Overexpression of SIRT1 alleviates oxidative damage and barrier dysfunction in CPB2 toxin-infected IPEC-J2 cells.

Clostridium perfringens (C. perfringens) beta2 (CPB2) toxin may induce necrotizing enteritis (NE) in pigs. Sirtuin1 (SIRT1) is involved in inflammatory intestinal diseases and affects intestinal barrier function. However, the effects of SIRT1 on piglet intestinal disease caused by CPB2 toxin are unclear. This study revealed the role of pig SIRT1 in CPB2 toxin-exposed intestinal porcine epithelial cells (IPEC-J2). Herein, we manifested that SIRT1 was dramatically decreased in IPEC-J2 cells infected with CPB2 toxin. Subsequently, we silenced and overexpressed SIRT1 using siRNA and a overexpression vector in CPB2 toxin-treated IPEC-J2 cells. The results indicated that overexpression of SIRT1 suppressed reactive oxygen species (ROS) generates, the expression tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and Bax, nuclear factor-kappa B (NF-κB p65), phospho (p)-NF-kB p65 and lactate dehydrogenase (LDH) activity and apoptosis in CPB2 toxin-treated IPEC-J2 cells, and increased IL-10, mitochondrial membrane potential (ΔΨm), Bcl-2, Claudin1 and Occludin levels and cell viability. These results indicated that SIRT1 protects IPEC-J2 cells against CPB2 toxin-induced oxidative damage and tight junction (TJ) disruption, which provides a theoretical basis for further study of the molecular regulatory mechanism of SIRT1 in C. perfringens-infected NE in piglets.

Identification of a Novel lncRNA LNC_001186 and Its Effects on CPB2 Toxin-Induced Apoptosis of IPEC-J2 Cells.

The Clostridium perfringens (C. perfringen) beta2 (CPB2) toxin produced by C. perfringens type C (CpC) can cause necrotizing enteritis in piglets. Immune system activation in response to inflammation and pathogen infection is aided by long non-coding RNAs (lncRNAs). In our previous work, we revealed the differential expression of the novel lncRNA LNC_001186 in CpC-infected ileum versus healthy piglets. This implied that LNC_001186 may be a regulatory factor essential for CpC infection in piglets. Herein, we analyzed the coding ability, chromosomal location and subcellular localization of LNC_001186 and explored its regulatory role in CPB2 toxin-induced apoptosis of porcine small intestinal epithelial (IPEC-J2) cells. RT-qPCR results indicated that LNC_001186 expression was highly enriched in the intestines of healthy piglets and significantly increased in CpC-infected piglets' ileum tissue and CPB2 toxin-treated IPEC-J2 cells. The total sequence length of LNC_001186 was 1323 bp through RACE assay. CPC and CPAT, two online databases, both confirmed that LNC_001186 had a low coding ability. It was present on pig chromosome 3. Cytoplasmic and nuclear RNA isolation and RNA-FISH assays showed that LNC_001186 was present in the nucleus and cytoplasm of IPEC-J2 cells. Furthermore, six target genes of LNC_001186 were predicted using cis and trans approaches. Meanwhile, we constructed ceRNA regulatory networks with LNC_001186 as the center. Finally, LNC_001186 overexpression inhibited IPEC-J2 cells' apoptosis caused by CPB2 toxin and promoted cell viability. In summary, we determined the role of LNC_001186 in IPEC-J2 cells' apoptosis caused by CPB2 toxin, which assisted us in exploring the molecular mechanism of LNC_001186 in CpC-induced diarrhea in piglets.

lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis.

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.

S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells.

BACKGROUND: As an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques. RESULTS: We identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited. CONCLUSION: In conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

Decreased S100A9 expression alleviates Clostridium perfringens beta2 toxin-induced inflammatory injury in IPEC-J2 cells.

Background: S100 calcium-binding protein A9 (S100A9) is a commonly known pro-inflammatory factor involved in various inflammatory responses. Clostridium perfringens (C. perfringens ) type C is known to cause diarrhea in piglets. However, the role of S100A9 in C. perfringens type C-induced infectious diarrhea is unclear. Methods: Here, the S100A9 gene was overexpressed and knocked down in the IPEC-J2 cells, which were treated with C. perfringens beta2 (CPB2) toxin. The role of S100A9 in CPB2 toxin-induced injury in IPEC-J2 cells was assessed by measuring the levels of inflammatory cytokines, reactive oxygen species (ROS), lactate dehydrogenase (LDH), cell proliferation, and tight junction-related proteins. Results: The results showed elevated expression of S100A9 in diarrhea-affected piglet tissues, and the elevation of S100A9 expression after CPB2 toxin treatment of IPEC-J2 was time-dependent. In CPB2 toxin-induced IPEC-J2 cells, overexpression of S100A9 had the following effects: the relative expression of inflammatory factors IL-6, IL8, TNF-α, and IL-1ß was increased; the ROS levels and LDH viability were significantly increased; cell viability and proliferation were inhibited; the G0/G1 phase cell ratio was significantly increased. Furthermore, overexpression of S100A9 reduced the expression of tight junction proteins in CPB2-induced IPEC-J2 cells. The knockdown of S100A9 had an inverse effect. In conclusion, our results confirmed that S100A9 exacerbated inflammatory injury in CPB2 toxin-induced IPEC-J2 cells, inhibited cell viability and cell proliferation, and disrupted the tight junctions between cells. Thus, decreased S100A9 expression alleviates CPB2 toxin-induced inflammatory injury in IPEC-J2 cells.

METTL3 Regulates the Inflammatory Response in CPB2 Toxin-Exposed IPEC-J2 Cells through the TLR2/NF-κB Signaling Pathway.

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.

miR-30d Inhibition Protects IPEC-J2 Cells Against Clostridium perfringens Beta2 Toxin-Induced Inflammatory Injury.

Clostridium perfringens beta2 (CPB2) toxin, one of the virulence factors of Clostridium perfringens (C. perfringens), can cause necrotizing enterocolitis in piglets. Accumulating pieces of evidence indicate that microRNAs (miRNAs) refer to the regulation of inflammatory processes. Previously, we have discovered that miR-30d was differentially expressed between the ileum of normal piglets and C. perfringens type C-infected diarrheal piglets. Here, we found that miR-30d expression was lowered in CPB2 toxin-treated intestinal porcine epithelial cells (IPEC-J2) at different time points. Subsequently, we determined that miR-30d inhibitor attenuated CPB2 toxin revulsive inflammatory damage in IPEC-J2 cells and promoted cell proliferation and cell cycle progression, whereas miR-30d mimic had opposite results. In addition, we confirmed that Proteasome activator subunit 3 (PSME3) was a downstream target gene of miR-30d via a dual luciferase reporter assay, qPCR, and western blot. We also found that overexpression of PSME3 suppressed CPB2 toxin-induced inflammatory damage and promoted cell proliferation and cycle progression. Our results demonstrate that miR-30d aggravates CPB2 toxin revulsive IPEC-J2 cells inflammatory injury via targeting PSME3, thereby providing a novel perspective for the prevention and treatment of piglet diarrhea at the molecular level.

FTO Regulates Apoptosis in CPB2-Treated IPEC-J2 Cells by Targeting Caspase 3 Apoptotic Protein.

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.

ssc-microRNA-132 targets DACH1 to exert anti-inflammatory and anti-apoptotic effects in Clostridium perfringens beta2 toxin-treated porcine intestinal epithelial cells.

Clostridium perfringens (C. perfringens) type C (CPC) is one of the chief pathogens that causes diarrhea in piglets, and C. perfringens beta2 (CPB2) toxin is the main virulence factor of CPC. Our previous research demonstrated that ssc-microR-132 was differentially expressed in ileal tissues of CPC-mediated diarrheic piglets and healthy piglets, which implied a potential role of ssc-microR-132 in this process. Here, we found that ssc-microR-132 was notably down-regulated in CPB2-exposed intestinal porcine epithelial cells (IPEC-J2), which was consistent with the ileal tissue expression. Moreover, ssc-microR-132 upregulation alleviated CPB2-induced inflammatory damage and apoptosis in IPEC-J2, whereas ssc-microR-132 knockdown presented the opposite effects. Furthermore, the dual-luciferase reporter assay indicated that ssc-microR-132 directly targeted Dachshund homolog 1 (DACH1). Moreover, DACH1 overexpression intensified CPB2-induced inflammatory injury and apoptosis in IPEC-J2. Remarkably, the introduction of DACH1 weakened the anti-inflammatory and anti-apoptotic effects of ssc-microR-132 in CPB2-exposed IPEC-J2. Overall, the results reveal that ssc-microR-132 targeted DACH1 to alleviate CPB2-mediated inflammation and apoptosis in IPEC-J2.

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin.

Background: The n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear. Methods: Here, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells. Results: We identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells. Conclusion: In summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Effects of miR-204 on apoptosis and inflammatory response of Clostridium perfringens beta2 toxin induced IPEC-J2 cells via targeting BCL2L2.

Clostridium perfringens beta2 (CPB2) toxin can cause intestinal damage and inflammatory responses in a variety of animals, which seriously endanger the healthy development of animal husbandry. Increasing evidence has demonstrated that microRNAs (miRNAs) can play an important regulatory role in the process of pathogenic infection. In our previous study, we found that miR-204 was highly expressed in the ileum tissues of the susceptible group diarrhea piglets after infection with Clostridium perfringens (C. perfringens) type C. In this study, we found that miR-204 was also up-regulated in different time points after CPB2 toxin treatment. Overexpression of miR-204 promoted apoptosis and inflammatory response of intestinal porcine epithelial cells (IPEC-J2), whereas the opposite results were displayed after transfected with miR-204 inhibitor. Furthermore, the luciferase reporter assays confirmed that BCL2L2 was a direct target gene of miR-204. Interestingly, we found that overexpression BCL2L2 attenuated the apoptosis and inflammatory response of CPB2 toxin induced IPEC-J2 cells. In conclusion, these results find that miR-204 promotes the apoptosis and intensify inflammatory response of CPB2 toxin induced IPEC-J2 cells via targeting BCL2L2. These data provide a valuable reference for the piglets resistance diarrhea at the molecular level.

Epigenetic upregulation of ssc-miR-124a following treatment with Clostridium perfringens beta2-toxin attenuates both apoptosis and inflammation in intestinal porcine epithelial cells.

Clostridium perfringens (C. perfringens) is a globally recognized zoonotic pathogen. It has been reported that the beta2-toxin produced by C. perfringens can cause a variety of gastrointestinal diseases and even systemic inflammation. MicroRNA-124a (miR-124a) has been reported to play important roles in the host response to pathogenic infection. Although C. perfringens beta2-toxin induced injury in intestinal porcine epithelial (IPEC-J2) cells has been established, the underlying molecular mechanism is not completely unraveled. Here we show that a significant upregulation of ssc-miR-124a in IPEC-J2 cells after beta2-toxin stimulation was associated with the MiR-124A-1 and MiR-124A-2 gene promoter demethylation status. Importantly, overexpression of ssc-miR-124a significantly increased cell proliferation and decreased apoptosis and cytotoxicity in beta2-toxin treated IPEC-J2 cells. Transfection of IPEC-J2 cells with ssc-miR-124a mimic suppressed beta2-toxin induced inflammation. On the contrary, ssc-miR-124a inhibitor promoted aggravation of cell apoptosis and excessive damage. Furthermore, rho-associated coiled-coil-containing protein kinase 1 (ROCK1) was identified as the direct target gene of ssc-miR-124a in IPEC-J2 cells and its siRNA transfection reversed the promotion of apoptosis and aggravation of cellular damage induced by ssc-miR-124a inhibitor. Overall, we speculated that the miR-124A-1/2 gene was epigenetically regulated in IPEC-J2 cells after beta2-toxin treatment. Upregulation of ssc-miR-124a may restrain ROCK1, and attenuate apoptosis and inflammation induced by beta2-toxin that prevent IPEC-J2 cells from severe damages. We discover a new molecular mechanism by which IPEC-J2 cells counteract beta2-toxin-induced damage through the ssc-miR-124a/ROCK1 axis partially.

ssc-miR-185 targets cell division cycle 42 and promotes the proliferation of intestinal porcine epithelial cell.

OBJECTIVE: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. METHODS: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC423' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyltetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. RESULTS: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. CONCLUSION: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.

MicroRNA-21-5p targets PDCD4 to modulate apoptosis and inflammatory response to Clostridium perfringens beta2 toxin infection in IPEC-J2 cells.

Clostridium perfringens (C. perfringens), a toxin-producing enteric pathogen, causes a variety of intestinal infections in humans and animals. C. perfringens beta2 (CPB2) toxin has been considered to be a strong virulence factor for C. perfringens infectious enteric diseases (CPED). Altered levels and functions of microRNA-21-5p (miR-21-5p) have been associated with apoptosis and inflammation response in pathological processes. However, little is known about its functional mechanism in CPED. Here, we found that miR-21-5p expressed in multiple tissues of pig, had a highest level in jejunum, and significantly upregulated in intestinal porcine epithelial cells (IPEC-J2) exposed to CPB2 toxin. Noteworthily, transfection of CPB2-treated IPEC-J2 cells with miR-21-5p mimic increased cell viability and Bcl2 expression, as well as reduced cytotoxicity, apoptosis rates and Bax level. Moreover, overexpression of miR-21-5p significantly suppressed the levels of interleukin (IL)-6, IL-8, TNF-α, IL-1ß and nuclear factor-kappa B (NF-κB p65) activity induced by CPB2 toxin, whereas that of the IL-10 was increased in IPEC-J2 cells. On the contrary, transfection of miR-21-5p inhibitor promoted CPB2-induced cell apoptosis and inflammation. Furthermore, we validated that programmed cell death 4 (PDCD4) was strikingly downregulated in CPB2-treated IPEC-J2 cells. PDCD4 exhibited opposing effects to those of miR-21-5p mimic on IPEC-J2 cells, and restoration of PDCD4 expression counteracted the suppressive effect of miR-21-5p on CPB2-induced apoptosis and inflammatory response. Collectively, our findings demonstrated that miR-21-5p was involved in regulating the immune response triggered by CPB2 toxin and contributed to protective effects in CPB2-induced CPED cell model by targeting PDCD4.

Inhibition of ssc-microRNA-140-5p ameliorates the Clostridium perfringens beta2 toxin-induced inflammatory response in IPEC-J2 cells via the ERK1/2 and JNK pathways by targeting VEGFA.

Piglet diarrhea and even death due to Clostridium perfringens (C. perfringens) type C infection have led to huge economic losses in the pig industry worldwide. C. perfringens beta2 (CPB2) toxin is the main virulence factor for this pathogen. MiR-140-5p can exacerbate toxin-induced toxicity of toxin to cells by promoting oxidative stress. However, the role of pig miR-140-5p (ssc-miR-140-5p) in piglet diarrhea caused by C. perfringens type C has not been studied. Here, we study investigated the function of ssc-miR-140-5p by generating an in vitro CPB2-induced injury model in intestinal porcine epithelial (IPEC-J2) cells. Our results revealed that transfection with an ssc-miR-140-5p inhibitor significantly increased the viability of CPB2-induced IPEC-J2 cells, decrease the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS), and inhibit inflammatory responses and apoptosis. In addition, vascular endothelial growth factor A (VEGFA) was identified as a direct target of ssc-miR-140-5p by luciferase reporter assay. Western blot analysis showed that inhibition of ssc-miR-140-5p could activate the ERK1/2 signaling pathway and inhibit the JNK signaling pathway. In summary, we showed that down-regulation of ssc-miR-140-5p ameliorated CPB2-induced inflammatory responses in IPEC-J2 cells via the ERK1/2 and JNK signaling pathways by targeting VEGFA.

Identification and Characterization of MAPK Signaling Pathway Genes and Associated lncRNAs in the Ileum of Piglets Infected by Clostridium perfringens Type C.

Clostridium perfringens type C (C. perfringens type C) is one of the main microbial pathogens responsible for piglet diarrhea worldwide, causing substantial economic losses for pig-rearing industries. The mitogen-activated protein kinase (MAPK) signaling pathway is a key regulator of inflammatory bowel disease, especially necrotic enteritis. However, whether and how the MAPK signaling pathway is involved in regulating the process of piglet diarrhea when challenged by C. perfringens type C are still unknown. Here, we screened 38 differentially expressed genes (DEGs) in piglets' ileum tissues experimentally infected with C. perfringens type C that were enriched in the Sus scrofa MAPK signaling pathway, based on our previous transcriptome data. Of these DEGs, 12 genes (TRAF2, MAPK8, and GADD45G, among others) were upregulated whereas 26 genes (MAPK1, TP53, and CHUK, among others) were downregulated in the infected group. Our results showed that MAPK1, TP53, MAPK8, MYC, and CHUK were in the core nodes of the PPI network. Additionally, we obtained 35 lncRNAs from the sequencing data, which could be trans-targeted to MAPK signaling pathway genes and were differentially expressed in the ileum tissues infected with C. perfringens. We used qRT-PCR to verify the expression levels of genes and lncRNAs related to the MAPK signaling pathway; their expression patterns were consistent with RNA sequencing data. Our results provide strong support for deeply exploring the role of the MAPK signaling pathway in diarrhea caused by C. perfringens type C.

Clostridium perfringens beta2 toxin induced in vitro oxidative damage and its toxic assessment in porcine small intestinal epithelial cell lines.

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 µg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30 µg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.

Effects of Clostridium perfringens beta2 toxin on apoptosis, inflammation, and barrier function of intestinal porcine epithelial cells.

Clostridium perfringens beta2 (CPB2) toxin is an important virulence factor that causes enteric diseases in both humans and animals. To investigate the underlying mechanism in CPB2-induced inflammation and damage in the small intestinal epithelium, intestinal porcine epithelial cells (IPEC-J2) were treated with recombinant CPB2 (rCPB2) toxin. The results showed that IPEC-J2 cell viability was decreased by rCPB2 toxin treatment in a dose- and time-dependent manner. Analysis of cell morphology and Annexin V-FTIC/PI staining revealed that rCPB2 toxin induces cell apoptosis. Indeed, the expression of caspase-3, caspase-8, and caspase-9 was significantly increased at both the mRNA and protein levels in IPEC-J2 cells treated with rCPB2 toxin. The caspase-3 inhibitor Ac-DEVD-CHO reduced rCPB2 toxin-induced cell apoptosis. Moreover, exposure to the toxin increased the expression of interleukin (IL)-6, IL-7, IL-12, and IL-1ß, while decreasing that of transforming growth factor beta 1 (TGFß1). Additionally, rCPB2 toxin treatment also induced intestinal barrier dysfunction, as evidenced by the degradation of zonula occludens (ZO)-1, claudin-1, and E-cadherin, as well as an increase in paracellular permeability. Overall, the results indicated that rCPB2 toxin induces apoptosis and inflammation, in addition to impairing intestinal barrier function in IPEC-J2 cells. Our findings provide a foundation to better understand the pathogenesis of C. perfringens infection and inform strategies to effectively prevent and treat C. perfringens-induced enteric diseases.

Transcriptional Regulation of HMOX1 Gene in Hezuo Tibetan Pigs: Roles of WT1, Sp1, and C/EBPα.

Heme oxygenase 1 (HMOX1) is a stress-inducing enzyme with multiple cardiovascular protective functions, especially in hypoxia stress. However, transcriptional regulation of swine HMOX1 gene remains unclear. In the present study, we first detected tissue expression profiles of HMOX1 gene in adult Hezuo Tibetan pig and analyzed the gene structure. We found that the expression level of HMOX1 gene was highest in the spleen of the Hezuo Tibetan pig, followed by liver, lung, and kidney. A series of 5' deletion promoter plasmids in pGL3-basic vector were used to identify the core promoter region and confirmed that the minimum core promoter region of swine HMOX1 gene was located at -387 bp to -158 bp region. Then we used bioinformatics analysis to predict transcription factors in this region. Combined with site-directed mutagenesis and RNA interference assays, it was demonstrated that the three transcription factors WT1, Sp1 and C/EBPα were important transcription regulators of HMOX1 gene. In summary, our study may lay the groundwork for further functional study of HMOX1 gene.