摘要
目的研究氧糖剥夺/恢复(oxygen-glucose deprivation/recovery,OGD/R)对A549肺泡上皮细胞凋亡与焦亡的作用,并探讨二者之间的相互影响。方法培养人肺泡A549细胞株,建立氧糖剥夺/恢复细胞损伤模型,分为对照组(control),氧糖剥夺/恢复组(OGD/R),氧糖剥夺/恢复复合焦亡抑制剂组(OGD/R+VX-765),氧糖剥夺/恢复复合凋亡抑制剂组(OGD/R+FMK),采用流式细胞术检测细胞凋亡率,采用ELISA法测定培养细胞上清中焦亡标志物IL-1β、IL-18水平,采用RT-qPCR和Western blot法测定细胞凋亡和焦亡关键分子的mRNA与蛋白质表达水平。结果与对照组比较,OGD/R组A549细胞凋亡率升高[(3.67±0.64)与(12.57±1.97),P<0.05]。与OGD/R组比较,OGD/R+VX-765组凋亡率降低[(5.97±1.28),P<0.05];与对照组比较,OGD/R组培养上清中焦亡标志物IL-1β、IL-18水平升高[(7.80±0.80)ng/L、(7.99±0.11)ng/L与(17.35±0.44)ng/L、(18.06±1.73)ng/L,P<0.05];与OGD/R组比较,OGD/R+FMK组IL-1β、IL-18水平降低[(4.72±0.59)ng/L、(10.24±1.42)ng/L,P<0.05];OGD/R可刺激A549细胞凋亡与焦亡关键分子的mRNA和蛋白表达;与OGD/R组比较,OGD/R+VX-765组BID的mRNA和蛋白相对表达量降低[(1.74±0.14)、(1.29±0.09)vs(1.12±0.18)、(1.01±0.10),P<0.05],OGD/R+FMK组GSDME的mRNA和蛋白相对表达量降低[(1.35±0.10)、(1.20±0.06)vs(0.94±0.05)、(0.52±0.03),P<0.05]。结论OGD/R可同时引发A549细胞发生焦亡与凋亡,且细胞焦亡与凋亡存在共享信号通路;两种抑制剂均可同时抑制OGD/R引起的A549细胞焦亡与凋亡。
Objective To investigate the effects of oxygen and glucose deprivation/recovery(OGD/R)on apoptosis and pyroptosis of A549 cells,and to explore the possibility of the mutual regulation in apoptosis and pyroptosis.Methods A549 cells were resuscitated and cultured,randomly divided into control group,OGD/R group,OGD/R+pyroptosis inhibitor group(OGD/R+VX-765)and OGD/R+apoptosis inhibitor group(OGD/R+FMK).The apoptotic rate was detected by flow cytometry,the levels of pyroptosis markers IL-1βand IL-18 in the supernatant were measured by ELISA,and the expression of key molecules in pyroptosis and apoptosis at mRNA and protein levels were detected by RT-qPCR and Western blotting,respectively.Results The apoptotic rate was significantly increased in the OGD/R group than the control group(12.57±1.97 vs 3.67±0.64,P<0.05),and the rate of the OGD/R+VX-765 group was decreased than that of the OGD/R group(5.97±1.28,P<0.05).OGD/R resulted in obvious increased levels of IL-1βand IL-18 in the supernatant(17.35±0.44 vs 7.80±0.80 ng/L,18.06±1.73 vs 7.99±0.11 ng/L,P<0.05),but FMK treatment notably reversed the increases of 2 inflammatory factors(4.72±0.59,10.24±1.42 ng/L,P<0.05).OGD/R enhanced the mRNA and protein expression levels of key molecules of apoptosis and proptosis in A549 cells.While,VX-765 decreased the mRNA and protein levels of BID(1.12±0.18 vs 1.74±0.14,1.01±0.10 vs 1.29±0.09,P<0.05),and FMK reduced those of GSDME(0.94±0.05 vs 1.35±0.10,0.52±0.03 vs 1.20±0.06,P<0.05)when compared with the cells from the OGD/R group.Conclusion OGD/R can induce pyroptosis and apoptosis in A549 cells simultaneously,and there exists shared signaling pathway in pyroptosis and apoptosis.Pyroptosis and apoptosis inhibitors can simultaneously inhibit the 2 types of OGD/R-induced cell death.
作者
安雨轩
肖宗懿
王小燕
宋娟
陈菲
王寿勇
AN Yuxuan;XIAO Zongyi;WANG Xiaoyan;SONG Juan;CHEN Fei;WANG Shouyong(Department of Anesthesiology,National Clinical Research Center for Child Health and Disorders,Key Laboratory of Child Development and Disorders of Ministry of Education,Chongqing Key Laboratory of Pediatrics,Children’s Hospital of Chongqing Medical University,Chongqing,400014,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第11期1119-1125,共7页
Journal of Army Medical University
基金
重庆市自然科学基金项目(cstc2020jcyj-msxmX0357)
关键词
氧糖剥夺
细胞焦亡
细胞凋亡
A549肺泡上皮细胞
oxygen-glucose deprivation
pyroptosis
apoptosis
hypotriploid alveolar basal epithelial A549 cells
作者简介
通信作者:王寿勇,E-mail:saulwang@126.com
