Inflammatory Effects Of Glycyrrhizin On LPS-induced Injury Of Goat Rumen Epithelial Cells

Rumen mucosa of ruminants is damaged by acid poisoning and imbalance of microfloracaused by unreasonable feed structure,which seriously affects the performance of ruminants and even leads to death.In order to study the effects of glycyrrhizin on inflammatory factors and the expression of NF-?B signaling pathway molecules in goat rumen epithelial cell injury model induced by LPS in vitro.To analyze the immunoregulatory mechanism of glycyrrhizin on LPS-induced inflammation in Goat Rumen in vitro,and provide new ideas and reference data for the prevention of rumen injury in ruminant breeding and the study of Glycyrrhiza extract feed additives.Experiment 1:Isolation and culture of goat rumen primary cells and identification of cell immunofluorescenceIn this experiment,goat rumen epithelial cells were isolated and cultured in vitro.Rumen epithelium of healthy Boer goats,weighing about 10 kg,aged 1.5 months,was successfully isolated by aseptic operation.Digested cells were digested with trypsin,and the cells digested were incubate in incubators at 37?and 5%CO₂.Microscopic observation showed that when the adherence of epithelial cells reached 80%-90%,the cells were subcultured until the 6th passage.Cell density was adjusted by cell counting method as follows:1×10³,1×10⁴,1×10⁵,1×10⁶?/mL.The cytokeratin?CK18?was used to identify the epithelial cells by immunofluorescence.The results showed that the cytoplasm of the cells showed green fluorescence by immunohistochemical identification of CK18,and the positive staining of CK18 showed that the cells were rumen epithelial cells.MTT assay showed that the cell growth activity of 1×10⁶/mL was the best,which was significantly higher than that of other groups?P<0.05?.Experiment 2:Establishment of LPS induced goat rumen epithelial cell injury model in vitro In this study,Using LPS to induce goat rumen epithelial cells,a subacute ruminal acidosis model was successfully established.The growth activity of rumen epithelial cells induced by LPS was detected by MTT assay,and the expression of inflammatory factors NF-?B,TNF-?,L-1?,IL-6,IL-8,IL-12 mRNA was detected by PCR.The morpHological of rumen epithelial cells were observed by scanning electron microscope,and the optimal concentration of LPS was 50 mg/L.Experiment 3:Intervention of saponin on inflammatory signaling pathway induced by LPS induced ruminal epithelial cell injury in vitroThe aim of this study was to investigate the effect of glycyrrhizin on inflammatory signaling pathways in LPS-induced injury of rumen epithelial cells in vitro.After successful establishment of the inflammatory model of rumen epithelial cells.The effect of glycyrrhizin on LPS-induced epithelial cells was analyzed.The results showed that glycyrrhizin significantly decreased the mRNA expression of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 in the supernatant of rumen epithelial cells in a concentration-dependent manner.The distribution of Occludin and Zo-1showed that the contents of Occludin and Zo-1 in epithelial cells increased with the increase of glycyrrhizin concentration after treatment with different concentrations of glycyrrhizin.Occludin and Zo-1 were mostly distributed at 125 mg/L.Western Blot assay showed that the expression of Occludin protein in rumen epithelium was significantly increased after glycyrrhizin treatment,and the activation of inflammatory factor TNF-?was inhibited.Finally,the expression of NF-?B,TNF-?,IL-1?,IL-6,IL-8 and IL-12 mRNA was detected by qRT-PCR.The results showed that the expression of inflammatory factor mRNA was the least when the concentration of glycyrrhizin was the highest,and the expression of gene was dose-dependent with the concentration of glycyrrhizin.In conclusion,rumen epithelial cells were successfully isolated and cultured in vitro,andSubacute Rumen Acidosis injury model could be established by LPS-induced goat rumen epithelial cells.Glycyrrhizin treatment could reduce the injury and intervention.This experiment further confirmed that glycyrrhizin can effectively control the occurrence of Subacute Rumen Acidosis and reduce the local inflammation of the rumen caused by Subacute Rumen acidosis.