miR-136-5p Regulates the Inflammatory Response by Targeting the IKKβ/NF-κB/A20 Pathway After Spinal Cord Injury

Background/Aims: miR-136-5p participates in recovery after spinal cord injury (SCI) via an unknown mechanism. We investigated the mechanism underlying the involvement of miR-136-5p in the inflammatory response in a rat model of SCI. Methods: Sprague-Dawley rat astrocytes were cultured in vitro to construct a reporter plasmid. Luciferase assays were used to detect the ability of miR-136-5p to target the IKKβ and A20 genes. Next, recombinant lentiviral vectors were constructed, which either overexpressed miR-136-5p or inhibited its expression. The influence of miR-136-5p overexpression and miR-136-5p silencing on inflammation was observed in vivo in an SCI rat model. The expression of IL-1β, IL-6, TNF-α, IFN-α, and related proteins (A20, IKKβ, and NF-κB) was detected. Results: In vitro studies showed that luciferase activity was significantly activated in the presence of the 3’ untranslated region (UTR) region of the IKKβ gene after stimulation of cells with miR-136-5p. However, luciferase activity was significantly inhibited in the presence of the 3’UTR region of the A20 gene. Thus, miR-136-5p may act directly on the 3’UTR regions of the IKKβ and A20 genes to regulate their expression. miR-136-5p overexpression promoted the production of related cytokines and NF-κB in SCI rats and inhibited the expression of A20 protein. Conclusion: Overexpression of miR-136-5p promotes the generation of IL-1β, IL-6, TNF-α, IFN-α, IKKβ, and NF-κB in SCI rats but inhibits the expression of A20. Under these conditions, inflammatory cell infiltration into the rat spinal cord increases and injury is significantly aggravated. Silencing of miR-136-5p significantly reduces the protein expression results described after miR-136-5p overexpression and ameliorates the inflammatory cell infiltration and damage to the spinal cord. Therefore, miR-136-5p might be a new target for the treatment of SCI

Differential Analysis of Gut Microbiota Correlated With Oxidative Stress in Sows With High or Low Litter Performance During Lactation

It has been suggested that gut microbiota play a critical role in maternal metabolic oxidative stress responses and offspring growth. However, whether the gut microbiota and oxidative stress status of the sows affect the litter performance during lactation is unclear. A total of 66 Yorkshire sows were identified as high (H) or low (L) litter performance sows based on litter weight at day 21 of lactation. Ten sows per group with similar parity, backfat thickness, and litter weight after cross-foster from the H or L group were collected randomly to analyze the oxidative stress and gut microbiota during lactation. The result showed that the serum total antioxidant capacity was higher in the H group, while 8-hydroxy-deoxyguanosine and thiobarbituric acid reactive substances were lower in the H group at farrowing. Four distinct clusters of bacteria were related to litter performance and reproductive periods of sows. Twelve differentially abundant taxa during gestation and 13 taxa during lactation were identified as potential biomarkers between the H group and the L group. Moreover, the litter performance and the antioxidant capacity of sows were positively correlated with Bacteroides_f__Bacteroidaceae but negatively with Phascolarctobacterium and Streptococcus. In conclusion, this study found that gut microbiota and oxidative stress were significantly correlated with the litter performance of sows during lactation

Exploring the effects of lysozyme dietary supplementation on laying hens: performance, egg quality, and immune response

An experiment was conducted to evaluate the dietary supplementation with lysozyme's impacts on laying performance, egg quality, biochemical analysis, body immunity, and intestinal morphology. A total of 720 Jingfen No. 1 laying hens (53 weeks old) were randomly assigned into five groups, with six replicates in each group and 24 hens per replicate. The basal diet was administered to the laying hens in the control group, and it was supplemented with 100, 200, 300, or 400 mg/kg of lysozyme (purity of 10% and an enzyme activity of 3,110 U/mg) for other groups. The preliminary observation of the laying rate lasted for 4 weeks, and the experimental period lasted for 8 weeks. The findings demonstrated that lysozyme might enhance production performance by lowering the rate of sand-shelled eggs (P < 0.05), particularly 200 and 300 mg/kg compared with the control group. Lysozyme did not show any negative effect on egg quality or the health of laying hens (P > 0.05). Lysozyme administration in the diet could improve intestinal morphology, immune efficiency, and nutritional digestibility in laying hens when compared with the control group (P < 0.05). These observations showed that lysozyme is safe to use as a feed supplement for the production of laying hens. Dietary supplementation with 200 to 300 mg/kg lysozyme should be suggested to farmers as a proper level of feed additive in laying hens breeding

Cecropin A Modulates Tight Junction-Related Protein Expression and Enhances the Barrier Function of Porcine Intestinal Epithelial Cells by Suppressing the MEK/ERK Pathway

Inflammatory bowel disease (IBD) in humans and animals is associated with bacterial infection and intestinal barrier dysfunction. Cecropin A, an antimicrobial peptide, has antibacterial activity against pathogenic bacteria. However, the effect of cecropin A on intestinal barrier function and its related mechanisms is still unclear. Here, we used porcine jejunum epithelial cells (IPEC-J2) as a model to investigate the effect and mechanism of cecropin A on intestinal barrier function. We found that cecropin A reduced Escherichia coli (E. coli) adherence to IPEC-J2 cells and downregulated mRNA expression of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). Furthermore, cecropin A elevated the transepithelial electrical resistance (TER) value while reducing the paracellular permeability of the IPEC-J2 cell monolayer barrier. Finally, by using Western blotting, immunofluorescence and pathway-specific antagonists, we demonstrated that cecropin A increased ZO-1, claudin-1 and occludin protein expression and regulated membrane distribution and F-actin polymerization by increasing CDX2 expression. We conclude that cecropin A enhances porcine intestinal epithelial cell barrier function by downregulating the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We suggest that cecropin A has the potential to replace antibiotics in the treatment of IBD due to its antibacterial activity on gram-negative bacteria and its enhancement effect on intestinal barrier function

Metabolic Profiling by UPLC–Orbitrap–MS/MS of Liver from C57BL/6 Mice with DSS-Induced Inflammatory Bowel Disease

Liver disorder often occurs in patients with inflammatory bowel disease (IBD); however, the changes in IBD-induced liver disorder at the intrinsic molecular level (chiefly metabolites) and therapeutic targets are still poorly characterized. First, a refined and translationally relevant model of DSS chronic colitis in C57BL/6 mice was established, and cecropin A and antibiotics were used as interventions. We found that the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the liver tissues of mice were highly increased in the context of DSS treatment but were lowered by cecropin A and antibiotics. Subsequently, an untargeted metabolomics analysis was performed by UPLC–Orbitrap–MS/MS to reveal the metabolic profile and attempt to find the potential therapeutic targets of the liver disorders that occur in IBD. Notably, 133 metabolites were identified by an integrated database. Metabolism network and pathway analyses demonstrated that the metabolic disturbance of the liver in IBD mice was mainly enriched in bile acid metabolism, arachidonic acid metabolism, amino acid metabolism, and steroid hormone biosynthesis, while those disturbances were regulated or reversed through cecropin A and antibiotic treatment. Furthermore, the top 20 metabolites, such as glutathione, maltose, arachidonic acid, and thiamine, were screened as biomarkers via one-way analysis of variance (one-way ANOVA, p1) of orthogonal partial least-squares discriminant analysis (OPLS-DA), which could be upregulated or downregulated with the cecropin A and antibiotics treatment. Spearman correlation analysis showed that the majority of the biomarkers have a significant correlation with cytokines (TNF-α, IL-1β, IL-6, and IL-10), indicating that those biomarkers may act as potential targets to interact directly or indirectly with cecropin A and antibiotics to affect liver inflammation. Collectively, our results extend the understanding of the molecular alteration of liver disorders occurring in IBD and offer an opportunity for discovering potential therapeutic targets in the IBD process

Implication of G Protein-Coupled Receptor 43 in Intestinal Inflammation: A Mini-Review

Short chain fatty acids (SCFAs, e.g., acetate, propionate, and butyrate) are a subset of fatty acids that are produced by gut microbiota during the fermentation of dietary fiber. They modulate different processes in the gastrointestinal tract and play various positive roles in mediating the intestinal health. Most beneficial roles of SCFAs in the gastrointestinal tract are mediated by directly activating its receptor, G protein-coupled receptor 43 (GPR43, also known as FFAR2). Various recent studies have demonstrated the role of GPR43 in intestinal inflammatory diseases, such as inflammatory bowel diseases. These SCFAs-mediated regulations of intestinal health are associated with neutrophil chemotaxis, T cell differentiation, activation, and subsequent cytokines production. Therefore, GPR43 could potentially be a drug target for intestinal inflammatory diseases. In this review, we review the current knowledge on the regulatory mechanisms associated with GPR43 in intestinal inflammation. The role of GPR43-mediated regulation of antibody responses is also discussed

Effects of Softening Dry Food with Water on Stress Response, Intestinal Microbiome, and Metabolic Profile in Beagle Dogs

Softening dry food with water is believed to be more beneficial to the intestinal health and nutrients absorption of dogs by some owners, but there appears to be little scientific basis for this belief. Thus, this study aimed to compare feeding dry food (DF) and water-softened dry food (SDF) on stress response, intestinal microbiome, and metabolic profile in dogs. Twenty healthy 5-month-old beagle dogs were selected and divided into two groups according to their gender and body weight using a completely randomized block design. Both groups were fed the same basal diet, with one group fed DF and the other fed SDF. The trial lasted for 21 days. The apparent total tract digestibility (ATTD) of nutrients, inflammatory cytokines, stress hormones, heat shock protein-70 (HSP-70), fecal microbiota, short-chain fatty acids (SCFAs), branch-chain fatty acids (BCFAs), and metabolomics were measured. Results showed that there was no significant difference in body weight, ATTD, and SCFAs between the DF and SDF groups (p > 0.05), whereas feeding with SDF caused a significant increase in serum cortisol level (p p = 0.062) and HSP-70 (p = 0.097) levels. Fecal 16S rRNA gene sequencing found that the SDF group had higher alpha diversity indices (p Streptococcus, Enterococcus, and Escherichia_Shigella, and lower levels of Faecalibacterium (p p < 0.05). Overall, feeding with SDF caused higher cortisol level and generated effects of higher intestinal microbial diversity in dogs, but it caused an increase in some pathogenic bacteria, which may result in intestinal microbiome disturbance and metabolic disorder in dogs. In conclusion, feeding with SDF did not provide digestive benefits but caused some stress and posed a potential threat to the intestinal health of dogs. Thus, SDF is not recommended in the feeding of dogs

Effects of dietary supplementation of probiotic Enterococcus faecium on growth performance and gut microbiota in weaned piglets

Abstract The adverse effects of antibiotics have attracted widespread attention, thus reducing the use of antibiotics in animal feed has become a very important issue in improving of the health of livestock. The effects of Enterococcus faecium (E. faecium) on growth performance and gut microbiota in weaned piglets were investigated in the present study. Piglets were randomly assigned to four treatments: a control group fed with a diet containing 75 mg/kg aureomycin (Diet 1 group) and three experimental groups fed with diets of 50 mg/kg aureomycin (Diet 2 group), 50 mg/kg aureomycin + 9 × 105 CFU/g E. faecium (Diet 3 group), or 50 mg/kg aureomycin + 1.2 × 106 CFU/g E. faecium (Diet 4 group). Their gut microbial communities were analyzed by sequencing the V3–V4 region of the 16SrRNA gene. The results showed that the final body weights and the average daily gain of the weaned piglets in the Diet 2 group were higher (P = 0.05) than those in the Diet 1 or Diet 3 group. Decreasing trends (P = 0.08) was observed in mortality rate in the Diet 3 and 4 group when compared with that in the Diet 1 group. Increases in the Sobs, Chao1, ACE, and Shannon indexes and a decrease in the Simpson index were observed at intervals from day 1 to 14 (P < 0.05). The Sobs, Chao1, and ACE indexes in the Diet 3 group were the lowest on day 14 (P < 0.05). The abundance of Bacteroidetes was increased and that of Proteobacteria was decreased from day 1 to 7, but both of them kept stable from day 7 to 14. Besides, the lowest abundance of Fusobacteria, Lentisphaerae, and Planctomycetes was observed on day 1 and the lowest abundance of Actinobacteria was observed on day 14 in the Diet 3 group (P < 0.05). Overall, these results suggest that the antibiotics and E. faecium interventions result in different changes in the gut microbiota, and a reduced antibiotics diet supplemented with 1.2 × 106 CFU/g E. faecium does not affect the growth performance in weaned piglets