Casp-8通过调控NLR...减轻脓毒症引起的急性肺损伤_翟延评.pdf

东南大学学报(医学版)J Southeast Univ(Med Sci Edi)2023，Feb;42(1):64-71thelial cells with autophagy-mediated protection against albu-min-induced injury J Exp Cell es，2018，370(2):198 15完颜萍萍 慢性间歇性缺氧对肾脏纤维化的影响及相关机理研究 D 甘肃:兰州大学，2020 16LIU M，NING X，LI，et al Signalling pathways involved inhypoxia-induced renal fibrosis J J Cell Mol Med，2017，21(7):1248-1259 17FENANDEZ-PATON C，LEUNG D Emergence of a metal-loproteinase/phospholipase A2 axis of systemic inflammation J Metalloproteinases Med，2015(2):29-38 18YU W，KE X，LI M，et al Downregulation of AB7 and cave-olin-1 increases MMP-2 activity in renal tubular epithelialcells under hypoxic conditions J Open Med(Wars)，2021，16(1):1428-1437 19XU Q，LIU L，YANG Y，et al Effects of ab7 gene up-regula-tion on renal fibrosis induced by unilateral ureteral obstruc-tion J Braz J Med Biol es，2020，53(4):e9220 20YANG Y，WANG J，ZHANG Y，et al Hypoxic tubular epithe-lial cells regulate the angiogenesis of HMEC-1 cells via medi-ation of ab7/MMP-2 axis J Aging，2021，13(20):23769-23779 21MIKI T，TAKEGAMI Y，OKAWA K，et al The reversion-in-ducing cysteine-rich protein with Kazal motifs(ECK)inter-acts with membrane type 1 matrix metalloproteinase andCD13/aminopeptidase N and modulates their endocytic path-ways J J Biol Chem，2007，282(16):12341-12352收稿日期2022-08-29 修回日期2022-11-12基金项目广东省医学科研基金项目(B2021139);广州市卫生健康科技一般引导项目(20221A010070)作者简介翟延评(1987 )，女，山东曲阜人，主治医师。E-mail:zhaiyanping0426163 com引文格式翟延评，何耀军，潘嘉宇，等 Casp-8 通过调控 NLP3 炎性小体介导的细胞焦亡减轻脓毒症引起的急性肺损伤J 东南大学学报(医学版)，2023，42(1):64-71 论著 Casp-8 通过调控 NLP3 炎性小体介导的细胞焦亡减轻脓毒症引起的急性肺损伤翟延评，何耀军，潘嘉宇，王志敏，张春云(广州医科大学附属第五医院 ICU，广东高校生物靶向诊治与康复重点实验室，广东 广州510700)摘要目的:分析半胱氨酸天冬氨酸蛋白水解酶-8(Casp-8)对脓毒症引起的急性肺损伤(ALI)的作用及其相关分子机制。方法:对 40 只 C57BL/6 小鼠使用盲肠结扎穿孔术(CLP)建立脓毒症诱导的小鼠 ALI 模型，将造模成功的小鼠随机分为模型组(CLP 组)和 CLP+Casp-8 抑制剂组(CLP+Z-IETD-FMK 组)，每组20 只。另取 20 只正常饲养的小鼠设为假手术组(sham 组)。采用称质量法测定小鼠肺组织湿干质量比值(W/D)，HE 染色观察小鼠肺组织病理变化，TUNEL 染色检测肺组织细胞凋亡情况，ELISA 检测小鼠支气管肺泡灌洗液(BALF)中炎症因子水平，qT-PC 检测肺组织中 Casp-8 mNA 表达，Western blotting 检测小鼠肺组织中Casp-8 蛋白、核苷酸结合寡聚化结构域样受体蛋白 3(NLP3)炎性小体及细胞焦亡相关蛋白表达。结果:与sham 组比较，CLP 组小鼠 24 h 存活率明显降低(P 0 05);小鼠肺组织充血水肿，肺泡结构严重破坏，肺间质内大量炎症细胞浸润;肺组织 W/D，细胞凋亡率，BALF 中肿瘤坏死因子-(TNF-)、白细胞介素(IL)-6、IL-1、IL-18 水平均显著升高(P 0 05);肺组织中 Casp-8 mNA 和蛋白表达水平，NLP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、切割蛋白 D(GSDMD)蛋白表达水平均显著升高(P 0 05)。与 CLP 组比较，CLP+Z-IETD-FMK 组小鼠存活率明显升高(P 0 05);小鼠肺泡结构破坏减轻，肺部炎症细胞浸润减少;肺组织 W/D，细胞凋亡率，BALF 中 TNF-、IL-6、IL-1、IL-18 水平均显著降低(P 460.05);肺组织中 Casp-8 mNA 和蛋白表达水平，NLP3、ASC、Caspase-1、GSDMD 蛋白表达水平均显著降低(P 0 05)。结论:Casp-8 通过调控 NLP3 炎性小体介导的细胞焦亡减轻脓毒症引起的 ALI。关键词Casp-8;脓毒症;急性肺损伤;NLP3 炎性小体;细胞焦亡;细胞凋亡 中图分类号631 2 文献标志码A 文章编号1671-6264(2023)01-0064-08doi:10 3969/j issn 1671-6264 2023 01 009Casp-8 attenuates sepsis-induced acute lung injury through regulation ofNLP3 inflammasome-mediated pyroptosisZHAI Yanping，HE Yaojun，PAN Jiayu，WANG Zhimin，ZHANG Chunyun(ICU，Key Laboratory of Biological Targeted Diagnosis，Treatment and ehabilitation，Guangdong University，the Fifth AffiliatedHospital of Guangzhou Medical University，Guangzhou 510700，China)AbstractObjective:To analyze the effect of cysteine aspartate proteolytic enzyme-8(Casp-8)on sepsis-inducedacute lung injury(ALI)and its related molecular mechanism Methods:Forty C57BL/6 mice were used to estab-lish sepsis-induced ALI model by cecal ligation and puncture(CLP)The successfully modeled mice were randomlydivided into model group(CLP group)and CLP+Casp-8 inhibitor group(CLP+Z-IETD-FMK group)，twenty ineach group Another twenty normal mice were divided into sham operation group(sham group)The wet/dry weightratio(W/D)of lung tissue was measured by weighting method，the pathological changes of lung tissue were ob-served by HE staining，the apoptosis of lung tissue cells was detected by TUNEL staining，the levels of inflammato-ry cytokines in bronchoalveolar lavage fluid(BALF)were detected by ELISA，the expression of Casp-8 mNA inlung tissue was detected by qT-PC，and the expression of Casp-8 protein，nucleotide binding oligomerization do-main like receptor protein 3(NLP3)inflammasome and apoptosis-related protein were detected by Western blot-ting esults:Compared with the sham group，the 24 h survival rate of mice in CLP group was significantly de-creased(P 0 05)The lung tissue of mice was congested and edematous，the alveolar structure was seriouslydamaged，and a large number of inflammatory cells were infiltrated in the lung stroma Lung W/D，apoptosis rate，the levels of tumor necrosis factor-(TNF-)，interleukin(IL)-6，IL-1 and IL-18 in BALF were significantly in-creased(P 0 05)The expression levels of Casp-8 mNA and protein，NLP3，apoptosis-associated speck-likeprotein containing a CAD(ASC)，cysteine aspartate proteolytic enzyme-1(Caspase-1)and gasdermin D(GSD-MD)protein in lung tissue were significantly increased(P 0 05)Compared with CLP Group，the 24 h survivalrate of mice in CLP+Z-IETD-FMK group was significantly increased(P 0 05)The destruction of alveolar struc-ture and the infiltration of inflammatory cells in lungs were reduced in mice Lung W/D，apoptosis rate，the levelsof TNF-，IL-6，IL-1 a