甲磺酸去铁胺抑制精索静脉曲张睾丸支持细胞模型铁死亡的研究

Deferoxamine mesylate inhibits ferroptosis in testicular Sertoli cell model of varicocele

目的探究甲磺酸去铁胺对精索静脉曲张睾丸支持细胞氧化应激模型的保护作用。方法细胞实验选择小鼠睾丸支持细胞TM-4,设置对照组(A组)、甲磺酸去铁胺+对照组(B组)、氯化钴组(C组)、甲磺酸去铁胺+氯化钴组(D组)。C组和D组加入400μmol/L氯化钴,随后培养细胞24 h构建精索静脉曲张氧化应激模型。B组和D组加入800μmol/L甲磺酸去铁胺。采用细胞计数试剂盒(CCK-8)和流式测定细胞存活和凋亡水平;采用酶联免疫吸附试验(ELISA)法测定细胞内活性氧(ROS)、十二烷硫酸钠(SDS)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(GSH)水平;采用蛋白质印迹法(Western blot)检测谷胱甘肽过氧化物酶-4(GPX4)的蛋白表达水平,组间比较采用t检验。结果CCK-8实验结果显示C组和D组细胞存活率低于A组[(45.75±12.08)%、(86.53±2.57)%比(100.00±0.00)%,t=11.000、12.810,P值均<0.05],差异有统计学意义,且D组细胞存活率高于C组[(86.53±2.57)%比(45.75±12.08)%,t=8.087,P<0.01],差异有统计学意义。流式实验结果显示D组凋亡细胞比例低于C组[(8.63±0.11)%比(34.19±0.35)%,t=121.300,P<0.01],差异有统计学意义。ELISA结果显示D组相对ROS、MDA表达水平低于C组[(239.17±23.18)%、(40.34±1.50)nmol/(L·mg)比(378.00±24.84)%、(49.18±2.68)nmol/(L·mg),t=10.010、7.053,P值均<0.01];超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)表达水平高于C组[(7.85±0.24)U/mg、(425.60±12.21)U/mg比(4.69±0.28)U/mg、(208.96±7.00)U/mg,t=20.650、37.710,P<0.01],差异均有统计学意义。蛋白质印迹法实验结果显示氯化钴缺氧诱导了铁死亡关键蛋白GPX4表达下调,而甲磺酸去铁胺(DFO)恢复了铁死亡关键蛋白GPX4的表达。结论甲磺酸去铁胺通过抑制铁死亡对精索静脉曲张睾丸支持细胞氧化应激模型起保护作用。

Objective To explore the protective effect of deferoxamine mesylate on oxidative stress model of testicular Sertoli cells with varicocele.Methods In the cell experiment,mouse testicular Sertoli cells TM-4 were selected,and control group(group A),deferoxamine mesylate+control group(group B),cobalt chloride group(group C),and deferoxamine mesylate+cobalt chloride group(group D)were set up.In groups C and D,400μmol/L cobalt chloride was added and the cells were cultured for 24 h to construct the oxidative stress model of varicocele.In groups B and D,800μmol/L deferoxamine mesylate was added.Cell counting kit-8(CCK-8)assay and flow cytometry were used to determine the survival and apoptosis of cells.The levels of reactive oxygen species(ROS),sodium dodecyl sulfate(SDS),malondialdehyde(MDA),and glutathione(GSH)-PX in cells were measured by enzyme linked immunosorbent assay(ELISA).Western blotting was used to detect the protein expression level of glutathione peroxidase-4(GPX4).The t-test was used for comparison between groups.Results The CCK-8 assay showed that the survival rate in group C and group D was lower than in group A[(45.75±12.08)%,(86.53±2.57)%vs.(100.00±0.00)%,t=11.000,12.810,P<0.05],and the difference was statistically significant,and the survival rate in group D was higher than that in group C[(86.53±2.57)%vs.(45.75±12.08)%,t=8.087,P<0.01].Flow cytometry showed that the proportion of apoptotic cells in group D was lower than that in group C[(8.63±0.11)%vs.(34.19±0.35)%,t=121.300,P<0.01].The results of ELISA showed that the relative expression levels of ROS and MDA in group D were lower than those in group C[(239.17±23.18)%,(40.34±1.50)nmol/(L·mg)ratio(378.00±24.84)%,(49.18±2.68)nmol/(L·mg),t=10.010,7.053,P<0.01].The expression levels of superoxide dismutase(SOD)and glutathione peroxidasc(GSH-Px)in group D were higher than those in group C[(7.85±0.24)U/mg,(425.60±12.21)U/mg ratio(4.69±0.28)U/mg,(208.96±7.00)U/mg,t=20.650,37.710,P<0.01],and the difference was statistically significant.Western blotting showed that cobalt chloride hypoxia induced the down-regulation of the expression of iron death key protein GPx4,while deferoxamine mesylate restored the expression of iron death key protein GPX4.Conclusion Deferoxamine mesylate has a protective effect on the oxidative stress model of testicular Sertoli cells in varicocele by inhibiting ferroptosis.