPEC-1

Figure 6

Fig. 5. Effects of EPA, DHA and/or DON on Fe balance in IPEC-1 cells. (a) The mRNA expression of TFR1, SLC7A11 and PRNP. (b) Effects of EPA, DHA and/or DON on TFR1 activity. (c) Effects of EPA, DHA and/or DON on Fe2+ concentration. (d) Effects of EPA, DHA and/or DON on MDA concentration. (e) Effects of EPA, DHA and/or DON on ROS concentration. Values are mean value and standard devaitions, n 5. abcdeMeans without a common letter difference, P < 0·05. , CON; , EPA; , DHA; , DON; , EPA + DON; , DHA + DON.

Figure 7

Fig. 6. The molecular mechanism of EPA and DHA protecting against DON-induced IPEC-1 cell damage.

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