摘要
目的:探讨香叶木素(DIO)对谷胱甘肽过氧化物酶(GSH-Px)抑制剂(1S,3R)-RSL3(RSL3)诱导小鼠精母细胞GC-2铁死亡的抑制作用,并阐明相关作用机制。方法:GC-2细胞分为对照组、RSL3组、RSL3+0.8 nmol·L^(-1)DIO组、RSL3+4.0 nmol·L^(-1)DIO组、RSL3+20.0 nmol·L^(-1)DIO组和RSL3+铁死亡抑制剂Ferrostain-1(Fer-1)组(200 nmol·L^(-1)Fer-1)。分别采用0、1、5、10、50、100、500和1000 nmol·L^(-1)RSL3溶液及0、0.1、0.5、1.0、5.0、10.0和50.0μmol·L^(-1)DIO溶液处理细胞。另取GC-2细胞,分为空白组、模型组和给药组,给药组GC-2细胞按照处理方式分为0.8、4.0和20.0 nmol·L^(-1)DIO组及RSL3+0.8 nmol·L^(-1)DIO组、RSL3+4.0 nmol·L^(-1)DIO组和RSL3+20.0 nmol·L^(-1)DIO组。噻唑蓝(MTT)法检测各组GC-2细胞存活率。采用100 nmol·L^(-1)RSL3分别作用GC-2细胞0、6、12、24、36和48 h,Western blotting法检测各组GC-2细胞中铁死亡相关蛋白表达水平。试剂盒检测各组GC-2细胞中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平及谷胱甘肽(GSH)/氧化型谷胱甘肽(GSSG)比值,免疫荧光法观察各组GC-2细胞中酰基辅酶A合成酶长链家族成员4(ACSL4)蛋白荧光强度。结果:MTT法检测,与0 nmol·L^(-1)RSL3组比较,50、100、500和1000 nmol·L^(-1)RSL3组GC-2细胞存活率均明显降低(P<0.01);与0μmol·L^(-1)DIO组比较,0.5、1.0、5.0、10.0和50.0μmol·L^(-1)DIO组GC-2细胞存活率均明显降低(P<0.01),后续实验中选择100 nmol·L^(-1)RSL3作用GC-2细胞,DIO作用浓度<0.1μmol·L^(-1)。与空白组比较,模型组GC-2细胞存活率明显降低(P<0.01);与模型组比较,RSL3+20.0 nmol·L^(-1)DIO组细胞存活率明显升高(P<0.01)。Western blotting法检测,与0 h比较,RSL3作用6 h时GC-2细胞中GPX4蛋白表达水平明显降低(P<0.01),RSL3作用12 h时GC-2细胞中HO-1蛋白表达水平明显升高(P<0.05),GPX4和FTH1蛋白表达水平均明显降低(P<0.05或P<0.01),RSL3作用24 h时GC-2细胞中GPX4和HO-1蛋白表达水平明显降低(P<
Objective:To discuss the inhibitory effect of diosmetin(DIO)on the ferroptosis induced by the glutathione peroxidase(GSH-Px)inhibitor(1S,3R)-RSL3(RSL3)in spermatocytes GC-2 of the mice,and to clarify the mechanism.Methods:The GC-2 cells were divided into control group,RSL3 group,RSL3+0.8 nmol·L⁻¹DIO group,RSL3+4.0 nmol·L⁻¹DIO group,RSL3+20.0 nmol·L⁻¹DIO group,and RSL3+ferroptosis inhibitor Ferrostain-1(Fer-1)group(200 nmol·L⁻¹Fer-1).The cells were treated with 0,1,5,10,50,100,500,and 1000 nmol·L⁻¹RSL3 solutions,and 0,0.5,0.1,1.0,5.0,10.0,and 50.0μmol·L⁻¹DIO solutions,respectively.Additionally,the GC-2 cells were divided into blank group,model group,and treatment group.The GC-2 cells in treatment group were further divided into 0.8,4.0,and 20.0 nmol·L⁻¹DIO groups,as well as RSL3+0.8 nmol·L⁻¹DIO group,RSL3+4.0 nmol·L⁻¹DIO group,and RSL3+20.0 nmol·L⁻¹DIO group.MTT method was used to detect the survival rates of the GC-2 cells in various groups.The GC-2 cells were treated with 100 nmol·L⁻¹RSL3 for 0,6,12,24,36,and 48 h;Western blotting method was used to detect the expression levels of ferroptosis-related proteins in the GC-2 cells in various groups;kits were used to detect the activities of superoxide dismutase(SOD),levels of malondialdehyde(MDA),and ratios of glutathione(GSH)to glutathione disulfide(GSSG)in the GC-2 cells in various groups;immunofluorescence method was used to detect the fluorescence intensities of acyl-CoA synthetase long-chain family member 4(ACSL4)protein in the GC-2 cells in various groups.Results:The MTT method results showed that compared with 0 nmol·L^(-1)RSL3 group,the survival rates of the GC-2 cells in 50,100,500,and 1000 nmol·L^(-1)RSL3 groups were significantly decreased(P<0.01);compared with 0μmol·L^(-1)DIO group,the survival rates of the GC-2 cells in 0.5,1.0,5.0,10.0,and 50.0μmol·L^(-1)DIO groups were significantly decreased(P<0.01),and 100 nmol·L^(-1)RSL3 with DIO concentration<0.1μmol·L^(-1)were selected for the subsequent exper
作者
马宝莲
胡晓雪
艾霄文
张永兰
MA Baolian;HU Xiaoxue;AI Xiaowen;ZHANG Yonglan(Department of Pharmacology,School of pharmacy and Bioengineering,Chongqing University of Technology,Chongqing 400054,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2024年第6期1481-1490,共10页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金青年基金项目(81803800)
重庆市科技局自然科学基金项目(CSTC2018jcyjAX0529,CSTB2023NSCQ-MSX0469)。
