【作者】 高瑞峰；

【导师】 张乃生；

【作者基本信息】 吉林大学 ， 临床兽医学， 2014， 博士

【摘要】 大肠杆菌等革兰氏阴性菌是乳腺炎常见的致病菌。这些革兰氏阴性菌中，能够使宿主机体组织器官受到损伤的主要致病因子是脂多糖（LPS）。LPS是革兰氏阴性菌细胞壁的特殊成分，其能够激活TLR4（Toll-like receptor4）信号转导通路，进而使宿主机体产生炎性反应，过氧化物酶增殖物激活受体（PPAR-γ）在这个过程中能够起到重要的调控作用。本课题以我国中草药中存在的天然成分绿原酸为研究对象，首先在小鼠乳腺炎模型上证实其对炎症的抑制效果，其次分别对绿原酸在小鼠乳腺上皮细胞和奶牛乳腺上皮细胞上抗炎机制进行研究，最后对其作用靶标进行初步确认，从而为兽医临床实践中抗炎治疗提供新思路，为相应抗炎药物的研发提供初步的实验数据和理论基础。首先，通过LPS诱发小鼠来建立乳腺炎损伤模型。我们选取18只Balb/C小鼠，雌雄比例2:1，混养后自由交配受孕，雌鼠在泌乳5～7天后，将其随机分为2组，LPS刺激组和空白对照组，6只/组。LPS刺激组雌鼠在其第四对乳头处分别灌注LPS50μL（0.2mg/mL），在灌注24h后将其脱颈处死；空白对照组的雌鼠不进行任何处理。通过肉眼观察LPS刺激组雌鼠的乳腺组织的病理学变化；对获得的雌鼠乳腺组织分别制作组织病理学切片和MPO的免疫组织化学切片，观察乳腺的病理组织学变化和MPO在组织中的分布及活性；通过ELISA方法检测乳腺组织中TNF-α因子的表达水平。实验结果表明，与空白对照组相比，肉眼观察到LPS刺激组雌鼠的乳腺组织出现明显的发红、肿胀、充血等病理学变化；组织病理学光学显微镜观察到乳腺的腺泡壁明显增厚、水肿，腺泡腔存在大量的嗜中性粒细胞；乳腺中MPO的分布与活性明显增加；乳腺中TNF-α因子的表达水平显著增多。以上获得的数据与典型乳腺炎的指标完全一致，说明本实验成功建立了LPS诱发的小鼠乳腺炎损伤模型。其次，利用所建立的LPS诱发小鼠乳腺炎损伤的模型，研究绿原酸对乳腺炎的保护效果及其作用机理。我们将获得的乳腺炎损伤模型雌鼠随机分为6组，12只/组，分别为LPS刺激组，LPS+绿原酸低、中、高（12.5、25、50mg/kg体重），LPS+DEX组（5.0mg/kg体重，阳性对照）和空白对照组。在LPS灌注前1h和灌注后12h分别通过腹腔注射绿原酸和DEX。观察乳腺组织的病理学变化和MPO的分布与活性；ELISA方法检测乳腺组织中TNF-α、IL-1β和IL-6等细胞因子的表达水平；Western blot方法检测绿原酸对TLR4和NF-κB信号通路的影响。实验结果显示，与LPS刺激组相比，绿原酸能够显著降低LPS诱发的小鼠乳腺组织的炎性损伤；减少MPO在乳腺组织中的分布，且MPO的活性也显著降低；TNF-α、IL-1β和IL-6细胞因子的蛋白表达水平也显著降低；TLR4和NF-κB信号通路的激活也受到绿原酸的抑制。以上实验结果证明绿原酸通过对TLR4和NF-κB信号通路的激活的抑制作用，实现对LPS诱发的小鼠乳腺炎损伤的保护作用。再次，我们为进一步验证绿原酸对乳腺炎损伤的保护效果，对小鼠乳腺上皮细胞进行了分离培养，研究绿原酸对其保护作用及其作用机理。利用MTT方法检测绿原酸对小鼠乳腺上皮细胞的毒性作用；ELISA方法检测绿原酸对小鼠乳腺上皮细胞中TNF-α和IL-6细胞因子表达水平的影响。Western blot方法检测绿原酸对小鼠乳腺上皮细胞中NF-κB信号通路的影响。实验结果表明，绿原酸在12.5、25、50μg/mL的浓度下不会对小鼠乳腺上皮细胞产生毒性作用；绿原酸预处理细胞后能够明显降低TNF-α和IL-6蛋白表达水平；Western blot证实绿原酸是通过抑制小鼠乳腺上皮细胞中NF-κB信号通路的激活而发挥其抗炎作用的。然后，我们又成功分离培养了奶牛乳腺上皮细胞，进一步研究绿原酸对乳腺炎本源动物细胞的保护作用及其作用机理。利用MTT方法检测绿原酸对奶牛乳腺上皮细胞的毒性作用； qRT-PCR方法检测绿原酸对奶牛乳腺上皮细胞中TNF-α、IL-1β和IL-6细胞因子表达水平的影响；Western blot方法检测绿原酸对奶牛乳腺上皮细胞中TLR4信号通路的影响。实验结果表明，绿原酸在12.5、25、50μg/mL的浓度下不会对奶牛乳腺上皮细胞产生毒性作用；绿原酸预处理细胞后能够明显降低TNF-α，IL-1β和IL-6蛋白表达水平；Western blot证实绿原酸是通过抑制小鼠乳腺上皮细胞中TLR4信号通路的激活而发挥其抗炎作用的。最后，我们对绿原酸的抗炎作用靶点进行了初步确定。通过PPAR-γ配体结合实验和Western blot方法检测绿原酸对PPAR-γ的结合和激活作用情况。选用PPAR-γ特异性拮抗剂GW9662来阻断PPAR-γ激活作用，再检测绿原酸对LPS诱发的TLR4表达的影响。实验结果显示，绿原酸能够与PPAR-γ结合并激活，同时发现绿原酸对LPS诱发的TLR4表达的影响也能够被PPAR-γ特异性拮抗剂GW9662所阻断，表明绿原酸通过结合并激活PPAR-γ而发挥抗炎作用。因此，通过实验我们初步确定PPAR-γ可能是绿原酸在体内的抗炎作用靶点。本实验对绿原酸的抗炎作用机理和作用靶标进行了初步探索性研究，发现绿原酸能够结合并激活PPAR-γ，从而使TLR4下调表达并对下游的NF-κB信号通路的激活起到抑制作用，最终使乳腺中TNF-α、IL-1β和IL-6等炎性因子的基因和蛋白表达水平均下降。以上实验结果提示，绿原酸在奶牛乳腺炎的治疗中可能具有潜在的应用前景。更多还原

【Abstract】 Gram-negative bacteria such as E.coli is the major pathogenic bacteria thatcauses bovine mastitis. LPS, the main component of the outer membranes ofGram-negative bacteria, plays a key role in producing an inflammatory response. LPScould activate TLR4signaling pathway and induce inflammatory response. Studiesshowed that PPAR-γ played an important role in inflammatory response. In this study,we first confirmed the anti-inflammatory effects of chlorogenic acid in LPS-inducedmastitis in mice. Secondly, we investigated the anti-inflammatory mechanism ofchlorogenic acid on LPS-stimulated bovine and mouse mammary epithelial cells. Inthe last, the molecular targets of the anti-inflammatory actions of chlorogenic acidwere identified. All the results will provide the reference and experimental basis forprevention and treatment of bovine mastitis by anti-inflammatory medicine inveterinary clinical practice.Firstly, the mouse mastitis model induced by LPS was established. The lactatingBalb/C mice of5-7d were random divided into two groups: control group and LPSgroup. The fourth pair mammary glands of the mice were infusion with LPS(0.2mg/ml,50μL) through the mammary duct.24h after LPS induction, the mice werekilled. The pathological and histopathological changes of mammary gland wereobserved. Meanwhile, the activity of MPO and the level of TNF-α was detected. Theresults showed that the mammary gland showed redness, swelling, and congestion inLPS group. Meanwhile, the mammary histological examination also indicated thatmammary tissues infused with LPS showed thickening and edema of the alveoluswalls and inflammatory cells infiltration. In addition, the level of TNF-α wassignificantly increased in LPS group. These results demonstrated that the mousemastitis model induced by LPS was successfully established.Secondly, the mouse model of mastitis was induced by injection of LPS throughthe duct of mammary gland. Chlorogenic acid was administered intraperitoneally withthe dose of12.5,25,50mg/kg respectively1h before and12h after induction of LPS. In this study, the effect of chlorogenic acid on LPS-induced mice mastitis wasassessed through histopathological examination, myeloperoxidase (MPO) activityanalysis, ELISA assay, and western blot analysis. The results showed that chlorogenicacid significantly attenuated the activity of MPO and reduced TNF-α, IL-1β, and IL-6production compared with LPS group. Besides, western blot analysis showed thatchlorogenic acid could inhibit the expression of TLR4and the phosphorylation ofNF-κB and IκB induced by LPS. These results suggested that anti-inflammatoryeffects of chlorogenic acid against LPS-induced mastitis may be due to its ability toinhibit TLR4-mediated NF-κB signaling pathway. Therefore, chlorogenic acid may bea potent therapeutic reagent for the prevention of mastitis.Thirdly, to further detect the protective effect of chlorogenic acid on mastitis, themouse mammary epithelial cells were used. The cells were stimulated with LPS in thepresence or absence of chlorogenic acid. The expression of pro-inflammartorycytokines TNF-α, IL-6and IL-1β were determined by ELISA. NF-κB and IκBα weredetermined by Western blotting. The results showed that chlorogenic aciddose-dependently inhibited the expression of TNF-α, IL-6and IL-1β inLPS-stimulated mouse mammary epithelial cells. Western blot analysis showed thatchlorogenic acid suppressed LPS-induced NF-κB activation, IκBa degradation.We also detected the protective effect of chlorogenic acid on LPS-stimulatedbovine mammary epithelial cells. The cells were stimulated with LPS in the presenceor absence of chlorogenic acid. The expression of pro-inflammartory cytokines TNF-α,IL-6and IL-1β were determined by qRT-PCR. TLR4, NF-κB and IκBα weredetermined by Western blotting. The results showed that chlorogenic aciddose-dependently inhibited the expression of TNF-α, IL-6and IL-1β inLPS-stimulated bovine mammary epithelial cells. Western blot analysis showed thatchlorogenic acid suppressed LPS-induced TLR4signaling pathway.In the last, molecular targets of chlorogenic acid was identified. The bindingaffinity and activating of PPAR-γ were detected by PPAR-γ ligand binding assays andWestern blotting. Meanwhile, to detect whether the effects of chlorogenic acid onTLR4expression is dependent on PPAR-γ activation, GW9662, the specific PPAR-γantagonist was used. Then the cells were stimulated by LPS and TLR4expression was detected. The results showed that chlorogenic acid could bind and activate PPAR-γ.Furthermore, the inhibition effects of TLR4by chlorogenic acid can be reversed byGW9662, a specific antagonist for PPAR-γ. These results suggest that PPAR-γ may bethe anti-inflammatory targets of chlorogenic acid.In conclusion, these results suggest that chlorogenic acid activates PPAR-γ,thereby attenuating TLR4expression and TLR4mediated NF-κB activation and therelease of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. These findings suggestthat chlorogenic acid may be a therapeutic agent against inflammatory diseases. Allthe results revealed that emodin may be used as a potential candidate in treatment ofmastitis.更多还原