Study Of Plant-derived Essential Oil Protection Injury Of Bovine Mammary Epithelial Cells Induced By Lipopolysaccharide

The mastitis was one of the disastrous diseases which effected development of the dairy industry. Recently years, many researchers focused on the cellular level of mastitis.The bovine mammary epithelial cells(b MEC) played an vital role in the bovine mammary immunity system. So b MEC was the important materials for pathogenesis study and effective drugs screening to treat mastitis. Plant-derived essential oil was aromatic substances owning small molecular weight and penetration ability extracted from plants, so it was easy to absorb for body. Many studies had suggested that plant-derived essential oil had a good effect on antibacterial and sterilization to a variety of pathogenic bacteriason mastitis. This study obtained the pure b MEC by primary culture system. Model of inflammation was established by Lipopolysaccharide(LPS) stimulating. That the effect of the essential oil to the inflammatory injury of b MEC was studied through the assay of cells activity and the morphology observation of cells. At the same time, this study had tested the gene expression of several key factors in the signaling pathway of NF-kappa B(NF-κB).The purpose of this study were to explore if the plant-derived essential oil could protect the b MEC from the inflammatory damage of LPS, which could provide the theoretical basis about the drug screening for the mastitis treatment.1. The separated and cultivation of b MEC Both the tissue block method and enzymatic digestion method were used to obtain the b MEC from a bovine udder without a history of lactation. The cell morphology features were conform to the b MEC, and the specificity skeleton egg- cell keratin18(CK-18) was expressed successfully. Cell growth curve showed typical “S” type in 2~8 d, which proved growth of cells were in in good condition.The number of the polarization cells within 10 generations was small While the cells that extend to 20 generations increased significantly.2. Cryopreserved of udder tissue The survival rate was 50% for the tissue mass(4~5mm3) by one-step cryopreservation(-80℃) followed by solution nitrogen storage for eight month with freeze-stored solution FBS: DMSO: DMEM/F12 = 7:2:1 while the survival rate was 56% with freeze-stored solution FBS: DMEM/F12: DMSO: HEPES/sodium pyruvate = 10:7:2:1.3. The establishment of b MEC inflammatory model The b MEC was stimulated by LPS dissolved in the basal medium at concentrations of 0 μg/m L、10 μg/m L、50 μg/m L、100 μg/m L、200 μg/m L separately. And the were OD values about the cell vitality detection were0.62±0.05、0.61±0.09、0.46±0.06、0.31±0.07、0.02±0.01 separately, which showed that the cell vitality decreased as the concentration of LPS increased. Subsequently, the cells were situmulated by LPS at the concentration of 0μg/m L、10 μg/m L、25 μg/m L、50 μg/m L、100μg/m L, and then analysisboth the change of the cell morphology and the gene expression of beta-defensins, IL~1 beta, IL~8, TNF-alpha, The result showed that the change of cell morphology was small and the expression level of inflammatory cytokines increased significantly when the cells stimulated by 50 μg/m L LPS.In a nutshell, 50 μg /m L was the best dose for establishment of b MEC inflammatory model.4. The effects of plant-derived essential oil to the b MEC inflammation model The cell morphology observation and the cell vitality determination were performed on the b MEC that stimulated by different plant-derived essential oil at different concentrations. It was founded that volume fraction of the thyme essential oil, tea tree oil, camphor oil, eucalyptus oil should not exceed 0.02%, 0.04%, 0.04% and 0.03% respectively.And then, the results of the RT-PCR proved that 0.01% thyme essential oil, 0.04% tea tree oil, 0.03% camphor oil and 0.02% eucalyptus oil could decreased the the expression of beta-defensins, IL-1 beta, IL-8, TNF-alpha and the key factor(TRAF6 Bp65, Bp50) of the NF-kappa B signaling pathway.To sum up, the udder tissue of the bovine without a history of lactation could be used to culture the b MEC after cryopreserved at-198℃ for eight months. The pure b MEC stimulated by LPS at 50 μg/m L for 12 h, which could be used to establish b MEC inflammation model.A certain concentration of thyme essential oil, tea tree oil, camphor oil, eucalyptus oil had the effect of reducing the injury of LPS to b MEC...