【摘要】 目的:基于溶质载体家族7成员11-谷胱甘肽过氧化酶4(SLC7A11-GPx4)信号通路探讨硒对环磷酰胺(CTX)致生精功能损伤(SI)模型小鼠精子发生的改善作用及机制。方法:将36只KM小鼠随机分为6组，每组6只，分别为SI模型对照组、缺硒模型(-Se SI)组、加硒模型(+Se SI)组，所有模型组均腹腔注射100 mg/kg CTX,正常对照组、-Se对照组、+Se对照组，对照组均注射生理盐水，注射周期为每周1次，连续6周。采用HE染色法观察各组小鼠睾丸组织形态学及病理学变化，并对附睾中精子进行计数，Western印迹试验检测GPx4和SLC7A11蛋白表达，RT-qPCR检测铁死亡相关基因变化。将GC2-spd细胞随机分为正常对照组，Ferrostatin(Fer-1)组，Erastin组，-Se组，亚硒酸钠(SeS)0.5、5.0μmol/L组，硒代蛋氨酸(SeM)5.0、50μmol/L组，SeS+Erastin组，SeS+Fer-1组，SeM+Erastin组，SeM+Fer-1组，观察GC2-spd细胞铁死亡相关蛋白和基因水平的变化。结果:CTX诱导的SI模型组小鼠睾丸和前列腺脏器指数明显低于正常对照组，生精细胞内层次减少，空洞增加，血清铁蛋白浓度下降(P<0.05),转铁蛋白浓度上升(P<0.05);+Se SI模型组、+Se对照组分别较-Se SI模型组、-Se对照组的脏器系数升高(P<0.05,P<0.01),且+Se组睾丸组织病理变化明显改善。SI模型对照组较正常对照组小鼠睾丸GPx4和SLC7A11基因表达量降低(P<0.01),而+Se SI模型组、+Se对照组分别较-Se SI模型组、-Se对照组小鼠睾丸GPx4和SLC7A11基因显著上升(P<0.01,P<0.05),但两种蛋白表达量却无明显差异。体外GC2-spd细胞实验结果显示，-Se组较正常对照组GPx4基因和GPx4蛋白水平均显著降低(P<0.05),SLC7A11基因水平下降(P<0.01);不同剂量的SeS和SeM均显著提高-Se组GPx4蛋白表达，低剂量SeM促进GPx4基因水平显著上升，高剂量SeS使SLC7A11基因和SLC7A11蛋白表达量增加(P<0.05,P<0.01)。-Se组较正常对照组，acsl4和ptgs2基因水平明显下降，SeM促进acsl4表达，SeS促进ptgs2和fth1的表达(P<0.01,P<0.05)。GC2-spd干预结果显示Erastin组较正常对照组ptgs2降低，SeS+Erastin、SeM+Erastin组较Erastin组ptgs2基因表达均增加，但Fer-1较正常对照组ptgs2表达降低，SeS+Fer-1、SeM+Fer-1组较Fer-1组ptgs2基因水平降低(P<0.05);SeM+Erastin、SeM+Fer-1组分别较Erastin、Fer-1组均GPx4的基因量增加(P<0.01,P<0.05);SeM+Erastin、SeS+Erastin较Erastin组和SeM+Fer-1、SeS+Fer-1组较Fer-1组SLC7A11均降低，且伴随着acsl4和fth1的升高(P<0.01)。结论:硒缺乏导致小鼠睾丸和精母细胞SLC7A11和GPx4基因水平降低，铁死亡相关基因表达异常，GPx4蛋白表达下降。硒促进GPx4基因和GPx4蛋白表达，提高SLC7A11基因表达量，改善生精功能损伤小鼠睾丸的精子发生，且有机硒SeM和无机硒SeS对铁死亡通路相关基因的调控存在差异。更多还原

【Abstract】 Objective: To investigate the effect of selenium on cyclophosphamide(CTX)-induced spermatogenic impairment(SI) in mice and its underlying mechanism. Methods: We equally randomized 36 male KM mice into 3 SI model and 3 control groups, the first 3 treated by intraperitoneal injection of CTX at 100 mg/kg(the SI model control group), CTX plus SI model control group, selenium deficient model group(-Se SI), selenium supplemented model group(+Se SI), while latter 3 by intraperitoneal injection of normal saline(the normal control), selenium deficiency control group(-Se control), selenium addition control group(+Se control), respectively, all once a week for 6 successive weeks. Then we observed the histopathological changes in the testes of all the mice by HE staining, obtained the sperm count in the epididymides, determined the expressions of glutathione peroxidase 4(GPx4) and SLC7A11 proteins by Western blot and ferroptosis-related genes by RT-qPCR, and examined the changes in the expressions of ferroptosis-related proteins and genes in the GC2-spd cells treated with ferroptosis inhibitors and inducers in combination with different concentrations of inorganic sodium selenite(SeS) and organic selenomethionine(SeM). Results: Compared with the normal controls, the SI model mice showed significantly decreased testicular and prostatic organ coefficients, reduced spermatogenic layers, increased voids, decreased serum ferritin concentration(P<0.05), and elevated transferrin concentration(P<0.05). The organ coefficients were significantly higher in the +Se SI and +Se control than in the-Se SI and-Se control groups(P<0.05, P<0.01), with evident pathological improvement of the testis tissue in the +Se controls. The expressions of the GPx4 and solute carrier family 7 members 11(SLC7A11) genes in the testis were dramatically down-regulated in the SI model controls(P<0.01), but up-regulated in the +Se SI and +Se control compared with those in the-Se SI and-Se control group(P<0.01 and P<0.05), but there were no statistically significant differences between their protein expressions. The results of in vitro GC2 spd cell experiments indicated that the GPx4 gene and GPx4 protein levels in the-Se group were significantly lower than those in the normal control group(P<0.05), while the SLC7A11 gene level decreased(P<0.01). Different doses of SeS and SeM significantly increased the GPx4 protein expression compared to the average Se group. Low doses of SeM promoted a significant increase in GPx4 gene levels, while high doses of SeS increased the expression levels of SLC7A11 gene and SLC7A11 protein(P<0.05, P<0.01). The Se group showed a significant decrease in the levels of acsl4 and ptgs2 genes compared to the normal control group. SeM promoted the expression of acsl4, while SeS promoted the expression of ptgs2 and fth1(P<0.01, P<0.05). The intervention results of GC2 spd showed that the Erastin group had a decrease in ptgs2 compared to the normal control group, while the SeS+Erastin and SeM+Erastin groups had an increase in ptgs2 gene expression compared to the Erastin group. However, the ptgs2 expression of Fer-1 was lower than that of the normal control group, and the ptgs2 gene level of SeS+Fer-1 and SeM+Fer-1 groups was lower than that of Fer-1 group(P<0.05); The gene quantity of GPx4 in the SeM+Erastin and SeM+Fer-1 groups increased compared to the Erastin and Fer-1 groups(P<0.01, P<0.05); SeM+Erastin and SeS+Erastin showed a decrease in SLC7A11 compared to the Erastin group, as well as SeM+Fer-1 and SeS+Fer-1 groups compared to the Fer-1 group, accompanied by an increase in acsl4 and fth1(P<0.01). Conclusion: Selenium deficiency causes the reduction of the SLC7A11 and GPx4 gene levels, disorder of ferroptosis-related genes and down-regulation of the GPx4 protein expression in the mouse testis and spermatocytes. Selenium can promote the expression of GPx4, up-regulate the level of SLC7A11, and improve spermatogenesis in the testis of the mouse with SI. There are differences between organic SeM and inorganic SeS in regulating the ferroptosis pathway-related genes.更多还原