摘要
目的探讨下调核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体表达对脓毒症小鼠急性肺损伤的保护作用及其介导线粒体自噬的作用机制。方法采用随机数字表法将18只BALB/c小鼠分为假手术组(sham组)、脓毒症模型组[盲肠结扎穿孔术(CLP)组]和MCC950(NLRP3特异性抑制剂)干预组(CLP+MCC950组),每组6只。采用CLP制作脓毒症模型。术前2 h,CLP+MCC950组小鼠腹腔注射50 mg/kg MCC950(用0.9%氯化钠溶液稀释至0.5 mL),sham组和CLP组腹腔注射等量0.9%氯化钠溶液。CLP术后24 h收集小鼠肺泡灌洗液及肺脏组织标本。将另外45只小鼠采用随机数字表法分为sham组、CLP组和CLP+MCC950组,记录小鼠7 d生存率。采用HE染色法观察肺损伤严重程度,并测量湿重/干重(W/D)比值。采用ELISA法检测肺泡灌洗液中IL-1β、IL-6、TNF-α、IL-10蛋白水平;Western blot法检测肺组织炎症相关蛋白[NLRP3、NF-κB p65、磷酸化NF-κB(p-NF-κB)p65]、凋亡相关蛋白[裂解的半胱氨酸天冬氨酸蛋白水解酶3(Cleaved-Caspase-3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)]和自噬相关蛋白[磷酸酶及张力蛋白同源基因诱导的激酶1(PINK1)、E3泛素连接酶(Parkin)、微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、选择性自噬接头蛋白p62]表达水平。结果sham组小鼠7 d生存率为100.0%。CLP组小鼠7 d累计死亡11只,7 d生存率为26.7%(4/15)。与sham组比较,CLP组小鼠7 d生存率明显降低(P<0.01);肺组织W/D比值升高(P<0.05);肺组织肺泡结构破坏严重,肺泡壁增厚,伴有明显的肺水肿和炎性细胞浸润;肺泡灌洗液中IL-1β、IL-6、TNF-α、IL-10蛋白水平升高(均P<0.05);肺组织中NLRP3、p-NF-κB p65、Cleaved-Caspase-3、Bax、PINK1、Parkin和LC3Ⅱ/Ⅰ蛋白表达水平升高,Bcl-2和p62蛋白表达水平降低(均P<0.05)。CLP+MCC950组小鼠7 d累计死亡8只,7 d生存率为46.7%(7/15)。CLP组和CLP+MCC950组小鼠7 d生存率比较,差异无统计学意义(P>0.05)。与CL
Objective To investigate the protective effect of down-regulating the expression of nucleotide-binding oligomerization domain like receptors family pyrin domain containing 3(NLRP3)inflammasome on acute lung injury of septic mice and its mechanism of modulating mitophagy.Methods Eighteen BALB/c male mice were randomly divided into sham group,cecal ligation and puncture(CLP)group and CLP+MCC950(NLRP3-specific inhibitor)group using a random number table method,with six mice in each group.The CLP procedure was used to establish the sepsis model.Mice in the CLP+MCC950 group were intraperitoneally injected with 50 mg/kg MCC950(diluted to 0.5 mL with 0.9%NaCl solution)2 h before the operation,while the sham and CLP groups were given equal amounts of 0.9%NaCl solution.Bronchoalveolar lavage fluid(BALF)and lung tissue were collected 24 h after CLP operation.Another 45 mice were divided into sham group,CLP group,and CLP+MCC950 group using the random number table method,and the 7 d survival rate of mice was recorded.HE staining was used to observe the severity of lung injury and the wet weight/dry weight(W/D)ratio was measured.Enzymelinked immunosorbent assay was used to measure the protein levels of IL-1β,IL-6,TNF-α,and IL-10 in BALF;Western blot was used to detect the expressions of lung tissue inflammation associated proteins[NLRP3,phosphorylated NF-κB(p-NF-κB)p65,and NF-κB-p65],apoptosis associated proteins[cleaved-cysteine-aspartic acid protease 3(Cleaved-Caspase-3),B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2 associated X protein(Bax)],and autophagy associated proteins[PTEN induced putative kinase 1(PINK1),E3 ubiquitin ligase(Parkin),microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),and selective autophagy adaptor protein p62].Results The 7 d survival rate of mice in the sham group was 100.0%.Eleven mice in the CLP group died cumulatively at 7 d,with the 7 d survival rate of 26.7%(4/15).Compared with the sham group,the 7 d survival rate of mice in the CLP group was significantly lower(P<0.05),the lu
作者
孙莹莹
祝晨烨
程志刚
骆敏
谢民民
SUN Yingying;ZHU Chenye;CHENG Zhigang;LUO Min;XIE Minmin(Department of Emergency,the First People's Hospital of Lin'an,Hangzhou 311300,China)
出处
《浙江医学》
CAS
2024年第22期2358-2365,共8页
Zhejiang Medical Journal
基金
杭州市临安区农业与社会发展立项项目(2021Y03)。
关键词
脓毒症
核苷酸结合寡聚化结构域样受体蛋白3
急性肺损伤
线粒体自噬
Sepsis
Nucleotide-binding oligomerization domain like receptors family pyrin domain containing 3
Acute lung injury
Mitophagy
