Effects Of VFA On Inflammatory Signaling Pathways Of Rumen Epithelial Cell In Beef Cattle With Subacute Rumen Acidosis

Subacute ruminal acidosis (SARA) is due to excessive intake of rapidfermentation of carbohydrates, resulting in VFAs accumulation, low pH environmentand the dysbacteria in rumen. Then the rumen epithelial cell structure and barrierfunction were destroyed by high concentration of VFAs in the rumen. Ruminalepithelium is not only the body’s first line of defense against pathogen infection, butalso is the vital organs to absorb the nutrient. Although there have been a largenumber of studies have reported SARA can trigger rumen location inflammatoryreaction and systemic inflammatory disease. Its inflammatory signaling pathway hasnot been reported.The experimental strategy combined with in vitro and in vivo experiments. Invivo experiments: to detect VFA concentration in rumen fluid, and the concentrationof plasma TNFα, IL-6and IL-1β in SARA beef cattle. Rumen morphologicalchanges were observed. The results showed that the inflammatory in rumenassociated with SARA. In vitro experiments: simulating subacute rumen acidosiswith different concentrations of VFAs in culturing rumen epithelial cells in vitro. Inorder to understand the mechanism that high VFAs induce inflammatory injury incalf rumen epithelial cells. Using RT-PCR and western blot techniques to detectNF-κB and p38signal pathway and its downstream inflammatory factor.This study was to develop a method for the primary culturing of RECs thatwere isolated from newborn calves by mechanical preparation combined with serialtrypsin digestion. The cells displayed monolayer cobblestone morphous and thestrong expression of cytokeratin18(CK18), which is a specific intermediatefilament protein in epithelial cells. The cells also exhibited typical epithelial cell characteristics, such as microvilli-like structures, desmosome and hemidesmosomeconnections, mosaic connections, and tight junctions. Results demonstrated that thecultured cells are RECs and their activities were highest after4days of culturing.These isolated RECs may be used as cell models for further studies of ruminalphysiology，pathology and molecular biology.The effects of low environmental pH on expression and activity of theinflammatory cytokines were investigated in REC cultured in vitro. After96h, thecells were treated with different pH medium (pH5.5or7.2) for10h, and each groupwith three treatments:0, VFA, and PDTC, respectively. The expression of NF-κBp65and IκB protein were assessed by western blot, and the result showed that theywere increased significantly in pH5.5group. The mRNA levels of were detectedwith RT-PCR, in pH5.5group, the mRNA levels of TNF-α, IL-6, NF-κB p65andIL-1β were significantly higher than those of the control group(P<0.05/P<0.01).Effects of VFA with different concentrations on inflammatory signalingpathways were verified in rumen epithelial cell. Setting treatment groups:0,×1,×5VFA, PDTC20Mm, PDTC20mM+×5VFA, SB203580, SB203580+×5VFA. The results showed that high concentrations of VFA activated NF-κB, p38inflammatory pathway and its downstream inflammatory factors.SARA activating inflammatory signaling pathways of rumen epithelial cells,was affected not only by low pH, also by the high concentration of VFA. In reality,which was the mixed results of the two factors.