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/lp/springer-journals/ferroptosis-is-involved-in-deoxynivalenol-induced-intestinal-damage-in-bN8ZEzU2vX
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- Springer Journals
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- Copyright © The Author(s) 2023
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- 2049-1891
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- 10.1186/s40104-023-00841-4
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Abstract
Background Deoxynivalenol (DON) is a widespread issue for feed and food safety, leading to animal and human health risks. The objective of this study was to determine whether ferroptosis is involved in DON-induced intestinal injury in piglets. Three groups of 21-day-old male weanling piglets (n = 7/group) were fed a control diet, or diet add- ing 1.0 or 3.0 mg DON/kg. At week 4, serum and small intestines were collected to assay for biochemistry, histology, redox status and ferroptosis-related genes expression. In addition, the involvement of ferroptosis and the role of FTL gene in DON-induced cell death were further verified in the IPEC-J2 cells. Results Compared to the control, dietary supplementation of DON at 1.0 and 3.0 mg/kg induced different degrees of damage in the duodenum, jejunum and ileum, and increased (P < 0.05) serum lipopolysaccharide concentration by 46.2%–51.4%. Dietary DON supplementation at 1.0 and (or) 3.0 mg/kg increased (P < 0.05) concentrations of malon- dialdehyde (17.4%–86.5%) and protein carbonyl by 33.1%–92.3% in the duodenum, jejunum and ileum. In addition, dietary supplemented with DON upregulated (P < 0.05) ferroptotic gene (DMT1) and anti-ferroptotic genes (FTL and FTH1), while downregulated (P < 0.05) anti-ferroptotic genes (FPN, FSP1 and CISD1) in the duodenum of the porcine. Furthermore, the in vitro study has demonstrated that deferiprone, a potent ferroptotic inhibitor, mitigated (P < 0.05) DON-induced cytotoxicity in porcine small intestinal IPEC-J2 cells. Additionally, deferiprone prevented or alleviated (P < 0.05) the dysregulation of ferroptosis-related genes (ACSL4 and FTL) by DON in IPEC-J2 cells. Moreover, specific siRNA knockdown FTL gene expression compromised the DON-induced cell death in IPEC-J2 cells. Conclusions In conclusion, this study revealed that ferroptosis is involved in DON-induced intestinal damage in porcine, and sheds a new light on the toxicity of DON to piglets. Keywords Deoxynivalenol, Ferroptosis, Intestine, Piglets, Toxicity *Correspondence: Hao Wu whao.1988@mail.hzau.edu.cn Lvhui Sun lvhuisun@mail.hzau.edu.cn State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China Newhope Liuhe Co. Ltd., Beijing 100102, China Department of Animal Biosciences, University of Guelph, Guelph, ON N1G2W1, Canada © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 2 of 10 of 1 piglet/pen. Piglets were allowed free access to water Introduction and a corn-soybean based diet (Control; Additional Deoxynivalenol (DON), is a type B trichothecene largely file 1: Table S1) formulated to meet the NRC nutritional generated by Fusarium graminearum and F. culmorum. requirements (NRC, 2012) [15], or the control diet spiked DON is one of the most widespread mycotoxins con- with 1.0 mg/kg DON or 3.0 mg/kg DON. Based on the taminates in cereal, including wheat, barley, oats, millet Chinese hygiene standard of 1.0 mg/kg for DON in pig and corn and their by-products [1–3]. Climate change compound feed and since it has been reported that diet and associated global warming is increasing crop sus- contaminated with 2.89 mg/kg DON decreased the per- ceptibility to fungal infection, which is further resulting formance and caused intestinal damage in piglets, we set in increased DON contamination of cereals [3]. It was the above doses of DON [16]. The DON was produced by reported that consumption of food and feed contami- the Fusarium graminearum strain W3008 and mixed into nated by DON leads to food refusal, emesis, diarrhea and the pig feed as in our previous study [16]. The concen - impaired intestinal and immune function in humans and trations of DON, aflatoxin B and zearalenone in the feed farm animals [4–6]. It also could be residual in the animal were measured by specific assay kits (COKAS4000W, food as the secondary environmental pollution to endan- COKAQ8000 and COKAS5000W) from the Romer Labs, ger human health [6, 7]. Therefore, in view of the harm - Singapore. The experiment lasted 28 d. At the end of the ful effects of DON, its toxic mechanisms to animals have experiment, all pigs were humanely euthanized by intra- drawn great attention during the past decades [8]. venous injection of sodium pentobarbital (40 mg/kg BW) DON is quickly and efficiently absorbed in the upper to harvest blood and intestine for either serology analy- part of the small intestine, which is the primary tar- sis or histology examination. Five-cm of mid-duodenum, get organ damaged by DON [9]. It is well documented mid-jejunum and mid-ileum were cut and washed with that the intes tinal toxic ity of DON is associated with ice-cold saline, then divided into aliquots and collected in impairment of the intestinal structure, epithelial barrier, Eppendorf tubes that were snap-frozen in liquid nitrogen intestinal mucosal immunity and gut micro biota home- and stored at −80 °C until use. ostasis [2, 10]. These DON-induced intestinal damages have been attributed primary to DON’s ability to binding Histology, serum biochemistry and redox status analysis to the eukaryotic 60S ribosomal subunit, blocking pep- The duodenum, jejunum, ileum tissues were microscopi - tidyl transferase, and inhibiting translation, but concur- cally examined after fixing in 10% neutral-buffered for - rently activating mitogen-activated protein kinase via the malin and processing for paraffin embedding, sectioning “ribotoxic stress response” and inducing inflammation at 5 μm, and then staining with hematoxylin and eosin and apoptosis [11]. Also, generation of DON-induced [17]. The concentrations of lipopolysaccharide (LPS), reactive oxygen species (ROS) can induce oxidative stress malondialdehyde (MDA), protein carbonyl (PC) and and apoptosis, which has been recognized as another reduced glutathione (GSH) and activity of diamine oxi- outcome of DON toxicity [10, 12]. dase (DAO), superoxide dismutase (SOD), total antioxi- Unlike apoptosis, ferroptosis is a newly discovered dant capacity (T-AOC) were measured by a colorimetric form of iron-dependent and ROS-reliant cell death with method with the use of specific assay kits (H255, A003- characteristics of lipid peroxide accumulation, and cyto- 1-2, A087-1-2, A006-1-1, A088-1-1, A001-1-2 and A015- logical change, including reduced or vanished mitochon- 1-2) from the Nanjing Jiancheng Bioengineering Institute drial cristae, and ruptured and condensed mitochondrial of China. Protein concentration was measured by the membrane [13]. However, whether ferroptosis is involved bicinchoninic acid assay (Beyotime Institute of Biotech- in DON-induced intestinal damage remains unclear. nology, Jiangsu, China). Among animal species, pigs are highly susceptible to DON [14]. Thus, in this study, pigs were selected to Porcine IPEC‑J2 cell culture and viability assay determine whether or not DON-induced intestinal injury The porcine IPEC-J2 cell line was cultured following the is associated with the regulation of ferroptosis signaling. method as previously described [16]. Briefly, the cells were grown in DMEM supplemented with 10% fetal Materials and methods bovine serum (FBS), 100 IU penicillin and 100 mg/mL Piglets, treatments, and sample collection streptomycin at 37 °C in a humidified 5% CO atmos- The animal protocol for this study was approved by phere, and medium was changed at 24 h intervals. For the Institutional Animal Care and Use Committee of DON exposure, IPEC-J2 cells were seeded in a 96-well Huazhong Agricultural University, China. In total, 21 plate (1 × 10 cells/well) and incubated for 24 h, then, castrated male crossbred [(Duroc × Landrace) × Large treated with DON (0–1000 μg/L; Additional file 2: Fig. White] weanling piglets (aged 3 weeks) were randomly S1A), or deferiprone (DFP, 10-80 μmol/L; Additional allocated to 3 groups; each group was assigned to 7 pens Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 3 of 10 file 2: Fig. S1B) for 24 h to determine the 30% inhibitory Results concentration (IC ) of DON or the safety dose of DFP Intestinal histology, serum biochemistry, and redox status by measuring cells viability with the CCK-8 kit following As shown in Fig. 1A, compared with the control, die- the manufacturer’s instruction (Beyotime biotechnology, tary supplementation of DON at 1.0 and (or) 3.0 mg/kg China). The determined IC of DON and safety dose of induced degeneration and necrosis of villous epithelium DFP were 500 μg/L and 20 μmol/L, respectively, which cell, and lamina propria edema in duodenum, degen- were used for the further analysis. 1) To verify whether eration and necrosis of villous epithelium cell in the ferroptosis involved in DON-induced cell death in IPEC- jejunum, lymphocyte hyperplasia in ileum. Meanwhile, J2 cells, the cells were treated with normal cell cultural dietary supplementation of DON at 1.0 and 3.0 mg/kg medium (control), control added with 500 μg/L DON increased (P < 0.05) the LPS concentration by 46.2% (DON), or control added with 500 μg/L DON plus 20 and 51.4% in the serum of piglets (Fig. 1B), while DON μmol/L DFP (DON+DFP) for 24 h. Then, calcein acetox - did not affect (P ≥ 0.05) the DAO activity in the serum ymethyl ester (Calcein AM, Abcam, ab141420) were used (Fig. 1C). Compared with the control, dietary supple- for assaying the cells viability. The fluorescence intensity mentation of DON at 1.0 mg/kg increased (P < 0.05) con- was observed and analyzed using fluorescence micro - centrations of MDA by 41.9% and 45.5% in the jejunum scope and software Image J. 2) To verify whether ferritin and ileum, while 3.0 mg/kg DON increased (P < 0.05) light chain (FTL) involved in DON-mediated ferroptosis concentrations of MDA by 49.1%–86.5% in the duode- in IPEC-J2 cells, the cells were treated with negative con- num, jejunum and ileum (Fig. 2A). In addition, dietary trol siRNA (NC), negative control siRNA plus 500 μg/L supplementation of DON at 1.0 increased (P < 0.05) DON (NC+DON), 50 nmol/L FTL siRNA (siRNA), or 50 concentrations of PC by 33.1%–79.8% and 3.0 mg/kg nmol/L FTL siRNA plus 500 μg/L DON (siRNA+DON). DON increased (P < 0.05) concentrations of PC by Briefly, the siRNA (Additional file 3: Table S2) was trans- 43.9%–170.0% (Fig. 2B) in the duodenum, jejunum and fected to the cells with 0.2% Lipofectamin 2000 reagent ileum. Meanwhile, dietary supplementation of DON at (Invitrogen, Shanghai, China) treatment for 48 h incuba- 3.0 mg/kg increased (P < 0.05) SOD activity in the duo- tion, then, 500 μg/L DON was added for 24 h treatment. denum and jejunum by 19.8%–61.5% and dietary sup- Then, the cells viability was measured by CCK-8 kit as plementation of DON at 1.0 mg/kg increased (P < 0.05) described above. The cells were also collected and stored GSH concentration by 32.5% in the ileum (Fig. 2C and at −80 °C until other assays. D). However, dietary supplementation of DON did not affect (P ≥ 0.05) the T-AOC in the intestinal samples (Fig. 2E). Real‑time q‑PCR and western blot analyses Real-time q-PCR analysis was conducted as previ- ously described [5]. Briefly, total mRNA was extracted Expression of ferroptosis‑related genes in duodenum from the jejunum with Trizol (Invitrogen) following the The expressions of 15 ferroptosis-related genes at instructions of the manufacturer. Primers for ferroptosis- mRNA and (or) protein levels in the duodenum are pre- related genes and the house keeping gene β-actin were sented in Fig. 3. Specifically, both dietary supplementa - designed using Primer Express 3.0 (Applied Biosystems) tion of DON at 1.0 mg/kg and 3.0 mg/kg increased (P -ddCt and are presented in Additional file 4: Table S3. The 2 < 0.05) the mRNA levels of divalent metal transporter method was used for the quantification of target genes, 1 (DMT1) and FTL and decreased (P < 0.05) ferropor- and the relative abundance of target genes was normal- tin (FPN), ferroptosis suppressor protein 1 (FSP1) and ized to β-actin. Western blot analyses of the jejunum six-transmembrane epithelial antigen of prostate 3 samples were performed as previously described [18], (STE AP3). Notably, dietary supplementation of DON and the primary antibody used for each gene is presented at 3.0 mg/kg also increased (P < 0.05) the mRNA lev- in Additional file 5: Table S4. The concentration of pro - els of ferritin heavy chain 1 (FTH1) and decreased (P tein was detected by the bicinchoninic acid assay (Beyo- < 0.05) CDGSH iron sulfur domain 1 (CISD1) in the time Institute of Biotechnology, Jiangsu, China). duodenum (Fig. 3A). Furthermore, dietary supple- mentation of DON at 1.0 and 3.0 mg/kg also increased (P < 0.05) DMT1, FTH1 and FTL and decreased (P < Statistical analysis 0.05) FPN and FSP1 at protein levels in the duodenum Statistical analysis was performed with the SPSS (version (Fig. 3B and C). However, the expression of the rest of 13, Chicago, IL, USA). Data were analyzed by a one-way 8 genes was not significantly affected (P ≥ 0.05) by the ANOVA with a significance level of P < 0.05, and the DON supplementation in the duodenum of porcine Tukey-Kramer method was used for multiple mean com- (Fig. 3A–C). parisons. Data are presented as means ± SD. Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 4 of 10 Fig. 1 Eec ff ts of DON on histology of intestine and serum biochemistry in weaned piglets. Histological sections of duodenum, jejunum and ileum (A). The content of LPS (B) and DAO (C) in serum. Values are expressed as means ± SD, n = 7. The sections were stained with hematoxylin and eosin; photomicrographs are shown at 200× magnification. Black arrow indicates degeneration, necrosis and desquamation of villous epithelial cells; Black arrowhead indicates lymphocyte hyperplasia; Red arrow indicates lamina propria edema. Labeled means without a common letter differ, P < 0.05. LPS, lipopolysaccharid, DAO, diamine oxidase; Control, base diet; 1.0 DON, basal diet supplemented with 1.0 mg/kg DON; 3.0 DON, basal diet supplemented with 3.0 mg/kg DON Fig. 2 Eec ff ts of DON on redox status of intestine in weaned piglets. Values are expressed as means ± SD, n = 7. Labeled means without a common letter differ, P < 0.05. MDA, malondialdehyde; PC, protein carbonyl, SOD, superoxide dismutase; GSH, reduced glutathione; T-AOC, total antioxidant capacity; Control, base diet; 1.0 DON, basal diet supplemented with 1.0 mg/kg DON; 3.0 DON, basal diet supplemented with 3.0 mg/kg DON Verification of ferroptosis involvement in DON‑induced cell reduced (P < 0.05) the IPEC-J2 cell viability (Fig. 4A–C) death in IPEC‑J2 cells by 25.3% and 34.5%, as evidenced by the Calcein AM Compared with the control, DON supplementation staining and CCK-8 analysis. Notably, DON-induced Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 5 of 10 Fig. 3 Eec ff ts of DON on the expression of ferroptosis-related genes in duodenum. The relative mRNA abundance of ferroptosis-related genes in duodenum (A). Values are expressed as means ± SD, n = 7. A representative image (B) and the relative density (C) of protein bands of ferroptosis-related proteins in duodenum. Values are expressed as means ± SD, n = 3. Labeled means without a common letter differ, P < 0.05. Control, base diet; 1.0 DON, basal diet supplemented with 1.0 mg/kg DON; 3.0 DON, basal diet supplemented with 3.0 mg/kg DON. ACSL4, acyl-CoA synthetase long chain family member 4; ALOX5, arachidonate 5-lipoxygenase; ALOX12, arachidonate 12-lipoxygenase, 12S type; ALOX15, arachidonate 15-lipoxygenase; CISD1, CDGSH iron sulfur domain 1; DMT1, divalent metal transporter 1; FTL, ferritin light chain; FPN, ferroportin; FTH1, ferritin heavy chain 1; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxidase 4; HSPB1, heat shock protein family B (small) member 1; SLC7A11, solute carrier family 7 member 11; STEAP3, six-transmembrane epithelial antigen of prostate 3; TFR1, transferrin receptor changes were alleviated (P < 0.05) by 15.1% and 20.5% cells by 26.2%, while knockdown of FTL mitigated (P < in the IPEC-J2 cells by the supplementation with DFP 0.05) these changes induced by DON (Fig. 6D). Addition- (Fig. 4A–C). Furthermore, DON supplementation upreg- ally, DON supplementation upregulated (P < 0.05) FTL ulated (P < 0.05) acyl-coenzyme A synthetase long-chain protein production by IPEC-J2 cells compared with the family member 4 (ACSL4), DMT1, FTL and STEAP3 control (Fig. 6E and F). Notably, upregulation of the FTL protein productions compared with the control (Fig. 5A protein production observed in the DON group was pre- and B). Notably, changes of the ACSL4 and FTL protein vented (P < 0.05) in the DON+siRNA group (Fig. 6E and productions observed in the DON group were attenuated F). (P < 0.05) in the DON+DFP group (Fig. 5A and B). Discussion Verification of the role of FTL involvement Dietary supplementation of DON at 1.0 and 3.0 mg/kg in DON‑mediated ferroptosis in IPEC‑J2 cells was shown to induce intestinal damage. Piglets that con- Compared with the control, the IPEC-J2 cells treated sumed DON manifested pathological signs of intestinal with FTL-specific siRNA had downregulated (P < 0.05) injury, including degeneration, necrosis and lymphocyte FTL expression at mRNA level (Fig. 6A). Furthermore, hyperplasia, and (or) lamina propria edema in duode- the FTL-specific siRNA treatment also downregulated num, jejunum and ileum. These outcomes were in agree - (P < 0.05) FTL protein production by IPEC-J2 cells ment with previous studies, which have reported that (Fig. 6B and C). Compared with the control, DON sup- pigs fed diets contaminated with 2.89 and 4.0 mg/kg plementation reduced (P < 0.05) the viability of IPEC-J2 DON exhibited gastrointestinal damage [16, 19, 20]. In Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 6 of 10 Fig. 4 Eec ff ts of DON and DFP on IPEC-J2 cell viability. The cells viability was assayed by calcein acetoxymethyl ester (Calcein AM; A) and the values shows the fluorescence intensity was analyzed by Image J (B). Cell viability was analyzed by CCK-8 (C). Values are expressed as means ± SD, n =6. Labeled means without a common letter differ, P < 0.05. Control, cell culture medium; DON, cell culture medium+500 μg/L DON; DON+DFP, cell culture medium+500 μg/L DON+20 μmol/L DFP Fig. 5 Eec ff ts of DON and DFP on the expression of ferroptosis-related proteins in IPEC-J2. A representative image (A) and the relative density (B) of protein bands of ferroptosis-related proteins in IPEC-J2 cells. Values are expressed as means ± SD, n = 3. Labeled means without a common letter differ, P < 0.05. ACSL4, acyl-coenzyme A synthetase long-chain family member 4; DMT1, metal transporter 1; FTL, ferritin light chain; STEAP3, six-transmembrane epithelial antigen of prostate 3. Control, cell culture medium; DON, cell culture medium+DON; DON+DFP, cell culture medium+ DON+DFP addition, LPS is a pivotal virulence factor and present supplementation than for the control, which would seem in the outer membrane of Gram-negative bacteria [21]; to confirm an impairment of the intestinal integrity and increased gut permeability or damage can be recognized injury. Notably, although the Chinese safety standard sets by the leakage of LPS into the blood [22, 23]. Our results 1.0 mg/kg for DON in pig compound feed [3], the cur- indicate a higher serum LPS concentration for DON rent study showed that 1.0 mg/kg of DON had caused Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 7 of 10 Fig. 6 Verification of the role of FTL in involvement in DON-mediated ferroptosis in IPEC-J2 cells. Relative mRNA (A), a representative image (B) and the relative density (C) of protein bands of FTL in IPEC-J2 cell after FTL siRNA transfection. IPEC-J2 cell viability was analyzed by CCK-8 after treated by FTL siRNA transfection and DON treatment (D). A representative image (E) and the relative density (F) of protein bands of FTL in IPEC-J2 cell after FTL siRNA transfection and DON treatment. Values are expressed as means ± SD, n = 3–6. Labeled means without a common letter differ, P < 0.05. NC, cells treated with negative control siRNA; siRNA, cells treated with FTL siRNA; NC+DON, cell treated with negative control siRNA plus DON; siRNA+DON, cell treated with FTL siRNA plus DON significant damage to the gastrointestinal tract of piglets. as a compensatory mechanism that piglets activated These findings warn us that suitable remediation strategy the antioxidant system as an adaptation to the DON- for DON need to be applied in the feed industry. induced oxidative damage in intestine [27]. This helps Impairment of redox balance is well-documented as piglets to maintain the redox homeostasis under DON- one of the common mechanisms for DON-triggered cell induced damage in intestine [27]. These outcomes are deaths in different organs of animals [24– 26]. Indeed, in agreement with previous reports, which reported the piglets exposed to 1.0 and 3.0 mg/kg DON suf- that 3.0 mg/kg and 10 μmol/L DON impaired the redox fered from intestinal oxidative stress, as indicated by homeostasis in mice and human intestinal cell line increase of the biomarkers of lipid oxidation (MDA) Caco-2 [28–30]. Taken together, these results implicate and protein oxidation (PC) to varying degrees in duo- that DON-induced oxidative stress as the cause of cell denum, jejunum and ileum. Intriguingly, SOD and GSH, death could be one of the major reasons for the intesti- which play pivotal roles in the antioxidant defense, were nal damage [21–33]. partly increased by DON in duodenum, jejunum and An interesting finding from the present study is (or) ileum in the current study. This might be explained that dysregulation of ferroptosis signaling expression Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 8 of 10 appears to be a novel mechanism for the DON-induced Conclusions intestinal injury damage in piglets. Specifically, dietary In summary, the present study found that consumption DON supplementation upregulated of DMT1, FTL of feed contaminated with ≥ 1.0 mg/kg DON caused and FTH1, and downregulated FPN, FSP1 and CISD1 piglet intestinal damage, as evidenced by changes in the at mRNA and (or) protein levels in the duodenum. histopathologic lesions and elevated serum LPS concen- 2+ Because DMT1 is responsible for F e import, which trations, presumably due to leaky gut. Meanwhile, the would result in lipid peroxidation and ferroptosis, and DON-induced intestinal injury was further evidenced 2+ FPN is responsible for the Fe export, which plays by the impairment of redox homeostasis and ferroptosis roles in inhibiting ferroptosis [34], both FSP1 and signaling. Furthermore, DFP, a potent ferroptosis inhibi- CISD1 can protect against mitochondrial lipid per- tor, alleviated DON-induced cell death in IPEC-J2 cells in oxidation, and thus inhibit ferroptosis [35, 36]. The the present study. This result provided further evidence upregulation of DMT1 and downregulation of FPN, that ferroptosis might be involved in the DON-induced FSP1 and CISD1 by DON may induce ferroptosis in cell death. Moreover, specific siRNA knockdown FTL this study. Ferritin, a protein complex represented by protein production compromised the DON-induced FTL and FTH1, plays roles in cytoplasmic iron storage cytotoxicity in IPEC-J2 cells. Overall, these findings and contributes to inhibiting ferroptosis [37]. Strik- helped us better understand the toxicity of DON and ingly, these two proteins were upregulated by DON provided novel target for the development remediation in the present study, which may be interpreted as a strategies to detoxify DON in piglets. complex feedback mechanism working against DON- induced ferroptosis. Abbreviations Furthermore, the in vitro study with IPEC-J2 cells ACSL4 Acyl-CoA synthetase long chain family member 4 confirmed that ferroptosis is involved in the DON- ALOX5 Arachidonate 5-lipoxygenase ALOX12 Arachidonate 12-lipoxygenase, 12S type induced cell death in IPEC-J2 cells [8]. Specifically, ALOX15 Arachidonate 15-lipoxygenase DON decreased the viability of IPEC-J2 cells, while CISD1 CDGSH iron sulfur domain 1 this change was alleviated by the supplementation of DAO Diamine oxidase DMT1 Divalent metal transporter 1 an iron chelator DFP, which is a potent inhibitor of DON Deoxynivalenol ferroptosis. These outcomes revealed that ferropto- FPN Ferroportin sis might involve in the DON-mediated cell death. FSP1 Ferroptosis suppressor protein 1 FTL Ferritin light chain Furthermore, DON upregulated 4 ferroptosis-related FTH1 Ferritin heavy chain 1 genes (DMT1, STE AP3, ACSL4 and FTL) [38–42]. GPX4 Glutathione peroxidase 4 Notably, the DFP treatment prevented or alleviated the GSH Glutathione HSPB1 Heat shock protein family B (small) member 1 changes on IPEC-J2 cell ACSL4 and FTL expression LPS Lipopolysaccharide that was induced by DON. These outcomes further MDA Malondialdehyde demonstrated that ferroptosis may be involved in the PC Protein carbonyl ROS Reactive oxygen species DON-induced cell death in the current study. SLC7A11 Solute carrier family 7 member 11 Because FTL was upregulated by DON in both the SOD Superoxide dismutase duodenum of piglets and IPEC-J2 cells, this study veri- STEAP3 Six -transmembrane epithelial antigen of prostate 3 T-AOC Total antioxidant capacity fied that DON mediates ferroptosis. Consistent with TFR1 Transferrin receptor previous findings, DON reduced the viability of IPEC- J2 cells, while compensatory feedback for the upregu- Supplementary Information lation of anti-ferroptosis protein FTL [43]. Notably, The online version contains supplementary material available at https:// doi. the present study showed that specific siRNA knock- org/ 10. 1186/ s40104- 023- 00841-4. down FTL protein production compromised the DON- induced cytotoxicity in IPEC-J2 cells. It is possible that Additional file 1: Table S1. Ingredients and nutrients composition of the Control diet. FTL knockdown leads to an iron-rich response, which Additional file 2: Fig. S1. Eec ff ts of DON (A) and DFP (B) on cell viability. would lead to decelerated iron uptake and accelerated iron efflux, resulting in the decrease in the intracellu- Additional file 3: Table S2. The sequences of siRNA for the knockdown analysis. lar bioactive iron, and thus mitigating ferroptotic cell Additional file 4: Table S3. List of primers used for q-PCR analysis. death in response to DON [13, 37]. However, the exact Additional file 5: Table S4. List of antibodies used for western blot functions and mechanism of FTL in DON-induced fer- analysis. roptosis need further exploration. Liu et al. Journal of Animal Science and Biotechnology (2023) 14:29 Page 9 of 10 Authors’ contributions 11. Hooft JM, Bureau DP. Deoxynivalenol: Mechanisms of action and its LHS and HW designed the research; ML, LZ, YXM, JHL, JCY and JW conducted effects on various terrestrial and aquatic species. Food Chem Toxicol. the experiments and analyzed the data; LM and LHS wrote the paper; NAK 2021;157:112616. and HW help review and edit the paper; LHS had the primary responsibility for 12. Chen J, Yang S, Li P, Wu A, Nepovimova E, Long M, et al. MicroRNA regu- the final content. All authors read and approved the final manuscript. lates the toxicological mechanism of four mycotoxins in vivo and in vitro. J Anim Sci Biotechnol. 2022;13(1):37. Funding 13. Jiang XJ, Stockwell BR, Conrad M. 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Journal
Journal of Animal Science and Biotechnology – Springer Journals
Published: Mar 16, 2023
Keywords: Deoxynivalenol; Ferroptosis; Intestine; Piglets; Toxicity