Casp-8通过调控NLRP3炎性小体介导的细胞焦亡减轻脓毒症引起的急性肺损伤-东南大学学报医学版

Casp-8通过调控NLRP3炎性小体介导的细胞焦亡减轻脓毒症引起的急性肺损伤

单位：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

分类号：R631.2

出版年·卷·期（页码）：2023·42·第一期（64-71）

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，qRT-PCR检测肺组织中Casp-8 mRNA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（NLRP3）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-α（TNF-α）、白细胞介素（IL）-6、IL-1β、IL-18水平均显著升高（P<0.05）；肺组织中Casp-8 mRNA和蛋白表达水平，NLRP3、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-α、IL-6、IL-1β、IL-18水平均显著降低（P<0.05）；肺组织中Casp-8 mRNA和蛋白表达水平，NLRP3、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NLRP3炎性小体介导的细胞焦亡减轻脓毒症引起的ALI。

Objective: To analyze the effect of cysteine aspartate proteolytic enzyme-8(Casp-8) on sepsis-induced acute lung injury(ALI) and its related molecular mechanism. Methods: Forty C57BL/6 mice were used to establish sepsis-induced ALI model by cecal ligation and puncture(CLP). The successfully modeled mice were randomly divided into model group(CLP group) and CLP+Casp-8 inhibitor group(CLP+Z-IETD-FMK group), twenty in each group. Another twenty normal mice were divided into sham operation group(sham group). The wet/dry weight ratio(W/D) of lung tissue was measured by weighting method, the pathological changes of lung tissue were observed by HE staining, the apoptosis of lung tissue cells was detected by TUNEL staining, the levels of inflammatory cytokines in bronchoalveolar lavage fluid(BALF) were detected by ELISA, the expression of Casp-8 mRNA in lung tissue was detected by qRT-PCR, and the expression of Casp-8 protein, nucleotide binding oligomerization domain like receptor protein 3(NLRP3) inflammasome and apoptosis-related protein were detected by Western blotting. Results: Compared with the sham group, the 24 h survival rate of mice in CLP group was significantly decreased(P<0.05). The lung tissue of mice was congested and edematous, the alveolar structure was seriously damaged, and a large number of inflammatory cells were infiltrated in the lung stroma. Lung W/D, apoptosis rate, the levels of tumor necrosis factor-α(TNF-α), interleukin(IL) -6, IL-1β and IL-18 in BALF were significantly increased(P<0.05). The expression levels of Casp-8 mRNA and protein, NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), cysteine aspartate proteolytic enzyme-1(Caspase-1) and gasdermin D(GSDMD) protein in lung tissue were significantly increased(P<0.05). Compared with CLP Group, the 24 h survival rate of mice in CLP+Z-IETD-FMK group was significantly increased(P<0.05).The destruction of alveolar structure and the infiltration of inflammatory cells in lungs were reduced in mice. Lung W/D, apoptosis rate, the levels of TNF-α, IL-6, IL-1β and IL-18 in BALF were significantly decreased(P<0.05). The expression levels of Casp-8 mRNA and protein, NLRP3, ASC, Caspase-1 and GSDMD protein in lung tissue were significantly decreased(P<0.05). Conclusion: Casp-8 attenuates sepsis-induced ALI by regulating NLRP3 inflammasome-mediated pyroptosis.

参考文献：

[1] KUMAR V.Pulmonary innate immune response determines the outcome of inflammation during pneumonia and sepsis-associated acute lung injury[J].Front Immunol, 2020, 11:1722-1743.
[2] JIANG J, HUANG K, XU S, et al.Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction[J].Redox Biol, 2020, 36:101638-101652.
[3] MOKRA D, MIKOLKA P, KOSUTOVA P, et al.Corticosteroids in acute lung injury:the dilemma continues[J].Int J Mol Sci, 2019, 20(19):4765-4789.
[4] GENGA K R, RUSSELL J A.Update of sepsis in the intensive care unit[J].J Innate Immun, 2017, 9(5):441-455.
[5] 林润, 罗凌青, 饶平, 等.lncRNA A_30_P01029806减轻脓毒症急性肺损伤的机制研究[J].现代医学, 2021, 49(8):897-903.
[6] CHENG K T, XIONG S, YE Z, et al.Caspase-11-mediated endothelial pyroptosis underlies endotoxemia-induced lung injury[J].J Clin Invest, 2017, 127(11):4124-4135.
[7] LI Z, JIA Y, FENG Y, et al.Methane alleviates sepsis-induced injury by inhibiting pyroptosis and apoptosis:in vivo and in vitro experiments[J].Aging(Albany NY), 2019, 11(4):1226-1239.
[8] ORNING P, LIEN E.Multiple roles of caspase-8 in cell death, inflammation, and innate immunity[J].J Leukoc Biol, 2021, 109(1):121-141.
[9] FRITSCH M, GVNTHER S D, SCHWARZER R, et al.Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis[J].Nature, 2019, 575(7784):683-687.
[10] JIANG R, XU J, ZHANG Y, et al.Ligustrazine alleviate acute lung injury through suppressing pyroptosis and apoptosis of alveolar macrophages[J].Front Pharmacol, 2021, 12:680512-680528.
[11] 张昊, 谭赟, 杨璐瑜.ZLN005通过上调PGC-1α表达诱导巨噬细胞的M2型分化减轻脓毒症引起的急性肺损伤[J].河北医学, 2022, 28(2):182-189.
[12] 李毅豪.Caspase-8/NF-κB信号通路在大鼠脑缺血再灌注损伤炎症机制的调控作用研究[D].广州:广州医科大学, 2021.
[13] MANDAL R, BARRóN J C, KOSTOVA I, et al.Caspase-8:the double-edged sword[J].Biochim Biophys Acta Rev Cancer, 2020, 1873(2):188357-188377.
[14] HAN J H, PARK J, KANG T B, et al.Regulation of caspase-8 activity at the crossroads of pro-inflammation and anti-inflammation[J].Int J Mol Sci, 2021, 22(7):3318-3335.
[15] NEWTON K, WICKLIFFE K E, DUGGER D L, et al.Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis[J].Nature, 2019, 574(7778):428-431.
[16] SCHWARZER R, JIAO H, WACHSMUTH L, et al.FADD and caspase-8 regulate gut homeostasis and inflammation by controlling MLKL-and GSDMD-mediated death of intestinal epithelial cells[J].Immunity, 2020, 52(6):978-993.e6.
[17] QIU Y L, CHENG X N, BAI F, et al.Inhibition of NLRP9b attenuates acute lung injury through suppressing inflammation, apoptosis and oxidative stress in murine and cell odels[J].Biochem Biophys Res Commun, 2018, 503(2):436-443.
[18] 马海军, 李肃, 仲盛年.miR-221通过靶向CDKN1B调节脓毒症血管内皮细胞细胞凋亡和炎症反应[J].现代医学, 2021, 49(9):1063-1069.
[19] JIA X, CAO B, AN Y, et al.Rapamycin ameliorates lipopolysaccharide-induced acute lung injury by inhibiting IL-1β and IL-18 production[J].Int Immunopharmacol, 2019, 67:211-219.
[20] LIU Z, GAN L, XU Y, et al.Melatonin alleviates inflammasome-induced pyroptosis through inhibiting NF-κB/GSDMD signal in mice adipose tissue[J].J Pineal Res, 2017, 63(1):12414-12453.
[21] ZHENG M, KANNEGANTI T D.The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis(PANoptosis)[J].Immunol Rev, 2020, 297(1):26-38.
[22] HUANG Y, XU W, ZHOU R.NLRP3 inflammasome activation and cell death[J].Cell Mol Immunol, 2021, 18(9):2114-2127.
[23] XUE Z, XI Q, LIU H, et al.miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock[J].Cell Death Dis, 2019, 10(6):461-474.
[24] ZHENG X, CHEN W, GONG F, et al.The role and mechanism of pyroptosis and potential therapeutic targets in sepsis:a review[J].Front Immunol, 2021, 12:711939-711957.
[25] GAO Y L, ZHAI J H, CHAI Y F.Recent advances in the molecular mechanisms underlying pyroptosis in sepsis[J].Mediators Inflamm, 2018, 2018:5823823-5823841.
[26] LIU Y, ZHOU J, LUO Y, et al.Honokiol alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome-mediated pyroptosis via Nrf2 activation in vitro and in vivo[J].Chin Med, 2021, 16(1):127-148.
[27] ZENG Y, QIN Q, LI K, et al.PKR suppress NLRP3-pyroptosis pathway in lipopolysaccharide-induced acute lung injury model of mice[J].Biochem Biophys Res Commun, 2019, 519(1):8-14.
[28] WANG R, WANG Y, HU L, et al.Inhibition of complement C5a receptor protects lung cells and tissues against lipopolysaccharide-induced injury via blocking pyroptosis[J].Aging(Albany NY), 2021, 13(6):8588-8598.
[29] ANTONOPOULOS C, RUSSO H M, EL SANADI C, et al.Caspase-8 as an effector and regulator of NLRP3 inflammasome signaling[J].J Biol Chem, 2015, 290(33):20167-20184.
[30] ZHENG M, WILLIAMS E P, MALIREDDI R K S, et al.Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection[J].J Biol Chem, 2020, 295(41):14040-14052.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录