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Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to neonatal piglets, particularly due to the limited efficacy of existing vaccines and the scarcity of efficacious therapeutic drugs. Gegen Qinlian Decoction (GQD) has been employed for over two millennia in treating infectious diarrhea. Nonetheless, further scrutiny is required to improve the drug's efficacy and elucidate its underlying mechanisms of action. In this study, a modified GQD (MGQD) was developed and demonstrated its capacity to inhibit the replication of PEDV. Animal trials indicated that MGQD effectively alleviated pathological damage in immune tissues and modulated T-lymphocyte subsets. The integration of network analysis with UHPLC-MS/MS facilitated the identification of active ingredients within MGQD and elucidated the molecular mechanisms underlying its therapeutic effects against PEDV infections. In vitro studies revealed that MGQD significantly impeded PEDV proliferation in IPEC-J2 cells, promoting cellular growth via virucidal activity, inhibition of viral attachment, and disruption of viral biosynthesis. Furthermore, MGQD treatment led to increased expression levels of IFN-α, IFN-ß, and IFN-λ3, while concurrently decreasing the expression of TNF-α, thereby enhancing resistance to PEDV infection in IPEC-J2 cells. In conclusion, our findings suggest that MGQD holds promise as a novel antiviral agent for the treatment of PEDV infections.

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Objective: This study aims to verify the protective effect of the Kelch-like ECH-associated protein1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways by studying the effect of plasmids containing Nrf2-small hairpin RNA (shRNA) interference down-regulation of Nrf2 on zearalenone (ZEA) -induced intestinal porcine epithelial cells (IPEC-J2) oxidative stress. Methods: We constructed an IPEC-J2 model that interferes with Nrf2 expression, set blank (Control), negative control group (Sh-control), positive control group (Sh-Nrf2), and added 10, 20, and 40 µmol/L ZEA experimental group (Sh-Nrf2+ZEA10, Sh-Nrf2+ZEA20, and Sh-Nrf2+ZEA40). Results: The study results showed that, compared with the Sh-Nrf2 group, ZEA significantly increased the apoptosis rate of IPEC-J2 in a time- and dose-dependent manner. Compared with the Sh-Nrf2 group, the activities of T-SOD and GSH-PX and relative expressions of Keap1 at mRNA and protein level in the Sh-Nrf2+ZEA20 and Sh-Nrf2+ZEA40 groups were significantly reduced, the MDA level, and the fluorescence intensity around and within the nucleus of ROS and Nrf2, and the relative expressions of Nrf2, Nqo1, and Ho1 at mRNA and protein level significantly increased. Conclusion: These results further prove that interfering with the expression of Nrf2 in IPEC-J2 cells affected the activation of the Keap1-Nrf2 signaling pathway and reduced the ability of cells to resist ZEA-induced oxidative stress. Therefore, the Keap1-Nrf2 signaling pathway had an important protective effect in ZEA-induced intestinal oxidative stress.

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Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that can damage various organizations and physiques through oxidative stress. Quercetin (Que) is a rich polyphenol flavonoid with good anti-inflammatory and antioxidant effects. However, the protection mechanism of Que against DEHP exposure-induced IPEC-J2 cell injury and the implication of autophagy, apoptosis and immunity are still unclear. In this experiment, we looked into the toxicity regime of DEHP exposure on IPEC-J2 cells and the antagonistic function of Que on DEHP. In the experiment, 135 µM DEHP and/or 80 µM Que were used to treat the IPEC-J2 cells for 24h. Experiments indicated that DEHP exposure can cause increased reactive oxygen species (ROS) levels leading to oxidative stress, decreased CAT, T-AOC and GSH-Px activities, increased MDA and H2O2 accumulation, activated the ASK1/JNK signalling pathway, and further increases in the levels of apoptosis markers Bax, Caspase3, Caspase9, and Cyt-c, while reduced the Bcl-2 expression. DEHP also increased the expression of genes linked to autophagy (ATG5, Beclin1, LC3), while decreasing the expression of P62. Additionally, DEHP exposure led to elevated levels of IL1-ß, IL-6, MCP-1, and TNF expression. When exposed to Que alone, there were no significant changes in cellular oxidative stress level, ASK1/JNK signalling pathway expression level, apoptosis, autophagy and cellular immune function. The combination of DEHP and Que treatment remarkably decreased the proportion of autophagy and apoptosis, and recovered cellular immunity. In summary, Que can attenuate DEHP-induced apoptosis and autophagy in IPEC-J2 cells by regulating the ROS/ASK1/JNK signalling pathway and improving the immune dysfunction of IPEC-J2 cells.

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BACKGROUND: De-oiled rice bran (DORB), a substantial yet underutilized byproduct of rice processing, boasts a rich composition of active ingredients but suffers from limited application. Previous studies have indicated that enzymatic or fermentation treatments enhanced these active components. In this study, lactobacilli and complex enzymes were employed to co-treat DORB, involving the determination of the changes in active components and functionalities of DORB extract (DORBE) before and after this treatment. RESULTS: Following fermentation-enzymolysis, the total phenol and total flavonoid contents in DORBE were significantly increased by 43.59% and 55.10%, reaching 19.66 and 34.34 g kg-1, respectively. Antioxidant tests in vitro demonstrated that the co-treatment enhanced the scavenging activities of DPPH, hydroxyl and ABTS radicals. Porcine intestinal epithelial cell experiments revealed that, compared to DORBE, the fermentation and enzymolysis DORBE (FDORBE) exhibited significantly improved cell viability and catalase activity as well as scavenging capacity for reactive oxygen species and malondialdehyde after induction by H2O2. Furthermore, FDORBE restored the decreased mRNA expression levels of Nrf2, HO-1 and NQO1 in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway stimulated by H2O2. CONCLUSION: Fermentation-enzymolysis co-treatment increases the contents of bioactive components of DORBE and enhances its antioxidant capacity, leading to a better protection against intestinal disorders induced by oxidative stress, suggesting that this co-treatment is a rational and effective strategy to increase the value of grains and promotes the use of DORB as a functional feed in animal production. © 2024 Society of Chemical Industry.

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Dihydroquercetin (DHQ), also known as Taxifolin (TA), is a flavanonol with various biological activities, such as anticancer, anti-inflammatory, and antioxidative properties. It has been found to effectively increase the viability of porcine intestinal epithelial cells (IPEC-J2). However, the precise mechanism by which DHQ increases the proliferation of IPEC-J2 cells is not entirely understood. This study aimed to explore the potential pathways through which DHQ encourages the proliferation of IPEC-J2 cells. The findings indicated that DHQ significantly improved the protein expression of tight junction proteins (ZO-1, Occludin, and Claudin1) and a molecular biomarker of proliferation (PCNA) in IPEC-J2 cells. Furthermore, DHQ was found to increase the Wnt/ß-catenin pathway-associated ß-catenin, c-Myc, and cyclin D1 mRNA expression, and promote the protein expression of ß-catenin and TCF4. To confirm the involvement of the Wnt/ß-catenin signaling pathway in the DHQ-promoted proliferation of IPEC-J2 cells, the inhibitor LF3, which targets ß-catenin/TCF4 interaction, was used. It was found that LF3 inhibited the protein expressions upregulated by DHQ and blocked the promotion of cell proliferation. These results indicate that DHQ positively regulates IPEC-J2 cell proliferation through the Wnt/ß-catenin pathway, providing constructive insights into the role of DHQ in regulating intestine development.

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Antimicrobial peptides (AMPs) function to extensively suppress various problematic factors and are considered a new alternative for improving livestock health and enhancing immunomodulation. In this study, we explored whether AMP regulation has positive influences on Ochratoxin A (OTA) exposure using a porcine intestinal epithelial cell line (IPEC-J2 cells). We constructed a beta-defensin 1 (DEFB1) expression vector and used it to transfection IPEC-J2 cells to construct AMP overexpression cell lines. The results showed that OTA induced cytotoxicity, decreased cell migration, and increased inflammatory markers mRNA in IPEC-J2 cells. In DEFB1 overexpressing cell lines, OTA-induced reduced cell migration and increased inflammatory markers mRNA were alleviated. Additionally, a natural product capable of inducing DEFB1 expression, which was selected through high-throughput screening, showed significant alleviation of cytotoxicity, cell migration, and inflammatory markers compared to OTA-treated IPEC-J2 cells. Our finding provides novel insights and clues for the porcine industry, which is affected by OTA exposure.

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Rice bran oil is a type of rice oil made by leaching or pressing during rice processing and has a high absorption rate after consumption. When oxidative rancidity occurs, it may cause oxidative stress (OS) and affect intestinal function. Meanwhile, the toxic effects of oxidised rice bran oil have been less well studied in pigs. Therefore, the IPEC-J2 cells model was chosen to explore the regulatory mechanisms of oxidised rice bran oil on OS and apoptosis. Oxidised rice bran oil extract treatment (OR) significantly decreased the viability of IPEC-J2 cells. The results showed that OR significantly elevated apoptosis and reactive oxygen species levels and promoted the expression of pro-apoptotic gene Caspase-3 messenger RNA levels. The activation of Nrf2 signalling pathway by OR decreased the cellular antioxidant capacity. This was further evidenced by the expression of kelch-like ECH-associated protein 1, heme oxygenase 1, NADH: quinone oxidoreductase 1, superoxide dismutase 2 and heat shock 70 kDa protein genes and proteins were all downregulated. In conclusion, our results suggested that oxidised rice bran oil induced damage in IPEC-J2 cells through the Nrf2 signalling pathway.

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Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.

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Intestinal infections caused by Escherichia coli and Salmonella enterica pose a huge economic burden on the swine industry that is exacerbated by the development of antimicrobial resistance in these pathogens, thus raising the need for alternative prevention and treatment methods. Our aim was to test the beneficial effects of the flavonoid luteolin in an in vitro model of porcine intestinal infections. We infected the porcine intestinal epithelial cell line IPEC-J2 with E. coli and S. enterica subsp. enterica serovar Typhimurium (106 CFU/mL) with or without previous, concurrent, or subsequent treatment with luteolin (25 or 50 µg/mL), and measured the changes in the reactive oxygen species and interleukin-6 and -8 levels of cells. We also tested the ability of luteolin to inhibit the adhesion of bacteria to the cell layer, and to counteract the barrier integrity damage caused by the pathogens. Luteolin was able to alleviate oxidative stress, inflammation, and barrier integrity damage, but it could not inhibit the adhesion of bacteria to IPEC-J2 cells. Luteolin is a promising candidate to be used in intestinal infections of pigs, however, further studies are needed to confirm its efficacy. The use of luteolin in the future could ultimately lead to a reduced need for antibiotics in pig production.

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Deoxynivalenol (DON) is a mycotoxin produced by Fusarium graminearum, and curcumin (CUR) is a natural polyphenolic compound found in turmeric. However, the combined treatment of CUR and DON to explore the mitigating effect of CUR on DON and their combined mechanism of action is not clear. Therefore, in this study, we established four treatment groups (CON, CUR, DON and CUR + DON) to investigate their mechanism in the porcine intestinal epithelial cells (IPEC-J2). In addition, the cross-talk and alleviating potential of CUR interfering with DON-induced cytotoxic factors were evaluated by in vitro experiments; the results showed that CUR could effectively inhibit DON-exposed activated TNF-α/NF-κB pathway, attenuate DON-induced apoptosis, and alleviate DON-induced endoplasmic reticulum stress and oxidative stress through PERK/CHOP pathways, which were verified at both mRNA and protein levels. In conclusion, these promising findings may contribute to the future use of CUR as a novel feed additive to protect livestock from the harmful effects of DON.

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Introduction: As a symbiotic probiotic for the host, Clostridium butyricum (CB) has the potential to strengthen the body's immune system and improve intestinal health. However, the probiotic mechanism of CB is not completely understood. The Clostridium butyricum CBX 2021 strain isolated by our team from a health pig independently exhibits strong butyric acid production ability and stress resistance. Therefore, this study comprehensively investigated the efficacy of CBX 2021 in pigs and its mechanism of improving pig health. Methods: In this study, we systematically revealed the probiotic effect and potential mechanism of the strain by using various methods such as microbiome, metabolites and transcriptome through animal experiments in vivo and cell experiments in vitro. Results: Our in vivo study showed that CBX 2021 improved growth indicators such as daily weight gain in weaned piglets and also reduced diarrhea rates. Meanwhile, CBX 2021 significantly increased immunoglobulin levels in piglets, reduced contents of inflammatory factors and improved the intestinal barrier. Subsequently, 16S rRNA sequencing showed that CBX 2021 treatment implanted more butyric acid-producing bacteria (such as Faecalibacterium) in piglets and reduced the number of potentially pathogenic bacteria (like Rikenellaceae RC9_gut_group). With significant changes in the microbial community, CBX 2021 improved tryptophan metabolism and several alkaloids synthesis in piglets. Further in vitro experiments showed that CBX 2021 adhesion directly promoted the proliferation of a porcine intestinal epithelial cell line (IPEC-J2). Moreover, transcriptome analysis revealed that bacterial adhesion increased the expression of intracellular G protein-coupled receptors, inhibited the Notch signaling pathway, and led to a decrease in intracellular pro-inflammatory molecules. Discussion: These results suggest that CBX 2021 may accelerate piglet growth by optimizing the intestinal microbiota, improving metabolic function and enhancing intestinal health.

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OBJECTIVE: The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. RESULTS: Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.

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Introduction: Porcine deltacoronavirus (PDCoV), an emerging swine enteropathogenic coronavirus with worldwide distribution, mainly infects newborn piglets with severe diarrhea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. However, the underlying pathogenic mechanisms of PDCoV infection and the effects of PDCoV infection on host transcripts and metabolites remain incompletely understood. Methods: This study investigated a combined transcriptomic and metabolomic analysis of porcine intestinal epithelial cells (IPEC-J2) following PDCoV infection by LC/MS and RNA-seq techniques. A total of 1,401 differentially expressed genes and 254 differentially accumulated metabolites were detected in the comparison group of PDCoV-infected vs. mock-infected. Results and discussion: We found that PDCoV infection regulates gene sets associated with multiple signaling pathways, including the neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, ras signaling pathway and so on. Besides, the metabolomic results showed that biosynthesis of cofactors, nucleotide metabolism, protein digestion and absorption, and biosynthesis of amino acid were involved in PDCoV infection. Moreover, integrated transcriptomics and metabolomics analyses revealed the involvement of ferroptosis in PDCoV infection, and exogenous addition of the ferroptosis activator erastin significantly inhibited PDCoV replication. Overall, these unique transcriptional and metabolic reprogramming features may provide a better understanding of PDCoV-infected IPEC-J2 cells and potential targets for antiviral treatment.

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BACKGROUND: The intestinal epithelium performs essential physiological functions, such as nutrient absorption, and acts as a barrier to prevent the entry of harmful substances. Mycotoxins are prevalent contaminants found in animal feed that exert harmful effects on the health of livestock. Zearalenone (ZEA) is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals. Here, we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium. RESULTS: Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin, which induced Snail1-mediated epithelial-to-mesenchymal transition (EMT). In addition, ZEA induces Snail-mediated EMT through the activation of TGF-ß signaling. The treatment of IPEC-J2 cells with atractylenolide III, which were exposed to ZEA, alleviated EMT. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epithelial cells and ways to mitigate it.

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The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

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BACKGROUND: The epithelia of the intestine perform various functions, playing a crucial role in providing a physical barrier and an innate immune defense against infections. By generating a "three-dimensional" (3D) model of cell co-cultures using the IPEC-J2 cell line and porcine blood monocyte-derived macrophages (MDMs), we are getting closer to mimicking the porcine intestine ex vivo.Methods: The effect of Limosilactobacillus reuteri B1/1 and Limosilactobacillus fermentum CCM 7158 (indicator strain) on the relative gene expression of interleukins (IL-1ß, IL-6, IL-8, IL-18 and IL-10), genes encoding receptors for TLR4 and TLR2, tight junction proteins such as claudin-1 (CLDN1), occludin (OCLN) and important antimicrobial proteins such as lumican (LUM) and olfactomedin-4 (OLMF-4) was monitored in this model. RESULTS: The results obtained from this pilot study point to the immunomodulatory potential of newly isolated L. reuteri B1/1, as it was able to suppress the enhanced pro-inflammatory response to lipopolysaccharide (LPS) challenge in both cell types. L. reuteri B1/1 was even able to up-regulate the mRNA levels of genes encoding antimicrobial proteins LUM and OLFM-4 and to increase tight junction (TJ)-related genes CLDN1 and OCLN, which were significantly down-regulated in LPS-induced IPEC-J2 cells. Conversely, L. fermentum CCM 7158, chosen as an indicator lactic acid bacteria (LAB) strain, increased the mRNA levels of the investigated pro-inflammatory cytokines (IL-18, IL-6, and IL-1ß) in MDMs when LPS was simultaneously applied to basally deposited macrophages. Although L. fermentum CCM 7158 induced the production of pro-inflammatory cytokines, synchronous up-regulation of the anti-inflammatory cytokine IL-10 was detected in both LAB strains used in both cell cultures. CONCLUSIONS: The obtained results suggest that the recently isolated LAB strain L. reuteri B1/1 has the potential to alleviate epithelial disruption caused by LPS and to influence the production of antimicrobial molecules by enterocytes.

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Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysate transport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-ß-cyclodextrin, significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

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Deoxynivalenol (DON) poses a serious global food safety risk due to its high toxicity and contamination rate. It disrupts the intestinal epithelial barrier, allowing exogenous toxins to enter the circulation and resulting in sepsis and systemic toxicity. In this research, 32 male Kunming mice and Porcine Small Intestinal Epithelial (IPEC-J2) cells were treated with DON at 0-4.8 mg/kg (7 d) and 0-12 µM (24 h), respectively. Histopathological results revealed that DON disrupted the intestinal epithelial barrier, causing apoptosis and tight junction (TJ) injury. Immunofluorescence and protein expression results showed that DON-induced p53-dependent mitochondrial pathway apoptosis and fibrillar actin (F-actin)-associated TJ injury and that the RhoA/ROCK pathway were activated in mice jejunal tissue and IPEC-J2 cells. Pretreatment with RhoA or ROCK inhibitors (Rosin or Y-27632) maintained DON-induced apoptosis and F-actin-associated TJ injury in IPEC-J2 cells. Thus, DON induces damage to the intestinal epithelial barrier through the RhoA/ROCK pathway-mediated apoptosis and F-actin-associated TJ disruption.

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BACKGROUND: Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), is derived from docosahexaenoic acid (DHA). It plays a key role in actively resolving inflammatory responses, which further reduces small intestinal damage. However, its regulation of the apoptosis triggered by endoplasmic reticulum (ER) stress in intestinal epithelial cells is still poorly understood. The intestinal porcine epithelial cells (IPEC-J2) were stimulated with tunicamycin to screen an optimal stimulation time and concentration to establish an ER stress model. Meanwhile, RvD1 (0, 1, 10, 20, and 50 nM) cytotoxicity and its impact on cell viability and the effective concentration for reducing ER stress and apoptosis were determined. Finally, the effects of RvD1 on ER stress and associated apoptosis were furtherly explored by flow cytometry analysis, AO/EB staining, RT-qPCR, and western blotting. RESULTS: The ER stress model of IPEC-J2 cells was successfully built by stimulating the cells with 1 µg/mL tunicamycin for 9 h. Certainly, the increased apoptosis and cell viability inhibition also appeared under the ER stress condition. RvD1 had no cytotoxicity, and its concentration of 1 nM significantly decreased cell viability inhibition (p= 0.0154) and the total apoptosis rate of the cells from 14.13 to 10.00% (p= 0.0000). RvD1 at the concentration of 1 nM also significantly reduced the expression of glucose-regulated protein 78 (GRP-78, an ER stress marker gene) (p= 0.0000) and pro-apoptotic gene Caspase-3 (p= 0.0368) and promoted the expression of B cell lymphoma 2 (Bcl-2, an anti-apoptotic gene)(p= 0.0008). CONCLUSIONS: Collectively, the results shed light on the potential of RvD1 for alleviating apoptosis triggered by ER stress, which may indicate an essential role of RvD1 in maintaining intestinal health and homeostasis.

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Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 µg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

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