咖啡酸对LPS诱导奶牛乳腺上皮细胞损伤的保护机制

Bovine mammary epithelial cells (BMEC) are susceptible to damage during bovine mastitis. Jin-Pu-Guan-Zhu-Ji is a pure Chinese medicinal preparation for treatment of acute mastitis. Previous research has shown that LPS could obviously decrease the mitochondrial membrane potential of BMEC, induce the accumulation of reactive oxygen species (ROS) and a large release of proinflammatory cytokine, resulting in the injury of cell morphological structure. Caffeic acid (the main active component of the preparation) can effectively alleviate the LPS-induced structural damage and apoptosis, but the mechanism is unclear. Based on the pre-established LPS-induced BMEC structural damage model, the cells were pretreated with the inhibitor of Nox and the scavenger of ROS to research the role of Nox/ROS signaling in BMEC injure induced by LPS. Then according to the use of specific signal transduction inhibitors and stimulators, immunoblot assays and real-time quantitative PCR analyses will be performed on BMEC to assess key signaling molecules expression levels in Nox/ROS signal, NF-κB and Nrf2 signaling pathways, cell apoptosis and the localization of nuclear transcription factors within the cell will be identified by flow cytometry and confocal fluorescent microscopy, respectively. Then to explore the intervention effect of caffeic acid. Therefore the molecular mechanism of BMEC injure induced by LPS and the targets of caffeic acid will be clarified. Furthermore, this project will contribute significantly to provide theoretical basis for the clinical application of the preparation.

奶牛乳腺上皮细胞（BMEC）在奶牛乳房炎发生过程中易受损害。“金蒲灌注剂”是申请者参与研发的治疗急性奶牛乳房炎的纯中药制剂。前期研究证实LPS诱导BMEC线粒体膜电位明显降低、胞内ROS蓄积和促炎性因子大量释放，破坏细胞形态结构；咖啡酸（该制剂主要活性成分）可有效减轻LPS诱导的细胞结构损伤和凋亡，但其作用机制尚不明确。本项目在前期建立的LPS诱导BMEC损伤模型的基础上，给予Nox抑制剂、ROS清除剂处理来研究Nox/ROS信号在LPS致细胞损伤中的作用；然后结合特异性信号转导抑制剂、激动剂的使用，采用免疫印迹、荧光定量PCR、流式细胞术及激光共聚焦等技术检测Nox/ROS信号、NF-κB信号通路、Nrf2信号通路中关键信号分子的表达、细胞凋亡及胞内核转录因子的定位情况，并探讨咖啡酸的干预效应，以此阐明LPS致BMEC损伤的分子机制及咖啡酸的作用靶点，为该制剂的临床应用提供一定理论依据。

本研究首先优化了原代奶牛乳腺上皮细胞（bMEC）的培养方法，采用“一步胶原酶法”简单、快捷的制备了具有良好细胞活力的原代奶牛乳腺上皮细胞。在前期建立的LPS诱导bMEC结构损伤模型的基础上，利用NADPH氧化酶（Nox）抑制剂DPI和ROS清除剂NAC探讨Nox-ROS信号在LPS诱导的细胞炎症、凋亡中的作用。结果表明，LPS刺激bMEC 12 h后，细胞内ROS蓄积显著增加，线粒体膜电位显著降低，但在LPS刺激初期细胞内ROS并未显著增加，随着细胞线粒体膜电位的下降，细胞内ROS生成不断增多，说明细胞内ROS的蓄积主要源于线粒体功能紊乱，且LPS诱导的bMEC凋亡作用主要通过线粒体凋亡途径。提前孵育NAC、DPI后，LPS诱导的细胞炎症、凋亡明显减轻，说明Nox-ROS信号在LPS诱导bMEC结构损伤过程中发挥了重要作用，ROS在反应初期发挥了第二信使的作用。相较于bMEC，LPS可引起RAW264.7细胞内ROS迅速生成、线粒体膜电位的迅速降低，炎性因子的大量释放，导致细胞凋亡；NAC、DPI可显著抑制细胞内ROS的蓄积，减少炎性因子的释放，减少细胞的凋亡，说明Nox-ROS信号在bMEC、RAW264.7内的功能有差异。.LPS激活了bMEC、RAW264.7的NF-κB、Nrf2信号通路，NF-κB活化可调控细胞产生大量的炎症介质及ROS，促进细胞炎症、氧化应激反应，而活化的Nrf2可调控细胞产生大量的抗炎、抗氧化物质，抑制细胞的炎症、氧化应激反应，但本研究中Nrf2的调控作用并未抑制NF-κB调控的炎症反应及细胞凋亡作用。本研究中咖啡酸（CA）能够显著抑制LPS诱导的bMEC、RAW264.7细胞内ROS的蓄积，抑制了细胞NF-κB信号通路活性、增强了Nrf2信号通路活性，从而抑制了LPS诱导的细胞炎症、氧化应激反应，保护了细胞线粒体膜结构及膜电位，抑制了细胞的凋亡作用，作用效果同NAC、DPI类似。说明Nox-ROS信号对细胞NF-κB、Nrf2信号通路具有调节作用；咖啡酸可抑制LPS激活的bMEC Nox-ROS信号，并调节细胞NF-κB、Nrf2信号通路的活化，发挥保护bMEC结构和功能的作用。通过试验证明，CA能够明显抑制LPS诱导的小鼠乳腺炎症、氧化应激，对乳腺结构发挥了保护作用，因此，该研究为CA在大肠杆菌型奶牛乳房炎的应用提供了参考依据。