LPS联合抗菌药诱发鸡肝细胞损伤的分子机制与药物修复研究

Numerous antibacterials promote the release of LPS when they are utilized to prevent or cure negative bacteria infections. LPS and some antibacterials are both considered as factors that cause liver injury when they functioned respectively in previous studies. We have confirmed that the damaged dose of LPS decreased when enrofloxacin, florfenicol or amoxicillin/clavulanate potassium existed in primary chicken hepatocytes, and then half dose of single LPS damage combined with one of this three antibacterials mentioned above induced compound primary hepatocytes injury. Apoptosis is a key link in hepatocellular damage. In this study, on the basis of this three compound hepatocytes injury, we try to investigate (1) the incidence rate of hepatocellular apoptosis and the morphological change; (2) the expression of cytochrome C, Bax, Bcl-2 and the activation of caspase-3, -9, evaluating the relationship between mitochondrial dysfunction and hepatocellular apoptosis; (3) the expression of phosphor-JNK, phosphor-ERK and phosphor-p38, demonstrating the role of MAPK signaling pathway mediated apoptosis; (4) the hepatoprotective effect and mechanism of compound ammonium glycyrrhetate or silymarin. The aim of the study is to represent the pathological process of liver injury induced by LPS in combination with one of this three antibacterials and the protective mechanism of liver-protective drugs in cellular and molecular level, providing theoretical basis and technical support for cognizing the reason why poultry liver injury happens frequently and using antibiotics and liver-protective drugs more scientifically.

多类抗菌药防治阴性菌感染时促进LPS释放，LPS、抗菌药以往均作为独立肝损伤诱因研究。借助培养的鸡原代肝细胞，我们已确认恩诺沙星、氟苯尼考、阿莫西林/克拉维酸钾均能降低LPS致肝损伤剂量，且用半量的LPS致损量联合适量上述药物已成功诱发复合式肝损伤。细胞凋亡是造成肝损伤最基本的中心环节。本项目拟研究上述三种LPS/抗菌药肝损伤时肝细胞凋亡发生率及形态变化；检测cytochrome C，Bax和Bcl-2表达量，caspase-9，-3活性及蛋白表达评价线粒体功能障碍与凋亡关系；检测JNK、ERK、p38表达量及其磷酸化蛋白水平评价MAPK介导的凋亡通路在此损伤中的作用；评价复方甘草酸单铵、水飞蓟素分别对上述过程的修复效应。从细胞、分子水平阐明LPS联合抗菌药诱发肝损伤的病理演变及药物修复规律，为认知家禽临床多发的肝损伤病因，科学使用抗菌和护肝药，提供理论依据和技术支撑。

多类抗菌药防治阴性菌感染时促进LPS释放，LPS、抗菌药以往均作为独立肝损伤诱因研究，二者共存致肝损伤的病理机制仍需探讨。研究显示：细胞凋亡是造成肝损伤基本的中心环节，细胞凋亡往往伴随线粒体损伤，MAPK通路能调控细胞凋亡程序。本项目以此为切入点，藉鸡原代肝细胞分离培养技术，探讨LPS联合阿莫西林克拉维酸钾（AC）、恩诺沙星（ENR）或氟苯尼考（FFC）致鸡肝细胞损伤的分子机制及护肝药保护效应。结果显示：1.LPS以1/2较佳致损剂量与低浓度的AC、ENR或FFC联合，即可诱发明显鸡原代肝细胞损伤，提示LPS与低剂量抗菌药共存有增加诱发肝损伤的风险。2.LPS/AC、ENR或FFC均能诱发鸡肝细胞广泛凋亡，其凋亡机制可能由线粒体途径介导：电镜可见线粒体损伤，cytochrome C、caspase-9、caspase-3，Bax、Bcl-2等基因蛋白表达量明显变化。3.LPS联合抗菌药通过MAPK信号通路诱导的细胞凋亡可能因药物不同而异：LPS/ENR或FFC致鸡肝细胞凋亡中检测到JNK、ERK、p38的基因及其磷酸化蛋白表达量变化明显，而在LPS/AC致损研究中无明显变化。4.复方甘草酸单胺和水飞蓟素均表现良好的护肝效应，能有效降低LPS/抗菌药诱发的细胞凋亡，改善线粒体途径和MAPK信号通路相关分子在细胞凋亡中的相应变化。通过该项目研究，深入了解了LPS/抗菌药致鸡肝损伤的病理机制及复方甘草酸单胺和水飞蓟素的保护效应，为认知家禽肝损伤多发病因和科学用药，提供了理论和技术支撑。