Bacillus subtilis JATP-3 Improves Nitrogen Metabolism by Regulating Intestinal Flora and AKG in Weaned Piglets.

Recently, it has been reported that oral probiotics improve the apparent digestibility of nitrogen in weaned piglets; however, the underlying mechanism is unclear. A total of 12 crossbred piglets (Yorkshire × Landrace; 28 days old) were randomly divided into two groups. The control (Con) group was fed with a basic diet + Luria-Bertani (LB; sterile; 10 mL), whereas the subject (Sub) group was fed with a basic diet + B. subtilis JATP-3 (1 × 109 CFU/mL; 10 mL). The results showed that feeding B. subtilis JATP-3 increased the final body weight and nitrogen deposition rate of weaned piglets (P < 0.05); while the daily weight gain showed an upward trend (P < 0.1). The abundance of Pedicoccus, Collinella, Turiciator, Veillonella, Clostridium, and Escherichia were significantly increased in the jejunum (P < 0.05). The abundance of Olsenella and Pediococcus were significantly increased in the ileum (P < 0.05). The metabolomics analysis showed that the levels of l-lactic acid and Alpha-ketoglutaric acid (AKG) in portal vein plasma were significantly increased (P < 0.05). In addition, the content of AKG in muscle and liver increased significantly (P < 0.01). The metagenomics analysis showed that Veillonella encoded the functional genes of 2-oxoglutarate synthase and promoted AKG production. The protein expression of eIF4E-binding protein 1 (4EBP1) phosphorylated in the skeletal muscle increased (P < 0.05). In summary, B. subtilis JATP-3 promotes dietary nitrogen metabolism and skeletal muscle synthesis by modulating the intestinal microbiota and its metabolites, in which AKG may be one of the main mediators of the therapeutic effects of B. subtilis JATP-3.

Probiotics Lactobacillus rhamnosus GG ATCC53103 and Lactobacillus plantarum JL01 induce cytokine alterations by the production of TCDA, DHA, and succinic and palmitic acids, and enhance immunity of weaned piglets.

Probiotics, including Lactobacillus rhamnosus GG ATCC53103 and Lactobacillus plantarum JL01, can improve growth performance and immunity of piglets, and relieve weaning stress-related immune disorders such as intestinal infections and inflammation. This study aimed to evaluate the ability of co-administration of the probiotics L. rhamnosus GG ATCC53103 and L. plantarum JL01 to stimulate immune responses and improve gut health during the critical weaning period in piglets. Forty-eight weaned piglets were randomly divided into four groups, and fed daily for 28 days either without, or with the two probiotics independently, or in combination. On day 28, we analyzed the cytokine and bacterial changes in intestinal mucosa and the hepatic portal vein blood metabolites of the weaned piglets. Our results showed that combined L. rhamnosus GG ATCC53103 and L. plantarum JL01 significantly increased (p < 0.05) the growth performance and expression of IL-10 and TGF-ß1 mRNAs. In contrast, this treatment significantly decreased (p < 0.05) IL-1ß mRNA level in the jejunum, ileum, and cecum. Furthermore, the secretion of IL-6 in the cecum, IL-1ß in the jejunum, ileum, and cecum, and TNF-α in the jejunum and ileum was significantly decreased (p < 0.05). The relative abundance of Prevotella_9 and Enterococcus in ileum and cecum was significantly increased (p < 0.05). The relative abundance of Ruminococcus_1 and Ruminococcaceae_UCG-005 in cecum was significantly decreased (p < 0.05). Prevotella_9 and Enterococcus may increase the accumulation of (4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA) and tauroursodeoxycholic acid (TCDA) in portal vein blood, while Ruminococcus_1 and Ruminococcaceae_UCG-005 may decrease the accumulation of succinic and palmitic acids. These results indicate that L. rhamnosus GG ATCC53103 and L. plantarum JL01 may regulate cytokine levels by reducing the accumulation of succinic and palmitic acids and increasing the accumulation of TCDA and DHA, thereby enhancing the immunity of weaned piglets.

Proteomic analysis reveals the molecular mechanism of Hippophae rhamnoides polysaccharide intervention in LPS-induced inflammation of IPEC-J2 cells in piglets.

Early weaning can cause intestinal disorders and dysfunction in piglets, and may induce intestinal diseases. Hippophae rhamnoides polysaccharide (HRP) has anti-inflammatory and immune promotion function. However, few studies have explored the change of differentially protein expression by lipopolysaccharide (LPS)-induced porcine intestinal epithelial cell (IPEC-J2) after HRP pre-treatment. In this study, proteomic analysis was used to explore the essential proteins and immune-related pathways that can be regulated by LPS-induced IPEC-J2 cells after HRP pre-treatment. The results indicate that by searching the Sus scrofa database, a total of 18,768 proteins was identified. Among recognized proteins, there are 2052 (1720 up-regulated and 332 down-regulated), 358 (262 up-regulated and 96 down-regulated) and1532 (314 up-regulated and 1218 down-regulated) proteins expressed differently in C vs. L, C vs. H6-L and L vs. H6-L, respectively. The Cluster of Orthologous Groups (COG) analysis divided the identified proteins into 23 categories. Gene Ontology (GO) enrichment analysis revealed that cellular process, cell, cell part, organelle and binding were the most enriched pathways for differentially expressed proteins. KEGG enrichment analysis indicated that the top 20 pathways in the L-vs-H6-L group related to immunity were the Tight junction, MAPK signaling pathway, PI3K-Akt signaling pathway, rap1 signaling pathway, HIF-1 signaling pathway, Ras signaling pathway and Fc gamma R-mediated phagocytosis. Moreover, we also found 42 key proteins related to these immune pathways in this study. Additionally, western blotting analyses confirmed that LPS reduced the levels of claudin2 (CLDN2), insulin-like growth factor 2 (IGF2) and increased phosphorylated mitogen-activated protein kinase 7 (MAPK7), phosphorylated transcription factor p65 (RELA), phosphorylated nuclear factor NF-kappa-B p105 subunit (NF-κB1) and phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (NF-κB2). Pre-treatment with HRP increased the levels of CLDN2, IGF2 and reduced the levels of the phosphorylated MAPK7, phosphorylated RELA, phosphorylated NF-κB1 and phosphorylated NF-κB2 in cells. These results also showed that HRP alleviated LPS-induced inflammation in IPEC-J2 cells by inhibiting the MAPK/NF-κB signaling pathway and its related differentially expressed proteins.

Hippophae rhamnoides polysaccharides protect IPEC-J2 cells from LPS-induced inflammation, apoptosis and barrier dysfunction in vitro via inhibiting TLR4/NF-κB signaling pathway.

Inflammatory response caused by early weaning stress in piglets is associated with various diseases. The Hippophae rhamnoides polysaccharide (HRP) exhibits anti-inflammatory activity and immunomodulatory properties. The mechanisms for the protective effects of HRP on barrier function, inflammatory damage and apoptosis in intestinal porcine epithelial cells (IPEC-J2) induced by the lipopolysaccharide (LPS) are unknown. In this study, we first demonstrated the cytotoxicity of HRP-induced IPEC-J2 cells by reducing cell viability. IPEC-J2 cells were treated with 0-800 µg/mL doses of HRP, and 0-600 µg/mL doses were used in further experiments. Upon exposure to LPS, the viability of IPEC-J2 cells, ROS production, immunoglobulin levels (immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin G (IgG)) and tight junction protein level (zonula occludens-1 (ZO-1), occluding, claudin-1) decreased. Inflammatory factors (interleukin-1beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α)) and apoptosis (Bcl-2, Bax, caspase-3, caspase-8 and caspase-9) were increased. Cell morphology and internal structure were damaged in the LPS treatment. Pre-treating cells with HRP (0-600 µg/mL) reduced inflammatory factors levels, apoptosis rate, increased immunoglobulins, tight junction protein levels and relieved cell surface morphology damage. Pre-treatment with HRP also reduced the levels of the Toll-like receptor 4 (TLR4) and Myeloid differentiation factor 88 (MyD88) and inhibited the phosphorylated NF-κB factor-kappa B (NF-κB) in cells induced by LPS. These results show that pre-treatment with HRP protected against LPS-induced IPEC-J2 cell damage through its anti-inflammatory activity.

Astragaloside IV Attenuates Acetaminophen-Induced Liver Injuries in Mice by Activating the Nrf2 Signaling Pathway.

Acetaminophen (APAP) is a well-known antipyretic and analgesic drug. However, the accidental or intentional APAP overdose will induce liver injury and even acute liver failure. Astragaloside IV (AS-IV), a bioactive compound isolated from Astragali Radix, has been reported to have protective effects on the digestive and immune systems because of its anti-oxidant and anti-inflammatory properties. This study aims to observe whether AS-IV pretreatment provides protection against APAP-induced liver failure. The results of serum alanine/aspartate aminotransferases (ALT/AST) analysis, hepatic glutathione (GSH), and malondialdehyde (MDA) amounts, and liver superoxide dismutase (SOD) activity showed that AS-IV protected against APAP-induced hepatotoxicity. Liver histological observation further evidenced this protection provided by AS-IV. AS-IV was found to reverse the APAP-induced increased amounts of pro-inflammatory cytokines, including interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Western-blot analysis showed that AS-IV increased the transcriptional activation of nuclear factor erythroid 2-related factor 2 (Nrf2), and enhanced the expression of heme oxygenase 1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H): quinone oxidoreductase 1 (NQO1) in the presence of APAP. AS-IV also decreased the expression of kelch-like ECH-associated protein-1 (Keap1). In conclusion, we demonstrated that AS-IV exerted a strong protection against APAP-induced hepatotoxicity by activating Nrf2 antioxidant signaling pathways.