摘要
目的评价富氢盐水减轻脓毒症小鼠肺损伤时自噬与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体的关系。方法清洁级健康雄性C57BL小鼠36只,体重20~25 g,6周龄,采用随机数字表法分为6组(n=6):假手术组(Sham组)、脓毒症组(Sep组)、脓毒症+富氢盐水组(Sep+H2组)、脓毒症+富氢盐水+雷帕霉素组(Sep+H2+Rap组)、脓毒症+富氢盐水+3-甲基腺嘌呤组(Sep+H2+3-MA组)和脓毒症+富氢盐水+MCC950组(Sep+H2+M组)。采用盲肠结扎穿孔法(CLP)制备小鼠脓毒症模型。Sep+H2组、Sep+H2+Rap组、Sep+H2+3-MA组和Sep+H2+M组于CLP术后1和6 h时腹腔注射富氢盐水5 ml/kg。于CLP术后1 h时,Sep+H2+Rap组腹腔注射自噬激动剂雷帕霉素10 mg/kg,Sep+H2+3-MA组腹腔注射自噬抑制剂3-甲基腺嘌呤15 mg/kg,Sep+H2+M组腹腔注射NLRP3炎症小体抑制剂MCC95050 mg/kg。造模后24 h时,收集支气管肺泡灌洗液(BALF),测定蛋白浓度;然后断颈法处死小鼠,取肺组织,光镜下观察病理学结果,并行病理学损伤评分,测定湿重/干重比值(W/D比值),采用分光光度计法测定MPO活性,采用ELISA法检测IL-1β、IL-18和TNF-α含量,采用Western blot法测定pro-caspase-1 P10、NLRP3和凋亡相关点状蛋白(ASC)的表达。结果与Sham组比较,Sep组和Sep+H2组BALF蛋白浓度、肺组织病理学损伤评分、W/D比值、MPO活性、IL-β、IL-18、TNF-α含量和pro-caspase-1 P10、NLRP3、ASC表达水平升高(P<0.05)。与Sep组比较,Sep+H2组、Sep+H2+Rap组和Sep+H2+3-MA组BALF蛋白浓度、肺组织病理学损伤评分、W/D比值、MPO活性、IL-β、IL-18、TNF-α含量和pro-caspase-1 P10、NLRP3、ASC表达水平降低(P<0.05)。与Sep+H2组比较,Sep+H2+Rap组和Sep+H2+M组BALF蛋白浓度、肺组织病理学损伤评分、W/D比值、MPO活性、IL-β、IL-18、TNF-α含量和pro-caspase-1 P10、NLRP3、ASC表达水平降低(P<0.05),Sep+H2+3-MA组BALF蛋白浓度、肺组织病理学损伤评分、W/D比值、MPO活性、IL-β、IL-18、TNF-α含量和pro-caspase-1 P10、NLRP3、ASC表达水平升高(P<0.05)。结论富氢盐水减轻脓毒症小鼠肺损伤的机制与抑制自噬介导的NLRP3炎症小体激活有关。
Objective To evaluate the relationship between autophagy and NOD-like receptor pyrin domain containing 3(NLRP3)inflammasome during hydrogen-rich normal saline-induced reduction of lung injury in mice.Methods Thirty-six clean-grade healthy male C57BL mice,aged 6 weeks,weighing 20-25 g,were divided into 6 groups(n=6 each)by a random number table method:sham operation group(group Sham),sepsis group(group Sep),sepsis plus hydrogen-rich normal saline group(group Sep+H2),sepsis plus hydrogen-rich normal saline plus rapamycin group(group Sep+H2+Rap),sepsis plus hydrogen-rich normal saline plus 3-methyladenine(3-MA)group(group Sep+H2+3-MA)and sepsis+hydrogen-rich normal saline plus MCC950 group(group Sep+H2+M).Sepsis was produced by cecal ligation and puncture(CLP)in mice anesthetized with chloral hydrate.Hydrogen-rich normal saline 5 ml/kg was intraperitoneally injected at 1 and 6 h after CLP in Sep+H2,Sep+H2+Rap,Sep+H2+3-MA and Sep+H2+M groups.At 1 h after CLP,autophagy agonist rapamycin 10 mg/kg was intraperitoneally injected in group Sep+H2+Rap,autophagy inhibitor 3-MA 15 mg/kg was intraperitoneally injected in group Sep+H2+3-MA,and NLRP3 inflammasome inhibitor MCC95050 mg/kg was intraperitoneally injected in group Sep+H2+M.At 24 h after establishing the model,bronchoalveolar lavage fluid(BALF)was collected to determine the protein concentration.The animals were then sacrificed,and the lung tissues were removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio(W/D ratio),activity of myeloperoxidase(MPO),contents of tumor necrosis factor-α(TNF-α),interleukin-1beta(IL-1β)and IL-18(by enzyme-linked immunosorbent assay),and expression of pro-caspase-1 P10,NLRP3 and apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain(ASC)(using Western blot).Results Compared with group Sham,the protein concentration in BALF,pathological score,W/D ratio,activity of MPO,contents of IL-β,IL-18 and TNF-αand expression of pro-caspase-1 P10,NLRP3 and ASC were significantly increased in Sep and Sep+H2 groups(P<0.05).Compared with group Sep,the protein concentration in BALF,pathological score,W/D ratio,activity of MPO,contents of IL-β,IL-18 and TNF-αand expression of pro-caspase-1 P10,NLRP3 and ASC were significantly decreased in Sep+H2,Sep+H2+Rap and Sep+H2+3-MA groups(P<0.05).Compared with group Sep+H2,the protein concentration in BALF,pathological score,W/D ratio,activity of MPO,contents of IL-β,IL-18 and TNF-αand expression of pro-caspase-1 P10,NLRP3 and ASC were significantly decreased in Sep+H2+Rap and Sep+H2+M groups(P<0.05),and the protein concentration in BALF,pathological score,W/D ratio,activity of MPO,contents of IL-β,IL-18 and TNF-αand expression of pro-caspase-1 P10,NLRP3 and ASC were significantly increased in group Sep+H2+3-MA(P<0.05).Conclusion The mechanism by which hydrogen-rich normal saline reduces lung injury is associated with inhibiting autophagy-mediated activation of NLRP3 inflammasome in septic mice.
作者
张杨
陈红光
谢克亮
于泳浩
Zhang Yang;Chen Hongguang;Xie Keliang;Yu Yonghao(Department of Anesthesiology,Tianjin Medical University General Hospital Tianjin Research Institute of Anesthesiology,Tianjin 300052,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2019年第11期1371-1375,共5页
Chinese Journal of Anesthesiology
基金
国家自然科学基金(81471842,81601667,81671888,8172043)。
