摘要
该研究探讨硫化氢(hydrogen sulfide,H_(2)S)预处理对大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cell typeⅡ,AECⅡ)缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的影响及其潜在作用机制。采用免疫荧光法进行细胞鉴定,检测各组细胞活性,超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)和乳酸(lactate)含量;ELISA检测IL-1β、IL-18含量;Western blot检测有氧糖酵解指标HK2、PKM2和细胞焦亡指标NLRP3、GSDMD-N、cleaved-Caspase1、cleaved-IL-1β、cleaved-IL-18蛋白水平;qRT-PCR检测HK2、PKM2、NLRP3 mRNA水平;免疫荧光法观察HK2、NLRP3的表达,评估H/R中有氧糖酵解和细胞焦亡的表达情况及H_(2)S对H/R损伤的影响。此外,为深入研究有氧糖酵解与细胞焦亡间的联系,使用HK2抑制剂2-脱氧葡萄糖(2-deoxyglucose,2-DG),并通过上述实验室方法检测有氧糖酵解和细胞焦亡指标表达情况。研究结果显示H/R可激活有氧糖酵解和细胞焦亡,使用H_(2)S后有氧糖酵解和细胞焦亡指标表达水平均下调且改善H/R诱导的细胞损伤;使用2-DG后细胞焦亡表达下调且细胞损伤得到改善。研究结果表明,H_(2)S可显著改善大鼠AECⅡ缺氧/复氧损伤,其机制可能与抑制HK2-NLRP3-GSDMD通路减轻细胞焦亡有关。
This study aims to investigate the effect of H_(2)S(hydrogen sulfide)preconditioning on H/R(hy-poxia/reoxygenation)injury in rat AECⅡ(alveolar epithelial cell typeⅡ)and the underlying mechanism.The cells were identified by immunofluorescence.Detection of each cell activity,SOD(superoxide dismutase)activity,MDA(malondialdehyde)and lactate content were measured.The levels of IL-1βand IL-18 were detected by ELISA.The protein levels of aerobic glycolysis markers HK2 and PKM2 and pyroptosis markers NLRP3,GSDMD-N,cleaved-Caspase1,cleaved-IL-1β,and cleaved-IL-18 were detected by Western blot.qRT-PCR was used to detect the mRNA levels of HK2,PKM2,and NLRP3.Immunofluorescence was used to observe the expression of HK2 and NLRP3,and to evaluate the expression of aerobic glycolysis and pyroptosis indicators in H/R and the effect of H_(2)S on H/R injury.In addition,to further investigate the link between aerobic glycolysis and pyroptosis,the HK2 inhibitor 2-DG(2-deoxyglucose)was used and its expression was detected by the laboratory methods described above.The results showed that H/R could activate aerobic glycolysis and pyroptosis.H_(2)S treatment down-regulated the expression of aerobic glycolysis and pyroptosis indicators and ameliorated H/R-induced cell injury.After using 2-DG,pyroptosis indicators expression was down-regulated and cell damage was improved.This study indicates that H_(2)S can signifi-cantly improve hypoxia/reoxygenation injury of AECⅡin rats,and its mechanism may be related to inhibiting the HK2-NLRP3-GSDMD pathway and reducing pyroptosis.
作者
王肖婷
骆珍珍
徐俊鹏
张淇昊
曹文傑
黄曼
田云娜
石璐
王万铁
WANG Xiaoting;LUO Zhenzhen;XU Junpeng;ZHANG Qihao;CAO Wenjie;HUANG Man;TIAN Yunna;SHI Lu;WANG Wantie(Institute of Ischemia/Reperfusion Injury,Wenzhou Medical University,Wenzhou 325035,China;College of Physical Education and Health,Yibin University,Yibin 644000,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2024年第7期1419-1431,共13页
Chinese Journal of Cell Biology
基金
浙江省介入肺脏病重点实验室建设项目(批准号:2019E10014)
宜宾学院科研培育项目(批准号:2022PY24)资助的课题。
