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

Casp-8 attenuates sepsis-induced acute lung injury through regulation of NLRP3 inflammasome-mediated pyroptosis

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.