沉默cFLIP在重症急性胰腺炎肺损伤中的作用机制研究  

Mechanism of silencing cFLIP in severe acute pancreatitis induced lung injury

作　　者：王宝枝[1] 彭和平[1] 张风华[1] 黄海霞 陈育宾[1] 杨学伟[1] WANG Baozhi;PENG Heping;ZHANG Fenghua;HUANG Haixia;CHEN Yubin;YANG Xuewei(Department of General Surgery,The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 511400,China)

机构地区：[1]广州医科大学附属第二医院番禺院区普外科,广东广州511400

出　　处：《肝胆胰外科杂志》2024年第3期161-167,共7页Journal of Hepatopancreatobiliary Surgery

基　　金：广东省医学科学技术研究基金项目(A2018087);西藏自治区自然科学基金组团式医学援藏项目(XZ2022ZR-ZY46(Z))。

摘　　要：目的探讨沉默细胞型Fas相关死亡区域蛋白样白介素-1β转换酶抑制蛋白(cFLIP)对重症急性胰腺炎(SAP)导致的肺损伤的影响及其可能的作用机制。方法分别取12只SD大鼠,随机分为对照组、cFLIPL(或cFLIPS)siRNA1组、cFLIPL(或cFLIPS)siRNA2组和cFLIPL(或cFLIPS)siRNA3组,筛选抑制率最高的cFLIPL siRNA和cFLIPS siRNA。50只SD大鼠随机分成假手术组、模型对照组、cFLIP siRNA-NC组、cFLIPS siRNA组、cFLIPL siRNA组,每组各10只。通过胰胆管内逆行注射3%牛磺胆酸钠溶液建立SAP模型,建模成功后,cFLIPS siRNA、cFLIPL siRNA和cFLIP siRNA-NC组大鼠尾静脉注射对应siRNA溶液,其余组注射等量0.9%NaCl溶液。HE染色检测肺组织病理学变化;ELISA检测IL-6、IL-1β和TNF-α含量;全自动分析仪检测静脉血白细胞数、中性粒细胞数;流式细胞术检测中性粒细胞凋亡;Western blotting检测中性粒细胞RIP1和caspase-8蛋白表达。结果cFLIPL siRNA2组以及cFLIPS siRNA1组干扰效率最明显,因此选择这2组进行后续实验(后续称为cFLIPL siRNA组和cFLIPL siRNA组)。与模型对照组大鼠相比,cFLIPL siRNA组和cFLIPS siRNA组大鼠肺泡结构损伤减轻,肺泡壁变薄,炎症细胞浸润减少;与模型对照组相比,cFLIPL siRNA组和cFLIPS siRNA组IL-6、IL-1β和TNF-α含量均明显降低,静脉血白细胞数、中性粒细胞数明显降低,中性粒细胞凋亡率明显升高,cFLIPL、cFLIPS和RIP1蛋白表达量明显降低,caspase-8蛋白表达量明显升高;以上差异均具有统计学意义(P<0.05)。结论本研究结果表明,靶向沉默cFLIP可能通过上调caspase-8和抑制RIP1的表达,促进中性粒细胞凋亡,减少炎症介质IL-6、IL-1β和TNF-α释放,抑制肺组织中的中性粒细胞浸润,缓解SAP导致的肺损伤,在SAP中发挥保护作用。Objective To explore the role and possible mechanism of silencing cellular FLICE-like inhibitory protein(cFLIP)in severe acute pancreatitis(SAP)induced lung injury.Methods Twelve SD rats were randomly divided into the control group,cFLIPL(cFLIPS)siRNA1 group,cFLIPL(cFLIPS)siRNA2 group,and cFLIPL(cFLIPS)siRNA3 group.The cFLIPL siRNA and cFLIPS siRNA with the highest inhibition rate were screened.Fifty SD rats were randomly divided into the sham surgery group,model control group,cFLIP siRNA-NC group,cFLIPS siRNA group,and cFLIPL siRNA group.A SAP model was established by retrograde injection of 3%sodium taurocholate solution into the pancreatic bile duct.After successful modeling,rats in the cFLIPS siRNA group,cFLIPL siRNA group,and cFLIP siRNA-NC group were injected with corresponding siRNA solution via the tail vein,while the remaining 2 groups were injected with an equal amount of 0.9%NaCl.HE staining was used to detect pathological changes in lung tissue;ELISA was used to detect the content of IL-6,IL-1βand TNF-α;The fully automatic analyzer was used to detect the number of white blood cells and neutrophils in venous blood;Neutrophil apoptosis was detected by flow cytometry;The expression of neutrophil RIP1 and caspase-8 proteins were detected by Western blotting.Results The cFLIPL siRNA2 group and cFLIPS siRNA1 group showed the most significant interference efficiency,so these 2 groups were selected for subsequent experiments.Compared with the model control group rats,the cFLIPL siRNA group and cFLIPS siRNA group rats showed reduced alveolar structural damage,thinning of alveolar walls,and reduced infiltration of inflammatory cells.Compared with the model control group,the cFLIPL siRNA group and cFLIPS siRNA group showed that the contents of IL-6,IL-1βand TNF-αwere significantly decreased,the numbers of white blood cells and neutrophils in venous blood were significantly decreased,the apoptosis rates of neutrophils were significantly increased,the expression levels of cFLIPL,cFLIPS,and RIP1 protein were si

关 键 词：细胞型Fas相关死亡区域蛋白样白介素-1β转换酶抑制蛋白(cFLIP) 重症急性胰腺炎 肺损伤 中性粒细胞 细胞凋亡 大鼠 

分 类 号：R576[医药卫生—消化系统] R563[医药卫生—内科学]