The Protective Effect And Molecular Mechanisms Of Caffeic Acid Against Inflammation Injury Of Primary Bovine Mammary Epithelial Cells Induced By Lipopolysaccharide

Jin-Pu-Guan-Zhu-Ji developed in our laboratory is a kind of Traditional Chinese Veterinary Medicine used for treating bovine mastitis, while caffeic acid acts as an important active ingredient. In order to provide theoretical and technological support for the clinical application of the herb formulation, the protective effect of caffeic acid against lipopolysaccharide (LPS)-induced inflammation injury of bovine mammary epithelial cells (bMEC) and the possible molecular mechanisms were studied in this dissertation.The effect of LPS on cell viability of bMEC was tested by MTT, then the LPS-injured bMEC modle was preliminarily established. The injury modle of bMEC caused by LPS was evaluated by scanning electron microscopy (SEM), transmission electron microscope (TEM) and flow cytometry (FCM). Changes of gene expression profile of bMEC induced by LPS were analyzed by deep sequencing technology. Through the multiple analysis of differential expression genes, we predicted the molecular mechanism of LPS-induced injury to bMEC. The levels of several key proteins and the expression of inflammatory cytokines were verified by the Western blot and RT-PCR, which related to the prediction of inflammatory signaling pathways. LPS caused inflammation injury of bMEC could be weakened by caffeic acid, then the molecular mechanisms of anti-inflammation injury of bmec induced by LPS were explored. The test results are as follows:Bovine mammary epithelial cells were treated with various concentrations (10,50,100, and200μg/mL) of LPS for3,6,12, and18h, the results showed that LPS significantly inhibited cell viability in a time-and dose-dependent manner. Cells were stimulated with a selected treatment,50μg/mL of LPS for12h, as the model conditions of inflammation injury of bMEC induced by LPS for further research. In model group, morphological changes including fuzzy cell boundary, cellular atrophy, disorderly or missing microvilli and different degrees of cell collapse were observed by SEM. Subcellular structure changes including swelling or rupture of microvilli, nuclear cavitation, extreme expansion of endoplasmic reticulum, hazy mitochondrial structures, loose cytoplasmic matrix structure were studied by means of TEM. The data of FCM showed that cell apoptosis was significantly increased after cells were stimulated with LPS, especially the early apoptosis rate. The above data revealed that LPS could destroy the cell morphology structures of bMEC, thus affecting the physiological function of bMEC.Caffeic acid could significantly reduce the decline of cell vitality induced by LPS, and obviously weaken the damage of cell morphological structure and subcellular structure induced by LPS. The effects of caffeic acid were in a dose-dependent manner.The molecular mechanisms of LPS-induced inflammtion injury of bMEC was analyzed by sequencing depth on transcriptome profiling.1435genes were sifted to be diffrently expressed, including1301up-regulated genes and134down-regulated genes. GO analysis revealed that the differential expression genes were mainly related to growth and differentiation, stress responses, signal transduction, material metabolism processes of bMEC, these results indicated that variations in physiologic functions. KEGG Pathway analysis obtained that several differential expression genes mainly related to nuclear transcription factor kappa B (NF-κB) signaling pathway and mitogen-activated protein kinases (MAPKs) signaling pathway. So the NF-κB signaling pathway and MAPKs signaling pathway of bMEC could be activated by LPS, then a large number of inflammatory mediators were released, which resulted in the inflammation injury of bMEC.The degradation of IκBα and phosphorylation of p65induced by LPS were partially inhibited by caffeic acid in a dose-dependent manner, which indicated a weakening activity of NF-κB. The LPS-induced phosphorylation of p38and JNK were partially inhibited by caffeic acid in a dose-dependent manner, the phosphorylation of ERK1/2was completely inhibited by all concentrations of caffeic acid, which indicated a weakening activity of MAPKs. The expression of IL-8, IL-1β, IL-6and TNF-α could be effectively inhibited by caffeic acid, which mainly regulated by NF-κB and MAPKs signaling pathway, thus significantly weakened LPS-induced inflammatory injury of bMEC.