甲磺酸去铁胺减轻氧化应激诱导的精原细胞铁死亡

Desferrioxamine mesylate attenuates the ferroptosis of mice’spermatogonia induced by oxidative stress

目的:探究甲磺酸去铁胺对氧化应激诱导的小鼠精原细胞铁死亡的作用。方法:将小鼠GC-1精原细胞分为设置对照组(A组)、甲磺酸去铁胺处理组(B组)、氯化钴处理组(C组)、甲磺酸去铁胺处理+氯化钴处理组(D组)。C组和D组加入200μmol/L氯化钴处理细胞24 h以构建小鼠精原细胞氧化应激模型。B组和D组加入350μmol/L甲磺酸去铁胺。采用细胞计数试剂盒(CCK-8)测定细胞活性;采用酶联免疫吸附试验(ELISA)法测定细胞内铁离子、丙二醛(MDA)、谷胱甘肽(GSH)水平;采用细胞荧光测定活性氧和线粒体膜电位水平;采用蛋白质印迹法检测谷胱甘肽过氧化物酶-4(GPX4)的表达水平,组间比较采取t检验。结果:C组和D组细胞活性显著低于A组[(49.79±2.86)%、(85.05±3.21)%比(100.00±0.00)%,t=42.99、11.42,P<0.05],且D组细胞活性高于C组[(85.05±3.21)%比(49.79±2.86)%,t=20.10,P<0.01],差异有统计学意义。ELISA结果示D组铁离子、MDA水平低于C组[(14.41±0.72)μmol/g、(7.025±0.127)nmol/mg比(19.18±0.61)μmol/g、(9.589±0.140)nmol/mg,t=12.43、33.27,P<0.01];GSH水平高于C组[(12.37±1.02)μmol/g比(6.98±0.70)μmol/g,t=10.66,P<0.01],差异均有统计学意义。细胞荧光结果显示A组、B组和D组ROS水平低于C组(27.17±2.44、25.15±2.28、41.46±1.75比71.20±1.78,t=32.58、35.55、26.61,P<0.01);A组和D组线粒体膜电位高于C组(14.98±0.73、8.46±1.69比0.61±0.09,t=43.55、10.82,P<0.01),差异均有统计学意义。蛋白质印迹法实验结果显示D组GPX4表达水平高于C组(0.457±0.014比0.386±0.012,t=6.556,P<0.05),差异有统计学意义。结论:甲磺酸去铁胺通过抑制铁死亡对小鼠精原细胞氧化应激模型起保护作用。

Objective To clarify the effect of deferoxamine mesylate on oxidative stress-induced ferroptosis of spermatogonia in mice.Methods Mouse Spermatogonia GC-1 cells(Pricella)were selected and divided into control group(group A),deferoxamine mesylate treatment group(group B),cobalt chloride treatment group(Group C),and deferoxamine mesylate+cobalt chloride treatment group(Group D).Groups Cand D were treated with 200μmol/L cobalt chloride for 24 hours to construct oxidative stress cell model of spermatogonia in mice.Groups B and D were treated with 350μmol/L deferoxamine mesylate.Cell counting kit-8(CCK-8)assay was used to measure the cell activity.The levels of Iron,malondialdehyde(MDA),and glutathione(GSH)in cells were measured by enzyme linked immunosorbent assay(ELISA);The levels of reactive oxygen species(ROS)and mitochondrial membrane potential were measured by cell fluorescence assays.Western blotting was used to detect the expression level of glutathione peroxidase-4(GPX4).Student’s t-test was used for comparison between the groups.Results The cell activity of group C and group D was significantly lower than that of group A[(49.79±2.86)%,(85.05±3.21)%vs.(100.00±0.00)%,t=42.99,11.42,P<0.05].The cell activity of group D was higher than that of group C[(85.05±3.21)%vs.(49.79±2.86)%,t=20.10,P<0.01].The results of ELISA showed that the relative expression levels of Iron and MDA in group D were lower than those in group C[(14.41±0.72)μmol/g,(7.025±0.127)nmol/mg ratio(19.18±0.61)μmol/g,(9.589±0.140)nmol/mg,t=12.43,33.27,P<0.01];the expression levels of GSH were higher than that in group C[(12.37±1.02)μmol/g vs.(6.98±0.70)μmol/g,t=10.66,P<0.01],the difference was statistically significant.The results of cell fluorescence showed that ROS levels in group A,B and D were lower than those in group C(27.17±2.44,25.15±2.28,41.46±1.75 vs.71.20±1.78,t=32.58,t=35.55,t=26.61,P<0.01);The mitochondrial membrane potential of group A and group D was higher than that of group C(14.98±0.73,8.46±1.69 vs.0.61±0.09,t=43.55,10.82,P<0.01).The results of Western blotting showed that the expression level of GPX4 in group D was higher than that in group C(0.457±0.014 vs.0.386±0.012,t=6.556,p<0.05),and the difference was statistically significant.Conclusion Deferoxamine mesylate can protect mouse spermatogonia from oxidative stress by inhibiting ferroptosis.