【作者】 刘明江；

【导师】 许剑琴； 刘凤华；

【作者基本信息】 中国农业大学 ， 临床兽医学， 2014， 博士

【摘要】 “金蒲”灌注剂是本实验室研制的一种治疗奶牛乳房炎的纯中药制剂,咖啡酸是该方剂中的一种主要活性成分,本文通过研究咖啡酸抗脂多糖(LPS)诱导的奶牛乳腺上皮细胞(bMEC)的炎性损伤作用及其分子机制,为该方剂的临床应用提供理论依据和技术支持。本研究通过MTT法检测LPS对bMEC细胞活力的影响,并筛选了LPS诱导bMEC损伤模型条件；利用扫描、透射电镜观察bMEC超微结构变化和流式细胞术检测bMEC的凋亡情况对损伤模型进行了评价；通过深度测序技术分析LPS诱导的bMEC基因表达谱变化,筛选差异基因并对其进行多重分析,预测了LPS诱导的bMEC损伤分子机制；利用Western Blot及RT-PCR技术对相关炎症信号通路中关键蛋白和炎性因子的表达进行了验证；并探讨了咖啡酸抗LPS诱导的bMEC结构损伤的分子机制。相关研究结果如下：不同浓度的LPS(10、50、100、200μg/mL)作用bMEC不同时间(3、6、12、18h)后,结果显示,LPS能够显著地降低bMEC的细胞活力,这种作用具有明显的时间、剂量依赖性。选择50μg/mL的LPS作用bMEC12h作为损伤模型条件。扫描电镜观察显示：模型组细胞界限不清,细胞明显固缩,细胞表面微绒毛排列紊乱或缺失,细胞出现不同程度塌陷；透射电镜观察显示：模型组细胞游离面微绒毛肿胀或断裂,细胞核空化,内质网极度扩张,线粒体结构模糊,细胞质基质结构疏松；流式细胞术检测结果显示：模型组细胞的细胞凋亡率明显升高,尤其是早期细胞凋亡率。说明LPS能够破坏bMEC的细胞形态结构。咖啡酸(10、25、50μg/mL)显著地降低了LPS诱导的bMEC细胞活力下降,明显减弱了LPS诱导的细胞形态结构损伤,显著降低了LPS诱导的细胞凋亡率,咖啡酸的功效具有明显剂量依赖性。基因转录组深度测序分析结果显示,模型组与对照组细胞共有1435个差异基因,其中上调基因1301个,下调基因134个。GO分析得出,差异基因主要富集于与细胞应激应答、信号转导、物质代谢、生长分化等生物进程,说明LPS对bMEC的生理功能产生了较大的影响。KEGG Pathway分析发现,多个差异基因富集于NF-κB、MAPKs信号转导通路中,因此初步阐明LPS通过激活bMEC的NF-κB、MAPKs信号通路,诱导大量的炎性介质释放,导致了bMEC的炎性损伤。咖啡酸(10、25、50μg/mL)通过减少IκBa的降解和降低p65的磷酸化水平,抑制了bMEC的NF-κB的活性；通过降低p38、JNK、ERK1/2的磷酸化水平,抑制了细胞MAPKs信号通路的转导,从而减少了LPS诱导的bMEC的IL-8、IL-1β、IL-6和TNF-a的表达,减弱了LPS诱导的bMEC炎性损伤作用。更多还原

【Abstract】 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.更多还原