NLRP3炎症小体介导的细胞焦亡在大鼠肺泡Ⅱ型上皮细胞缺氧/复氧损伤中的作用机制研究  

Molecular mechanism of NLRP3 inflammasome-mediated pyroptosis in hypoxia/reoxygenation injury of rat type II alveolar epithelial cells

作　　者：周卓琳 刘秀洁 王新雨 王淑远 宋正阳 田云娜 张赛[1] 黄丹娜 王万铁[1] Zhou Zhuo-lin;Liu Xiu-jie;Wang Xin-yu;Wang Shu-yuan;Song Zheng-yang;Tian Yun-na;Zhang Sai;Huang Dan-na;Wang Wan-tie(Institute of Ischemia-Reperfusion Injury,Wenzhou Medical University,Wenzhou 325035,China;Respiratory Medicine,Wenzhou people’s hospital,Wenzhou 325035,China)

机构地区：[1]温州医科大学缺血-再灌注损伤研究所,浙江温州325035 [2]温州市人民医院呼吸内科,浙江温州325035

出　　处：《中国病理生理杂志》2021年第10期1822-1827,共6页Chinese Journal of Pathophysiology

基　　金：温州市高层次人才创新技术重点资助项目(No.2016-7);温州市引进国外专家智力资助项目(No.Z2015-68);温州市基础性科研项目(No.Y20210083)。

摘　　要：目的:探讨核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体介导的细胞焦亡在大鼠肺泡Ⅱ型上皮细胞(AEC Ⅱ)缺氧/复氧(H/R)损伤中的作用及其机制。方法:常氧培养AEC Ⅱ,并建立H/R损伤模型,随机分为:正常组(Con组,不做处理)、H/R组、NLRP3炎症小体激动剂(脂多糖和ATP,LPS+ATP)组(在H/R组的基础上用NLRP3炎症小体激动剂)及NLRP3炎症小体抑制剂(MCC950)组(在H/R组的基础上用NLRP3炎症小体抑制剂)。实验结束后,收集细胞,采用CCK-8法检测各组细胞活力;试剂盒检测乳酸脱氢酶(LDH)和caspase-1活性;免疫荧光染色法观察各组细胞NF-κB和caspase-1表达变化;Western blot检测细胞焦亡相关蛋白NF-κB、NLRP3、gasdermin D(GSDMD)和白细胞介素1β(IL-1β)的表达水平。结果:与Con组相比,其余3组细胞活力显著下降(P<0.05),LDH释放显著增加(P<0.05),caspase-1活性显著升高(P<0.05),NF-κB和caspase-1荧光表达显著增加(P<0.05),细胞焦亡相关蛋白NF-κB、NLRP3、GSDMD-N和IL-1β表达显著增加(P<0.05);与H/R组相比,MCC950组细胞活力显著升高(P<0.05),LDH释放显著减少(P<0.05),caspase-1活性显著下降(P<0.05),NF-κB和caspase-1荧光表达显著减少(P<0.05),NF-κB、NLRP3、GSDMD-N和IL-1β蛋白表达显著减少(P<0.05),而LPS+ATP组与H/R组相比,结果则与MCC950组呈相反趋势(P<0.05)。结论:H/R可激活NF-κB信号通路,上调细胞焦亡相关蛋白表达,导致大鼠AEC Ⅱ损伤;NLRP3炎症小体抑制剂MCC950可减轻H/R引起的大鼠AEC Ⅱ损伤,其机制可能与其抑制NF-κB,减轻细胞焦亡有关。AIM:To investigate the molecular mechanism of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome-mediated pyroptosis in hypoxic/reoxygenation(H/R)injury of rat type Ⅱ alveolar epithelial cells(AEC Ⅱ).METHODS:The AEC Ⅱ were cultured in normoxia,and the model of H/R injury was established. The cells were randomly divided into control(Con)group,H/R group,NLRP3 inflammasome agonist(lipopolysaccharide+adenosine triphosphate,LPS+ATP)group and NLRP3 inflammasome inhibitor(MCC950)group. The cells in the latter groups were pretreated with LPS+ATP and MCC950 before H/R,respectively. CCK-8 was used to analyze the cell viability. The activity of lactate dehydrogenase(LDH)and caspase-1 was measured by kits. Immunofluorescence staining was used to observe the changes of NF-κB and caspase-1 in the cells. Western blot was used to determine the expression of NF-κB,NLRP3,gasdermin D(GSDMD)and interleukin-1β(IL-1β).RESULTS:Compared with Con group,the cell viability was significantly reduced in H/R,LPS+ATP and MCC950 groups(P<0. 05),while the release of LDH,the cell damage,the fluorescence expression of NF-κB and caspase-1,the caspase-1 activity,the pyroptosis level,and the relative protein levels of NF-κB,NLRP3,GSDMD-N and IL-1β were all significantly increased(P<0. 05). Compared with H/R group,the cell viability was significantly increased in MCC950 group(P<0. 05),while the release of LDH,the cell damage,the fluorescence expression of NF-κB and caspase-1,the caspase-1 activity,the pyroptosis level,and the relative protein levels of NF-κB,NLRP3,GSDMD-N and IL-1β were all significantly decreased(P<0. 05). The cells in LPS+ATP group showed the opposite results(P<0. 05).CONCLUSION:The H/R can activate NF-κB,up-regulate the levels of pyroptosis-related proteins,and trigger the damage of rat AEC Ⅱ. The NLRP3 inflammasome inhibitor MCC950 can alleviate rat AEC Ⅱ injury caused by H/R,which may be related to inhibiting NF-κB and attenuating pyroptosis.

关 键 词：NLRP3炎症小体 细胞焦亡 缺氧/复氧 肺泡II型上皮细胞 

分 类 号：R563[医药卫生—呼吸系统] R363.2[医药卫生—内科学]