硫化氢预处理改善大鼠肺泡Ⅱ型上皮细胞缺氧/复氧损伤的机制研究
Mechanism of Hydrogen Sulfide in Improving Hypoxia/Reoxygenation Injury in Rat Alveolar Epithelial Cell Type Ⅱ
王肖婷 1骆珍珍 1徐俊鹏 1张淇昊 1曹文傑 1黄曼 1田云娜 1石璐 2王万铁1
作者信息
- 1. 温州医科大学缺血/再灌注损伤研究所,温州 325035
- 2. 宜宾学院体育与大健康学院,宜宾 644000
- 折叠
摘要
该研究探讨硫化氢(hydrogen sulfide,H2S)预处理对大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cell type Ⅱ,AEC Ⅱ)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H2S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-deoxy-glucose,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H2S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,H2S可显著改善大鼠AEC Ⅱ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.
Abstract
This study aims to investigate the effect of H2S(hydrogen sulfide)preconditioning on H/R(hy-poxia/reoxygenation)injury in rat AECⅡ(alveolar epithelial cell type Ⅱ)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-1β and IL-18 were detected by ELISA.The protein levels of aerobic glycolysis markers HK2 and PKM2 and pyroptosis markers NLRP3,GSDMD-N,cleaved-Caspasel,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 H2S 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.H2S 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 H2S can signifi-cantly improve hypoxia/reoxygenation injury of AEC Ⅱ in rats,and its mechanism may be related to inhibiting the HK2-NLRP3-GSDMD pathway and reducing pyroptosis.
关键词
硫化氢/大鼠肺泡Ⅱ型上皮细胞/缺氧/复氧损伤/HK2-NLRP3-GSDMD通路Key words
hydrogen sulfide/rat alveolar epithelial cell type Ⅱ/hypoxia/reoxygenation injury/the HK2-NLRP3-GSDMD pathway引用本文复制引用
基金项目
浙江省介入肺脏病重点实验室建设项目(2019E10014)
宜宾学院科研培育项目(2022PY24)
出版年
2024
NETL
NSTL
万方数据