摘要
目的探讨田蓟苷对脂多糖(LPS)诱导的肺泡上皮细胞铁死亡的影响及潜在机制。方法将培养的人肺泡上皮细胞BEAS-2B分为对照组(正常培养)、LPS组、田蓟苷低剂量组(20μmol/L)、田蓟苷中剂量组(50μmol/L)、田蓟苷高剂量组(100μmol/L)、Compound C(ComC)抑制剂组[100μmol/L田蓟苷+10μmol/L腺苷酸活化蛋白激酶(AMPK)的抑制剂ComC]。四甲基偶氮唑盐(MTT)法检测BEAS-2B细胞活力,流式细胞仪检测细胞凋亡,DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平,分光光度法检测细胞内丙二醛(MDA)、还原型谷胱甘肽(GSH)、Fe^(2+)水平;Western blotting法检测谷胱甘肽过氧化物酶4(GPX4)、转铁蛋白(Tf)、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶3(caspase-3)及AMPK/沉默信息调节因子2相关酶1(SIRT1)/过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)信号通路蛋白表达。结果与对照组相比,LPS组BEAS-2B细胞的A 490值、还原型GSH、GPX4、PCNA、磷酸化AMPK(p-AMPK)/AMPK、SIRT1、PGC-1α蛋白表达显著降低(P<0.05),凋亡率、ROS、MDA、Fe^(2+)水平,以及Tf、Bax、caspase-3蛋白表达显著升高(P<0.05);与LPS组比较,田蓟苷低、中、高剂量组BEAS-2B细胞的A 490值、还原型GSH水平、GPX4、PCNA、p-AMPK/AMPK、SIRT1、PGC-1α蛋白表达显著升高(P<0.05),凋亡率、ROS、MDA、Fe^(2+)水平、Tf、Bax、caspase-3表达显著降低(P<0.05);与田蓟苷高剂量组比较,ComC抑制剂组BEAS-2B细胞的A 490值、还原型GSH水平、GPX4、PCNA、p-AMPK/AMPK、SIRT1、PGC-1α蛋白表达显著降低(P<0.05),凋亡率、ROS、MDA、Fe^(2+)水平,以及Tf、Bax、caspase-3蛋白表达显著升高(P<0.05)。结论田蓟苷通过激活AMPK/SIRT1/PGC-1α信号通路抑制LPS诱导的肺泡上皮细胞铁死亡。
Objective To investigate the effect of Tilianin on lipopolysaccharide(LPS)-induced ferroptosis in alveolar epithelial cells and its potential mechanism.Methods Human alveolar epithelial cells BEAS-2B were grouped into control group(normal culture),LPS group(10μg/mL),low-dose Tilianin group(20μmol/L),middle-dose Tilianin group(50μmol/L),high-dose Tilianin group(100μmol/L),and Compound C(ComC)inhibitor group(100μmol/L Tilianin+10μmol/L AMPK inhibitor).The viability of BEAS-2B cells was detected by MTT assay,cell apoptosis was detected by flow cytometry,the levels of intracellular reactive oxygen species(ROS)were detected by DCFH-DA fluorescence probe,and the levels of malondialdehyde(MDA),reduced glutathione(GSH)and Fe^(2+)were detected by spectrophotometry.Western blotting was used to detect the expressions of glutathione peroxidase 4(GPX4),transferrin(Tf),proliferating cell nuclear antigen(PCNA),Bcl-2-associated X protein(Bax),cysteine protease 3(caspase-3),and AMPK/SIRT1/PGC-1αsignaling pathway proteins in cells.Results Compared with the control group,the A 490 value,reduced GSH,GPX4,PCNA,p-AMPK/AMPK,SIRT1,PGC-1αprotein expressions in BEAS-2B cells in LPS group significantly decreased(P<0.05),while the apoptosis rate,ROS,MDA,Fe^(2+)levels and Tf,Bax and caspase-3 protein expressions significantly increased(P<0.05).Compared with LPS group,the A 490 value,reduced GSH level,GPX4,PCNA,p-AMPK/AMPK,SIRT1 and PGC-1αprotein expressions in BEAS-2B cells in low-dose,medium-dose and high-dose Tilianin groups significantly increased(P<0.05),while the apoptosis rate,ROS,MDA,Fe^(2+)levels,Tf,Bax and caspase-3 protein expressions decreased(P<0.05).Compared with high-dose group,the A 490 value,reduced GSH level,GPX4,PCNA,p-AMPK/AMPK,SIRT1,PGC-1αprotein expressions of BEAS-2B cells in ComC inhibitor group significantly decreased(P<0.05),and the apoptosis rate,ROS,MDA,Fe^(2+)levels and Tf,Bax and caspase-3 protein expressions significantly increased(P<0.05).Conclusion Tilianin inhibits LPS-induced ferroptosis in alveolar epithelial cells by activating AMPK/SIRT1/PGC-1αsignaling pathway.
作者
陈云
胡立杰
王婷婷
郝明明
CHEN Yun;HU Lijie;WANG Tingting;HAO Mingming(Department of General Pediatrics,the No.4 People′s Hospital of Hengshui,Hengshui,Hebei 053000,China;Department of Pharmacy,the No.4 People′s Hospital of Hengshui,Hengshui,Hebei 053000,China;Department of Pediatrics,Hengshui People′s Hospital,Hengshui,Hebei 053000,China)
出处
《国际检验医学杂志》
CAS
2023年第19期2349-2353,共5页
International Journal of Laboratory Medicine
基金
河北省衡水市重点研发计划项目(2017014015Z)。