基于PI3K/Akt信号通路介导的细胞自噬探讨纳米铁抑制鸡肠炎沙门氏菌增殖的机制研究

How to suppress the proliferation of salmonella in chicks is a challenging issue in poultry industry. Our previous studies demonstrated that Fe3O4 nanoparticles (Fe3O4 NPs) could significantly decrease the content of Salmonella enterica serovar Enteritidis(S.Enteritidis) in hepatic cells of chicken(LMH). However, the molecular mechanism remains unknown. Autophagy is reported to be bounded up with the proliferation of bacteria in organism, while some studies revealed the degree of autophagy could be affected by Fe3O4 NPs via PI3K/Akt signal pathways. Therefore, we infer that Fe3O4 NPs regulate autophagy in LMH cells mediated by PI3K/Akt pathways, as a consequence, execute its modulatory effect on the suppression of S.Enteritidis. In order to ascertain this hypothesis, autophagy and PI3K/Akt signal pathways will be determined accompany with the changes in S.Enteritidis suppression that modulated by Fe3O4 NPs in LMH cells . The effect of signal pathways in above process will also be expounded in the PI3K/Akt inhibition/activation model. In vivo, the effect of Fe3O4 NPs on the suppression of S.Enteritidis in the liver of chicken will also be validated . This study will help to provide supportive and novel information on the prevention of salmonella in poultry production.

如何抑制鸡体内沙门氏菌的增殖仍然是当前家禽业一大科学难题，申请人前期研究发现纳米铁可有效抑制肠炎沙门氏菌（S.Enteritidis）在鸡肝细胞内的增殖，但其分子机制尚未明确。近年来研究表明沙门氏菌的增殖与细胞自噬密切相关，而纳米铁也被报道可通过PI3K/Akt信号通路对自噬程度进行调节。因此，项目组推测纳米铁通过PI3K/Akt信号通路对细胞自噬进行调节，进而发挥抑制S.Enteritidis增殖的作用。为验证该假说，本项目通过体外细胞试验研究纳米铁抑菌过程中细胞自噬和PI3K/Akt信号通路的变化规律，并通过构建鸡肝细胞的PI3K/Akt信号通路抑制/激动模型明确该通路的作用；再利用在体动物试验验证纳米铁对雏鸡肝脏感染S.Enteritidis的影响和PI3K/Akt信号通路介导的自噬在此过程中的作用，从而为禽类沙门氏菌的防治提供新思路。

本试验旨在研究Fe3O4-NPs对鸡肠炎沙门氏菌（S.Enteritidis）感染的抑制作用，以及PI3K/Akt/mTOR信号通路在该抑制过程中的作用。.体外试验：检测Fe3O4-NPs体外抑菌效果，观察S.Enteritidis形态。结果显示：Fe3O4-NPs可显著抑制培养基中S.Enteritidis的增殖，使其细胞壁明显皱缩。.细胞试验：建立鸡肝癌细胞系（LMH）感染S.Enteritidis模型，检测Fe3O4-NPs添加后LMH细胞中自噬、ROS及信号通路变化，并分别使用抑制剂后检测其抑菌效果。结果显示：（1）Fe3O4-NPs的添加未升高自噬水平，显著提高ROS水平，使用自噬抑制剂和ROS清除剂后Fe3O4-NPs的抑菌效果减弱；（2）共定位技术拍摄到Fe3O4-NPs和S.Enteritidis共同存在于自噬泡中；（3）PI3K/Akt/mTOR信号通路表达升高，使用抑制剂后Fe3O4-NPs的抑菌效果未变化，但细胞活性减弱。.动物试验：建立雏鸡感染S.Enteritidis模型，检测Fe3O4-NPs的抑菌效果，对肝脏进行病理分析，检测肝脏抗氧化酶和炎症因子的活性，检测肝脏自噬和ROS的变化规律，检测信号通路的变化规律。结果显示：（1）Fe3O4-NPs能有效抑制S.Enteritidis的增殖；（2）有效缓解感染S.Enteritidis后肝脏的病理损伤、氧化应激状态和炎症反应；（3）抑菌过程中肝脏自噬和ROS表达升高；（4）PI3K/Akt/mTOR信号通路表达量升高。.综上所述，Fe3O4-NPs 可通过提高胞内ROS水平来提高胞内自噬泡的抑菌作用，并缓解S.Enteritidis感染导致的机体损伤。PI3K/Akt/mTOR信号通路在此过程中起关键作用。Fe3O4-NPs可作为目前应对胞内S.Enteritidis感染的潜在替抗产品。