芳樟醇通过激活Nrf-2信号通路抑制小鼠多杀性巴氏杆菌肺炎

发布时间：2018-05-05 10:07

本文选题：芳樟醇 + 细胞因子　；参考：《吉林大学》2015年硕士论文

【摘要】：多杀性巴氏杆菌肺炎是在兽医临床实践中极为常见的一种疾病，并会导致非常高的发病率和死亡率。为解决这一全球性难题，过去数年中一些新发现的抗生素被陆续投入到临床使用，但是细菌性肺炎的致死率并没有降低。对此一个重要的原因就是病原菌引发的炎症反应会失去控制以致急性肺损伤或急性呼吸窘迫症的发生。虽然炎症是机体对抗病原菌刺激时的保护防御反应，但是大量的白细胞与肺组织固有细胞激活会导致严重的肺组织损伤，甚至导致多器官功能衰竭，对人类和动物的生命健康都造成了极大地危害。目前传统的化学合成药物毒副作用大、不能长期连续使用而且细菌耐药性也在不断增加。因此，急需寻找和开发疗效好并且副作用小的抗炎药物。而中药有着来源广、毒性小和作用范围广等优点，将其开发应用到医学临床已经成为当今热点课题。芳樟醇，几乎存在于所有的天然植物香料中，属于链状萜烯醇类物质。已有研究表明芳樟醇能够抑制脂多糖引起的急性肺损伤，但是关于其对多杀性巴氏杆菌肺炎作用的相关报道较少。本实验从体外和体内两个方面研究芳樟醇通过调节Nrf-2信号转导通路从而在一定程度上抑制多杀性巴氏杆菌引起的肺炎。首先，将不同浓度的芳樟醇加入到人肺腺癌上皮细胞（A549细胞）中，Western Blot检测不同浓度的芳樟醇是否能够激活Nrf-2信号通路；并借助凝胶迁移或电泳迁移率实验（EMSA)进一步验证上述结果。然后，我们应用RNA干扰技术敲降Nrf-2的表达，研究Nrf-2是否参与到多杀性巴氏杆菌引起的炎症反应中。在此基础上，构建多杀性巴氏杆菌诱发的革兰氏阴性菌肺炎模型，检测不同浓度的芳樟醇对肺炎小鼠的治疗效果；并收集肺组织以检测湿重干重（W/D）比率、髓过氧化物酶（MPO）活性、超氧化物歧化酶（SOD)活性、活性氧（ROS）含量、炎性细胞因子浓度和观察病理组织学的变化。体外实验结果表明，芳樟醇能够增加细胞核中Nrf-2的含量，促进抗氧化因子血红素加氧酶-1（HO-1）的分泌，，并呈浓度依赖性增强；多杀性巴氏杆菌感染A549细胞后，促炎性细胞因子TNF-a和IL-6分泌量显著增加，而芳樟醇能够较明显的抑制它们的分泌量，但这种抑制效果在Nrf-2敲降的细胞中极显著的下降，这表明在多杀性巴氏杆菌诱发的炎症中，Nrf-2是一个重要的调控因子，并且芳樟醇的治疗作用也依赖于对Nrf-2的作用。体内实验结果显示，在多杀性巴氏杆菌诱发的革兰氏阴性菌肺炎模型中，芳樟醇能够减少小鼠的死亡率，治疗效果呈浓度依赖性增强；并且能够显著降低小鼠肺组织中的MPO和ROS的活性以及小鼠肺脏的湿重干重比率，抑制TNF-a和IL-6的分泌，增加SOD的活性，病理切片表明芳樟醇明显地减弱了多杀性巴氏杆菌导致的肺损伤。综上所述，芳樟醇可以通过其抗氧化活性发挥抗炎作用，并有可能作为一种新的抗炎药物来治疗与炎症相关的疾病。
[Abstract]:Many new antibiotics have been put into clinical use in the past few years , but the death rate of bacterial pneumonia has not been reduced .

Lincamphol , which is almost present in all natural plant spice , belongs to a chain - like terpene alcohol substance , and has been found to be capable of inhibiting acute lung injury caused by lipopolysaccharide . However , there are few related reports about its effect on multi - kill pasteurellosis .

In vitro and in vivo two aspects were studied in vitro and in vivo by regulating the pathway of Nrf - 2 signal transduction pathway to some extent to inhibit the pneumonia caused by multocida . First , the different concentrations of linalyl alcohol were added to human lung adenocarcinoma epithelial cells ( A549 cells ) , and the Western Blot was used to detect whether linalyl alcohol in different concentrations could activate the Nrf - 2 signal pathway .
The results were further verified by means of gel migration or electrophoretic mobility experiment ( EMSA ) . Then , we used RNA interference technique to knock down Nrf - 2 expression and investigate whether Nrf - 2 was involved in the inflammatory reaction caused by Pasteurellosis .
The lung tissue was collected to detect the ratio of wet weight dry weight ( W / D ) , myeloperoxidase ( MPO ) activity , superoxide dismutase ( SOD ) activity , reactive oxygen ( ROS ) content , inflammatory cytokine concentration and observed pathological histology .

In vitro experiment , the content of Nrf - 2 in the nucleus was increased , the secretion of heme oxygenase - 1 ( HO - 1 ) was promoted and the concentration - dependent enhancement was promoted .
The results showed that Nrf - 2 was an important regulator in the treatment of Gram - negative bacteria - induced inflammation .
and the activity of TNF - a and IL - 6 can be remarkably reduced , the secretion of TNF - a and IL - 6 is inhibited , the activity of SOD is increased , and the pathological section shows that the linalyl alcohol obviously reduces the lung injury caused by the multocida . Therefore , the linalyl alcohol can exert anti - inflammatory effect through the antioxidant activity of the mouse , and can be used as a new anti - inflammatory drug for treating diseases related to inflammation .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7

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