程序性坏死参与油酸诱导的大鼠急性呼吸窘迫综合征的发病过程（英文）  

Activation of necroptosis in a rat model of acute respiratory distress syndrome induced by oleic acid

作　　者：潘龙[1,2] 姚敦琛 余雨中 陈秉钧[1,2] 李晟杰 胡桂和 奚畅 王梓晖 李建华[1] 龙捷[5] 涂永生[1] PAN Long YAO Dun-Chen YU Yu-Zhong CHEN Bing-Jun LI Sheng-Jie HU Gui-He XI Chang WANG Zi-Hui LI Jian-Hua LONG Jie TU Yong-Sheng(Department of Physiology, School of Basic Sciences The Third Clinical Medical College The First Clinical Medical College The Second Clinical Medical College Department of Pathology, School of Basic Sciences, Guangzhou Medical University, Guangzhou 511436, China)

机构地区：[1]广州医科大学基础学院生理教研室,广州511436 [2]广州医科大学第三临床学院,广州511436 [3]广州医科大学第一临床学院,广州511436 [4]广州医科大学第二临床学院,广州511436 [5]广州医科大学基础学院病理教研室,广州511436

出　　处：《生理学报》2016年第5期661-668,共8页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.81470205);National College Students’Innovation Entrepreneurship Training Plan Program of China(No.2015105700010);the Guangzhou Medical University College Students Science and Technology Innovation Project,China(No.2014A015);the Medical Scientific Research Foundation of Guangdong Province,China(No.A2016382);Guangzhou City-belonged Universities Scientific Research Program,China(No.2012C130);the Foundation for Excellent Teachers by Guangzhou Medical University,China

摘　　要：本研究旨在明确程序性坏死在急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)发病中的作用。通过尾静脉注射油酸(oleic acid,OA)制备大鼠ARDS模型,并观察4 h。通过动脉血气分析、肺干湿重比(lung wet-dry weight ratio,W/D)、肺组织HE染色及肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中总蛋白测定、白细胞计数及分类计数来评估ARDS模型。通过ELISA检测BALF中肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)水平。通过免疫组化和蛋白免疫印迹观察受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)、RIPK3、mixed lineage kinase domain-like protein(MLKL)在肺组织中的表达水平。通过免疫沉淀观察RIPK1和RIPK3之间的相互作用。结果显示,OA注射4 h后,与对照组比较,OA组大鼠肺泡-动脉氧分压差[P(A-a)O2]、W/D、BALF中白细胞总数、中性粒细胞比例、蛋白浓度及TNF-α水平均显著上升,而氧合指数(Pa O2/Fi O2)下降;OA组大鼠肺组织中RIPK1、RIPK3、MLKL表达明显增加,且RIPK1与RIPK3之间的相互作用显著增强。以上结果表明,在ARDS的发生、发展过程中,TNF-α分泌增加,RIPK1/RIPK3/MLKL信号通路被激活并表达上调,提示程序性坏死可能在ARDS的发病机制中发挥作用,这可能为治疗ARDS的新药开发提供新思路。型。通过ELISA检测BALF中肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)水平。通过免疫组化和蛋白免疫印迹观察受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)、RIPK3、mixed lineage kinase domain-like protein(MLKL)在肺组织中的表达水平。通过免疫沉淀观察RIPK1和RIPK3之间的相互作用。结果显示,OA注射4 h后,与对照组比较,OA组大鼠肺泡-动脉氧分压差[P(A-a)O_2]、W/D、BALF中白细胞总数、中性粒细胞比例、蛋白浓度及TNF-α水平均显著上升,而氧合指数(Pa O_2/Fi O_2)下降;OA组大鼠肺组织中RIPK1、RIPK3、MLKL表达明显增加,且RIPK1�The present study was aimed to investigate the role of necroptosis in the pathogenesis of acute respiratory distress syn- drome （ARDS）. The rat model of ARDS was induced by intravenous injection of oleic acid （OA）, and observed for 4 h. The lung injury was evaluated by arterial blood gas, lung wet-dry weight ratio （W/D） and histological analyses. Simultaneously, bronchoalveolar lavage fluid （BALF） was collected for total and differential cell analysis and total protein determination. Tumor necrosis factor alpha （TNF-α） level in BALF was determined with a rat TNF-α ELISA kit. Expressions of receptor interacting protein kinase 1 （RIPK1）, RIPK3 and mixed lineage kinase domain-like protein （MLKL） in lung tissue were determined by Western blot and immunohistochemical staining. The interaction between RIPK1 and RIPK3 was explored by immunoprecipitation. The results showed that, compared with those in control group, total white blood cells count （WBC）, polymorphonuclear percentage （PMN%）, total protein concentration, TNF-α level in BALF, W/D, and the alveolar-arterial oxygen tension difference （P（A-a）O2） in OA group were significantly increased at 4 h after OA injection. Western blot and immunostaining further showed remarkably increased expressions of RIPK1, R/PK3 and MLKL in lung tissue from OA group. Additionally, immunoprecipitation results indicated an enforced interaction between RIPK1 and RIPK3 in OA group. Collectively, the TNF-α level in BALF and the RIPKI-RIPK3-MLKL signaling pathway in lung tissue were found to be upregulated and activated with the process of ARDS. These findings implicate that RIPK1/RiPK3-mediated necroptosis plays a possible role in the pathogenesis of ARDS, which may provide a new idea to develop novel drugs for the therapy of ARDS.

关 键 词：急性呼吸窘迫综合征 程序性坏死 肿瘤坏死因子Α 受体相互作用蛋白激酶1 受体相互作用蛋白激酶3 MLKL 

分 类 号：R563.8[医药卫生—呼吸系统]