Agar oligosaccharides mitigate lipopolysaccharide-induced apoptosis and inflammatory damage in rumen epithelial cells via the tlr4-myd88-nf-κb signaling pathway

<title>Abstract</title> <p>Agar oligosaccharide (AOS) is an oligosaccharide with a degree of polymerization (DP) of agarose, which is degraded by hydrolysis, chemical degradation, and other methods, and has an oligosaccharide with a DP of less than 20. It has anti-inflammatory and anti-apoptotic effects, but its intervention effect and mechanism on rumen epithelial damage are still unclear. Therefore, this study intends to study and analyze the regulatory effect of AOS on LPS-induced rumen epithelial cell damage in sheep through in vitro LPS-induced rumen epithelial cell injury model, so as to provide a reference idea for AOS as a green feed additive to alleviate ruminant Subacute rumen acidosis (SARA) or other metabolic diseases. First, the ovine rumen epithelial cells were divided into a control group and four AOS concentration groups (1, 15, 50, 100 μg/mL), and three independent biological replicates were set up in each group to stimulate the cells for 6, 12, 18, and 24 h respectively to determine the optimal AOS treatment time and concentration. The results showed that the content of pro-inflammatory cytokines and the relative expression of pro-apoptotic genes in rumen epithelial cells cultured with 15 µg/mL AOS for 18 h were significantly reduced (p&lt;0.05), and the relative expression of cell viability and inhibition of apoptosis genes were significantly increased (p&lt;0.05), and there were no adverse effects on the cells under the intervention of the concentration of this treatment and the time of the treatment. Second, establish a cell injury model. The cells were divided into a control group and four Lipopolysaccharide (LPS) concentration groups (1, 15, 50, 100 µg/mL), with three independent biological replicates in each group, and the cells were stimulated for 6, 12, and 24 hours. The results showed that after culturing rumen epithelial cells with 50 µg/mL LPS for 24 hours, reactive oxygen species (ROS), pro-inflammatory cytokine levels, and the relative expression of pro-apoptotic genes were significantly increased (p&lt;0.05), while cell viability and the relative expression of anti-apoptotic genes were significantly decreased (p&lt;0.05). Therefore, this condition was determined as the injury model. Third, based on the above experimental results, the cells were divided into a control group (CON group), LPS group, and AOS+LPS group (A+L group) to further explore the effect of AOS on LPS-induced rumen epithelial cell injury. The results showed that 50 µg/mL LPS incubation for 24 h could cause suitable damage to the cells. Cell morphology was improved and cell viability was significantly higher in the A+L group (p&lt;0.05). The A+L group significantly decreased the reactive oxygen species (ROS) fluorescence intensity and interleukin-6 (IL-6) , IL-8, IL-1β and tumor necrosis factor-α (TNF-α) contents and increased the immunoglobulin A (IgA) , IgM, and IgG contents (p&lt;0.05). The apoptosis rate were significantly reduced (p&lt;0.05). In addition, the A+L group significantly inhibited the expression of genes and proteins related to the Toll-like Receptor 4-Myeloid Differentiationfactor 88-Nuclear Factor-κB (TLR4-MyD88-NF-κB) signaling pathway. In summary, AOS attenuated LPS-induced apoptosis and inflammatory injury in rumen epithelial cells in vitro via the TLR4-MyD88-NF-κB signaling pathway.</p>