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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-1β), 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. • HRP protected IPEC-J2 cells from LPS-induced inflammation damage and apoptosis. • HRP protected the barrier function integrity of IPEC-J2 cells induced by LPS. • HRP-inhibited LPS-induced inflammation is related to the TLR4/NF-κB pathway. [ABSTRACT FROM AUTHOR]

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. • Proteomic analysis identifies differentially expressed proteins involved in the immune system and associated pathways in IPEC-J2 cells. • HRP alleviated LPS-induced inflammation in IPEC-J2 cells, which is related to MAPK/NF-κB pathway. [ABSTRACT FROM AUTHOR]