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Defensins have attracted considerable research interest worldwide because of their potential to serve as a substitute for antibiotics. In this study, we characterized a novel porcine β-defensin (pBD129) and explored its role in alleviating bacterial endotoxin-induced inflammation and intestinal epithelium atrophy. The pBD129 gene was cloned and expressed in Escherichia coli. A recombinant pBD129 protein was also purified. To explore its role in alleviating the endotoxin-induced inflammation, mice, with or without lipopolysaccharide (LPS) challenge were treated by pBD129 at different doses. The recombinant pBD129 showed significant antimicrobial activities against the E. coli and Streptococcus with a minimal inhibitory concentration (MICs) of 32 μg/mL. Hemolytic assays showed that the pBD129 had no detrimental impact on cell viabilities. Interestingly, we found that pBD129 attenuated LPS-induced inflammatory responses by decreasing serum concentrations of inflammatory cytokines, such as the IL-1β, IL-6, and TNF-α (P < 0.05). Moreover, pBD129 elevated the intestinal villus height (P < 0.05) and enhanced the expression and localization of the major tight junction-associated protein ZO-1 in LPS-challenged mice. Additionally, pDB129 at a high dose significantly decreased serum diamine oxidase (DAO) concentration (P < 0.05) and reduced intestinal epithelium cell apoptosis (P < 0.05) in LPS-challenged mice. Importantly, pBD129 elevated the expression level of Bcl-2-associated death promoter (Bcl-2), but down-regulated the expression levels of apoptosis-related genes such as the B-cell lymphoma-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and caspase-9 in the intestinal mucosa (P < 0.05). These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of pBD129 may allow it a potential substitute for conventionally used antibiotics or drugs.

Background β-defensins have attracted considerable research interest because of their roles in protecting hosts from various pathogens. This study was conducted to investigate the expression profiles of the porcine β-defensin 114 ( PBD114 ) in different breeds and in response to infections. Moreover, the function of PBD114 protein was partially investigated. Methods Six Tibetan pigs (TP) and six DLY (Duroc×Landrace×Yorkshire) pigs were slaughtered to explore the expression profiles of PBD114 in different breeds and tissues. For infection models, sixteen DLY pigs were divided into two groups and challenged either with sterile saline or E. coli K88. The recombinant protein PBD114 (rPBD114) was obtained by using a heterologous expression system in E. coli . Results PBD114 gene was highly expressed in tissues such as the intestine, liver, spleen, and thymus. Interestingly, the expression level of PBD114 gene was higher in the TP pigs than in the DLY pigs ( P < 0.05), and was significantly elevated upon E. coli K88 challenge ( P < 0.05). The nucleotide sequences of PBD114 from Tibetan and DLY pigs was identical, and both showed a 210-bp open reading frame encoding a 69-amino acid mature peptide. To explaore the function of PBD114 protein, PBD114 gene was successfully expressed in E. coli Origami B (DE3) and the molecular weight of the rPBD114 was estimated by SDS-PAGE to be 25 kDa. The rPBD114 was purified and mass spectrometry verified the protein as PBD114. Importantly, rPBD114 showed antimicrobial activities against E. coli DH5α and E. coli K88, and the minimal inhibitory concentrations (MICs) were 64 and 128 μg/mL, respectively. Hemolytic and cytotoxicity assays showed that rPBD114 did not affect cell viability under physiological concentrations. Conclusions PBD114 is an infection response gene that is differentially-expressed between different porcine breeds and tissues. The antimicrobial activity of PBD114 protein, against pathogens such as the E. coli K88, suggested that it may serve as a candidate for the substitution of conventionally used antibiotics. Electronic supplementary material The online version of this article (10.1186/s40104-019-0367-0) contains supplementary material, which is available to authorized users.

Intestinal inflammation is a major threat to the health and growth of young animals such as piglets. As a next-generation probiotics, limited studies have shown that Akkermansia muciniphila could alleviate inflammation of intestinal epithelial cells (IECs). In this study, a TNF-α-induced inflammatory model of IPEC-J2 cells, the intestinal porcine enterocytes, was built to evaluate the effects of active or inactive A. muciniphila on the inflammation of IECs. The viability of IPEC-J2 cells was the highest when treated with active (108 copies/mL) or inactive (109 copies/mL) A. muciniphila for 7.5 h (P &lt; 0.01). Treated with 20 ng/mL of TNF-α and followed by a treatment of A. muciniphila, the mRNA level of proinflammatory cytokines (IL-8, IL-1β, IL-6 and TNF-α) was remarkably reduced (P &lt; 0.05) along with the increased mRNA level of tight junction proteins (ZO-1 and Occludin, P &lt; 0.05). Flow cytometry analysis showed that active or inactive A. muciniphila significantly suppressed the rate of the early and total apoptotic of the inflammatory IPEC-J2 cells (P &lt; 0.05). According to results of transcriptome sequencing, active and inactive A. muciniphila may decline cell apoptosis by down-regulating the expression of key genes in calcium signaling pathway, or up-regulating the expression of key genes in cell cycle signaling pathway. And the bacterium may alleviate the inflammation of IECs by down-regulating the expression of PI3K upstream receptor genes. Our results indicate that A. muciniphila may be a promising NGP targeting intestinal inflammation.

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