Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3.

BACKGROUND: Acute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells. RESULTS: We performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways. CONCLUSIONS: We identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

Analysis of the roles of the Notch1 signalling pathway in modulating deoxynivalenol cytotoxicity.

Deoxynivalenol (DON) is a trichothecenes produced by fungi that is widespread and poses a threat to human and animal health. The Notch1 signalling pathway is tightly involved in cell fate determination. The aim of this study was to investigate the role of the Notch1 signalling pathway in DON exposure. Herein, we found that the Notch1 signalling pathway was significantly activated after DON exposure, while Notch1 expression was negatively regulated by DON-induced ROS. Then, the Notch1 signalling pathway was blocked by the γ-secretase inhibitor DAPT in DON exposure. Flow cytometry analysis and antioxidant parameter measurements revealed that DAPT treatment significantly aggravated the oxidative stress induced by DON. The detection of apoptosis showed that DAPT treatment increased the cell apoptotic rate. Further analysis revealed that inhibiting the Notch1 signalling pathway reduced autophagy upon DON exposure. RT-qPCR and Western blot analysis showed that inhibiting the Notch1 signalling pathway aggravated cellular inflammation and activated the MAPK pathway, indicating that the MAPK pathway may be the downstream signalling pathway. Taken together, our research revealed that the Notch1 signalling pathway is essential for protection against DON. Inhibition of Notch1 signalling increases oxidative stress, causes cell apoptosis, reduces autophagy and aggravates cell inflammation after DON exposure. This study investigated the role of the Notch1 signalling pathway in DON exposure and provided a basis for exploring the mechanism of DON.

Correction to: Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

The original article has been published with an incorrect grant number in the Funding section.

Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

Animal melanin has an important role in the formation of animal fur and skin, which is determined by its quantities, character, and distribution. To identify the effect of melanin on the formation of multi-colored Rex rabbits (Black, Chinchilla, Beaver, Protein cyan, Protein yellow, White), the structure of hair follicles and melanin content in multi-colored Rex rabbit skins were observed by Hematoxylin and Eosin (H&E) staining and melanin staining, respectively. The melanin granules were primarily found in the epidermis and hair follicle roots. The melanin content of skin was measured by extracting melanin from skin tissue. The results demonstrated that the melanin content was the highest in the skin of black Rex rabbit. Additionally, we measured the mRNA and protein expression levels of melanin-related key genes (MITF and TYR) in the skin of different hair color by quantitative real-time PCR and Wes assay, respectively. The results revealed that the mRNA expression levels in the skin of black Rex rabbit was highly expressed when as compared with other Rex rabbit skin (P < 0.01), and they were the lowest in the skin of white Rex rabbit. Finally, correlation analysis was conducted between melanin content and the expression levels of mRNA and protein. The results indicated a significant correlation between melanin content and the mRNA expression of MITF (P < 0.05), but it was not correlated with the mRNA expression of TYR (P > 0.05). In summary, melanin deposition has important economic value, and the coat color of fur-bearing animals is partly determined by the melanin-related genes.

Lycopene alleviates Deoxynivalenol-induced toxicity in Porcine intestinal epithelial cells by mediating mitochondrial function.

Deoxynivalenol (DON) is widely found in food and feed, posing a threat to human and animal health. Lycopene (Lyc) is a natural plant extracts with significant antioxidant properties. This study was conducted to investigate the protective effects of Lyc on IPEC-J2 cells upon DON exposure. The detection of cell viability and trypan blue staining showed that Lyc alleviated cell damage and decreased cell apoptotic rate induced by DON. The analysis of reactive oxygen species (ROS) level and antioxidant parameter measurements showed that Lyc significantly down-regulated the content of ROS and restored antioxidant enzyme activity. Furthermore, mitochondrial membrane potential (ΔΨm) detection, mitochondrial DNA copy number (mtDNAcn) assay and adenosine triphosphate (ATP) concentration detection showed Lyc improved mitochondrial function after DON exposure. The results of transcriptome analysis, ROS detection and CCK8 assay suggested that Lyc may activated the oxidative phosphorylation (OXPHOS) to improve mitochondrial function. Conclusively, our results suggested that Lyc alleviated DON-induced oxidative stress by improving mitochondrial function through OXPHOS signaling pathway.

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Mechanisms in Porcine Intestinal Epithelial Cells under Zearalenone Stress.

Zearalenone (ZEA) is a mycotoxin that frequently occurs in agricultural crops and related products and seriously threatens both animal feed and human food safety. To identify key metabolites and regulators involved in ZEA toxicological processes, we performed metabolomic and transcriptomic analyses of porcine IPEC-J2 intestinal epithelial cells upon ZEA exposure using liquid chromatography-mass spectrometry (LC-MS)/MS and RNA-seq techniques. A total of 325 differential metabolites and 5646 differentially expressed genes were detected. Integrated analyses of metabolomic and transcriptomic data indicated that metabolic processes including lipid metabolism, amino acid metabolism, and carbohydrate metabolism were most affected. Exogenous addition of the key metabolite l-arginine significantly facilitated ZEA metabolism and ameliorated ZEA-induced reactive oxygen species levels and cell apoptosis. Furthermore, l -arginine contributed to the expression of phase II detoxification genes (SULT2B1, GSTA1, GSTM3, and GPX4). l-Arginine addition also increased the protein levels of LC3-II and Beclin 1, and downregulated p62/SQSTM1 levels, indicating its regulatory roles in autophagic flux activation upon ZEA exposure. This study provided global insights into metabolic and transcriptional changes as well as key metabolites and regulators underlying the cellular response to ZEA exposure, and paved the way for the identification of metabolic and molecular targets for biomonitoring and controlling contamination by ZEA.

Analysis of the Roles of the ISLR2 Gene in Regulating the Toxicity of Zearalenone Exposure in Porcine Intestinal Epithelial Cells.

Zearalenone (ZEN) is one of the mycotoxins that pose high risks for human and animal health, as well as food safety. However, the regulators involved in ZEN cellular toxicity remain largely unknown. Herein, we showed that cell viability of porcine intestinal epithelial cells (IPEC-J2) tended to decrease with increasing doses of ZEN by the cell counting kit-8 assay. Expression of the ISLR2 (immunoglobulin superfamily containing leucine-rich repeat 2) gene in IPEC-J2 cells was significantly downregulated upon ZEN exposure. Furthermore, we found the dose-effect of ZEN on ISLR2 expression. We then overexpressed the ISLR2 gene and observed that overexpression of ISLR2 obviously reduced the effects of ZEN on cell viability, apoptosis rate and oxidative stress level. In addition, ISLR2 overexpression significantly decreased the expression of TNF-α and IFN-α induced by ZEN. Our findings revealed the effects of ZEN on the ISLR2 gene expression and indicated the ISLR2 gene as a novel regulator of ZEN-induced cytotoxicity, which provides potential molecular targets against ZEN toxicity.

Genome-wide transcriptional profiling and functional analysis reveal miR-330-MAPK15 axis involving in cellular responses to deoxynivalenol exposure.

Deoxynivalenol (DON) is one of the mycotoxins that is toxic to agricultural environment, which poses high risks to human and farm animal health. Noncoding RNAs have been shown to be crucial regulators of toxicological processes and as promising biomarkers for toxicity monitoring and prevention of mycotoxin contamination. Herein, we characterized genome-wide transcriptional profiling of porcine intestinal epithelial cells upon DON exposure and illustrated a subset of miRNAs and lncRNAs involved in the cellular processes by targeting genes associated with stress responses. A total of 110 differential expression miRNAs and 143 differential expression lncRNAs were identified between the DON exposure and control cell samples. Interactive network analysis showed that miR-330 was one hub noncoding RNA, expression of which was significantly increased upon DON exposure. Functional enrichment analysis indicated that the genes involved in the networks were mainly enriched in the terms of plasma membrane bounded cell projection assembly, mRNA processing, and regulation of mitochondrion organization. Further functional analysis revealed that high expression of miR-330 inhibits the reactive oxygen species production, cell apoptosis, and autophagic flux in cells upon DON exposure. Luciferase assay further indicated that miR-330 could directly target MAPK15. Knockdown of MAPK15 resulted in decreased reactive oxygen species level and cell apoptosis induced by DON, indicating the existence of miR-330-MAPK15 regulatory axis in regulating DON toxicity. Our work shed novel insights into the mode of action of DON at cellular level and indicated the potential of miR-330 as a biomarker for toxicity monitoring of DON contamination, which contributes to the development of effective biomonitoring and prevention strategies to reduce the toxicological effects of DON.

Effect of Promoter Methylation on the Expression of Porcine MUC2 Gene and Resistance to PEDV Infection.

Integrity of the intestinal mucosal barrier is closely related to the occurrence of diarrhea. As an important component protein of the intestinal mucosal barrier, Mucin 2 (MUC2) plays a critical role in preventing the invasion of pathogens, toxins, and foreign bodies. In the present study, we preliminary verified the function of the porcine MUC2 gene in resisting porcine epidemic diarrhea virus (PEDV) infection and investigated the effect of DNA methylation in the promoter region on MUC2 gene expression. The results showed that after PEDV infection, the intestinal mucosal barrier was damaged. Moreover, MUC2 expression was significantly higher in PEDV-infected piglets than in healthy piglets (P < 0.01). The mRNA expression of MUC2 was significantly higher in PEDV-infected IPEC-J2 cells than in non-infected IPEC-J2 cells (P < 0.05). Methylation of the mC-5 site in the MUC2 promoter inhibited the binding of Yin Yang 1 (YY1) to the promoter, down regulated the expression of MUC2 and increased the susceptibility of piglets to PEDV. In conclusion, this study suggests that MUC2 plays an essential regulatory role in PEDV infection. High MUC2 expression improves the resistance of pigs to PEDV infection. The binding of YY1 to the MUC2 promoter is hindered by the methylation of the mC-5 site, which downregulates MUC2 expression and ultimately affects the resistance of pigs to PEDV infection.

Systematic Analysis of Non-coding RNAs Involved in the Angora Rabbit (Oryctolagus cuniculus) Hair Follicle Cycle by RNA Sequencing.

The hair follicle (HF) cycle is a complicated and dynamic process in mammals, associated with various signaling pathways and gene expression patterns. Non-coding RNAs (ncRNAs) are RNA molecules that are not translated into proteins but are involved in the regulation of various cellular and biological processes. This study explored the relationship between ncRNAs and the HF cycle by developing a synchronization model in Angora rabbits. Transcriptome analysis was performed to investigate ncRNAs and mRNAs associated with the various stages of the HF cycle. One hundred and eleven long non-coding RNAs (lncRNAs), 247 circular RNAs (circRNAs), 97 microRNAs (miRNAs), and 1,168 mRNAs were differentially expressed during the three HF growth stages. Quantitative real-time PCR was used to validate the ncRNA transcriptome analysis results. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses provided information on the possible roles of ncRNAs and mRNAs during the HF cycle. In addition, lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks were constructed to investigate the underlying relationships between ncRNAs and mRNAs. LNC_002919 and novel_circ_0026326 were found to act as ceRNAs and participated in the regulation of the HF cycle as miR-320-3p sponges. This research comprehensively identified candidate regulatory ncRNAs during the HF cycle by transcriptome analysis, highlighting the possible association between ncRNAs and the regulation of hair growth. This study provides a basis for systematic further research and new insights on the regulation of the HF cycle.