Breast milk, an indispensable source of immunological and nutrient components, is essential for the growth and development of newborn mammals. MicroRNAs (miRNAs) are present in various tissues and body fluids and are selectively packaged inside exosomes, a type of membrane vesicle. Milk exosomes have potential regulatory effects on the growth, development, and immunity of newborn piglets. To explore the differences in milk exosomes related to the breed and milk type, we isolated exosomes from colostrum and mature milk from domestic Bamei pigs and foreign Landrace pigs by using density gradient centrifugation and then characterized them by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, the profiles and functions of miRNAs in the two types of pig milk exosomes were investigated using miRNA-seq and bioinformatics analysis. We identified a total of 1081 known and 2311 novel miRNAs in pig milk exosomes from Bamei and Landrace pigs. These differentially expressed miRNAs (DE-miRNAs) are closely associated with processes such as cell signaling, cell physiology, and immune system development. Functional enrichment analysis showed that DE-miRNA target genes were significantly enriched in endocytosis, the T cell receptor signaling pathway, and the Th17 cell differentiation signaling pathway. The exosomal miRNAs in both the colostrum and mature milk of the two pig species showed significant differences. Based on related signaling pathways, we found that the colostrum of local pig breeds contained more immune-system-development-related miRNAs. This study provides new insights into the possible function of milk exosomal miRNAs in the development of the piglet immune system.

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3′s involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens.

In mammals, exposure to low temperatures induces white adipose tissue (WAT) browning and alters lipid metabolism to promote thermogenesis, thereby maintaining body temperature. However, this response varies across different adipose depots. In this study, Hezuo pigs were exposed to either room temperature (23 ± 2 °C) or low temperature (−15 ± 2 °C) for periods of 12 h, 24 h, 48 h, 5 d, 10 d, and 15 d. Inguinal fat (IF) and perirenal fat (PF) were collected and analyzed using hematoxylin and eosin (HE) staining, transmission electron microscopy, RT-qPCR, and RNA-seq. Following cryoexposure, our results demonstrated a significant increase in adipocyte number and a corresponding decrease in cross-sectional area in both IF and PF groups from 24 h to 10 d. While adipocyte numbers were elevated at 12 h and 15 d, these changes were not statistically significant. Moreover, lipid droplets and mitochondria were more abundant, and the mRNA expression levels of thermogenic genes UCP3 and PGC-1α were significantly higher compared to the control group during the 24 h-10 d cold exposure period. No significant changes were observed in the other groups. RNA-seq data indicated that the lipid metabolism of IF and PF peaked on day 5 of low-temperature treatment. In IF tissue, lipid metabolism is mainly regulated by genes such as FABP4, WNT10B, PCK1, PLIN1, LEPR, and ADIPOQ. These genes are involved in the classical lipid metabolism pathway and provide energy for cold adaptation. In contrast, in PF tissue, genes like ATP5F1A, ATP5PO, SDHB, NDUFS8, SDHA, and COX5A play roles within the neurodegenerative disease pathway, and PF tissue has a positive impact on the process related to degenerative diseases. Further investigation is needed to clarify the functions of these candidate genes in lipid metabolism in Hezuo pigs and to explore the genetic mechanisms underlying the cold-resistance traits in local pig populations.

Inflammatory diseases of the intestinal tract in piglets severely impair the economic performance of pig farms. Pig milk exosomes can encapsulate miRNAs which can then enter the piglet intestine to play an immunomodulatory role. Previously, we comparatively analyzed and identified exosomal miRNAs in the colostrum and mature milk of Bamei and Landrace pigs, and we screened for ssc-miR-22-3p, which is associated with inflammation and immune response; however, the role played by ssc-miR-22-3p in the immune response in IPEC-J2 cells is not yet clear. In this study, we first constructed a pig intestinal inflammatory response model using Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly (I:C)), and we investigated the role of ssc-miR-22-3p targeting MAPK14 in the regulation of LPS and Poly (I:C)-induced inflammatory injury in IPEC-J2 cells by RT-qPCR, cell counting kit-8 (CCK-8), EdU staining, lactate dehydrogenase (LDH) activity assay, and dual luciferase reporter gene assay. We successfully established LPS and Poly (I:C)-induced cell damage models in IPEC-J2 cells. The immune response of IPEC-J2 cells was stimulated by induction of IPEC-J2 cells at 10 μg/mL LPS and 20 μg/mL Poly (I:C) for 24 h. Overexpression of ssc-miR-22-3p decreased cytokine expression and promoted cell viability and proliferation. The functional enrichment analysis revealed that ssc-miR-22-3p targets genes enriched in the pathways of negative regulation of inflammatory response and bacterial invasion of epithelial cells. The validity of the binding site of ssc-miR-22-3p to MAPK14 was tested by a dual luciferase reporter gene. Pig milk exosome ssc-miR-22-3p promotes cell viability and proliferation by targeting MAPK14, and it alleviates LPS and Poly (I:C)-induced inflammatory responses in IPEC-J2 cells.

Long non-coding RNAs (lncRNAs) modified by n6-methyladenosine (m6A) have been implicated in the development and progression of several diseases. However, the mechanism responsible for the role of m6A-modified lncRNAs in Clostridium perfringens type C piglet diarrhea has remained largely unknown. We previously developed an in vitro model of CPB2 toxin-induced piglet diarrhea in IPEC-J2 cells. In addition, we previously performed RNA immunoprecipitation sequencing (MeRIP-seq), which demonstrated lncRNA EN_42575 as one of the most regulated m6A-modified lncRNAs in CPB2 toxin-exposed IPEC-J2 cells. In this study, we used MeRIP-qPCR, FISH, EdU, and RNA pull-down assays to determine the function of lncRNA EN_42575 in CPB2 toxin-exposed IPEC-J2 cells. LncRNA EN_42575 was significantly downregulated at different time points in CPB2 toxin-treated cells. Functionally, lncRNA EN_42575 overexpression reduced cytotoxicity, promoted cell proliferation, and inhibited apoptosis and oxidative damage, whereas the knockdown of lncRNA EN_42575 reversed these results. Furthermore, the dual-luciferase analysis revealed that METTL3 regulated lncRNA EN_42575 expression in an m6A-dependent manner. In conclusion, METTL3-mediated lncRNA EN_42575 exerted a regulatory effect on IPEC-J2 cells exposed to CPB2 toxins. These findings offer novel perspectives to further investigate the function of m6A-modified lncRNAs in piglet diarrhea.

N6-methyladenosine (m6A) modification can accommodate mRNA processing, stability, and translation in mammals, and fat mass and obesity associated protein (FTO) is a vital demethylase in the m6A modification pathway. Clostridium perfringens type C (C. perfringens type C) causes diarrhea in piglets and has a serious impact on the pig industry. However, our understanding of the effect of m6A in the process of C. perfringens type C infectious piglet diarrhea (CPTCIPD) is limited. Here, an in vitro model of CPTCIPD was constructed by treating the intestinal porcine epithelial cell line-J2 (IPEC-J2) with Clostridium perfringens beta2 (CPB2) toxin, and the role of FTO was analyzed using quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry. The results revealed that the overall RNA m6A contents at the tissue and cell levels were significantly up-regulated after C. perfringens infection (p < 0.05). FTO expression was significantly reduced in CPB2-treated IPEC-J2 cells. Functionally, FTO knockdown in the treated cells inhibited their proliferation and promoted apoptosis and the inflammation phenotype, whereas FTO overexpression had the opposite effects. Inhibiting FTO prolonged the half-life and up-regulated the expression of Caspase 3, leading to apoptosis. Therefore, this work explored the regulation of FTO in IPEC-J2 cells after CPB2 treatment and enhanced our understanding of the effect of the m6A modification in CPTCIPD.