吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (5): 1217-1226.doi: 10.13481/j.1671-587X.20240504

• 基础研究 • 上一篇    

硫唑嘌呤对RSL3诱导小鼠精母细胞铁死亡的影响

叶严珏1,汤子怡1,谭钰培1,阳诗盈1,刘永2,尹俐1()   

  1. 1.重庆理工大学药学与生物工程学院生物制药系,重庆 400054
    2.陆军军医大学新桥医院肾内科,重庆 400037
  • 收稿日期:2023-08-07 出版日期:2024-09-28 发布日期:2024-10-28
  • 通讯作者: 尹俐 E-mail:yinli@cqut.edu.cn
  • 作者简介:叶严珏(1998-),女,重庆市人,在读硕士研究生,主要从事分子药理学方面的研究。
  • 基金资助:
    国家自然科学基金青年基金项目(81402655);重庆市科技局自然科学基金面上项目(cstc2019jcyj-msxmX0427)

Effect of azathioprine on ferroptosis in spermatocytes of mice induced by RSL3

Yanjue YE1,Ziyi TANG1,Yupei TAN1,Shiying YANG1,Yong LIU2,Li YIN1()   

  1. 1.Department of Biopharmaceutical Sciences,School of Pharmacy and Bioengineering,Chongqing University of Technology,Chongqing 400054,China
    2.Department of Nephrology,Xinqiao Hospital,Army Military Medical University,Chongqing 400037,China
  • Received:2023-08-07 Online:2024-09-28 Published:2024-10-28
  • Contact: Li YIN E-mail:yinli@cqut.edu.cn

摘要:

目的 探讨硫唑嘌呤(AZA)对还原型谷胱甘肽(GSH)过氧化物酶4抑制剂RSL3诱导的小鼠精母细胞铁死亡的影响,并阐明其可能的作用机制。 方法 小鼠精母GC-2细胞随机分为对照组(不进行处理)、RSL3组(给予10 nmol·L-1 RSL3处理24 h)、RSL3+铁死亡抑制剂(Fer-1)组(给予10 nmol·L-1 RSL3处理24 h+2 μmol·L-1 Fer-1处理12 h)、RSL3+低剂量AZA组(给予10 nmol·L-1 RSL3处理24 h+5 μmol·L-1 AZA处理12 h)、RSL3+中剂量AZA组(给予10 nmol·L-1 RSL3处理24 h+10 μmol·L-1 AZA处理12 h)和RSL3+高剂量AZA组(给予10 nmol·L-1 RSL3处理24 h+20 μmol·L-1 AZA处理12 h)。MTT法检测不同浓度AZA和不同浓度RSL3作用后GC-2细胞活性,采用GSH和氧化型谷胱甘肽(GSSG)检测试剂盒检测GC-2细胞中GSH和GSSG水平,采用丙二醛(MDA)试剂盒检测各组GC-2细胞中MDA水平,采用Western blotting法检测各组GC-2细胞中长链脂酰CoA合成酶4(ACSL4)、血红素氧合酶1(HO-1)和谷胱甘肽过氧化物酶4(GPX4)蛋白表达水平,采用免疫荧光法检测各组GC-2细胞中ACSL4蛋白表达情况。 结果 与对照组比较,5、10和20 μmol·L-1AZA组GC-2细胞活性差异无统计学意义(P>0.05),30和40 μmol·L-1 AZA组GC-2细胞细胞活力明显降低(P<0.01),因此AZA作用浓度选择为20 μmol·L-1以内。与对照组比较,1、5和10 nmol·L-1 RSL3组GC-2细胞活性差异无统计学意义(P>0.05),50、100、500和1 000 nmol·L-1 RSL3组GC-2细胞活性明显降低(P<0.05或P<0.01),因此将RSL3作用浓度定为10 nmol·L-1以内。GSH和MDA试剂盒检测,与对照组比较,RSL3组GC-2细胞中GSSG和MDA水平明显升高(P<0.05),GSH水平明显降低(P<0.05);与RSL3组比较,RSL3+Fer-1组和RSL3+AZA组GC-2细胞中GSSG和MDA水平明显降低(P<0.01),GSH水平明显升高(P<0.01)。Western blotting法检测,与对照组比较,RSL3组GC-2细胞中ACSL4和HO-1蛋白表达水平明显升高(P<0.05),GPX4蛋白表达水平明显降低(P<0.01);与RSL3组比较,RSL3+Fer-1组和RSL3+AZA组GC-2细胞中GPX4蛋白表达水平明显升高(P<0.05),ACSL4和HO-1蛋白表达水平明显降低(P<0.01)。免疫荧光检测,与对照组比较,RSL3组GC-2细胞中ACSL4蛋白表达量明显增多;与RSL3组比较,RSL3+Fer-1组和RSL3+AZA组ACSL4蛋白表达量明显降低。 结论 AZA可以减轻RSL3诱导的小鼠精母细胞铁死亡。

关键词: 硫唑嘌呤, 铁死亡, 精母细胞, 谷胱甘肽过氧化物酶4, 酯酰辅酶A合成酶长链家族成员4

Abstract:

Objective To discuss the effect of azathioprine(AZA) on ferroptosis in spermatocytes of the mice induced by reduced glutathione(GSH) peroxidase 4 inhibitor RSL3,and to clarify the potential mechanism. Methods The spermatogonia GC-2 cells of the mice were randomly divided into control group (no treatment), RSL3 group (treated with 10 nmol·L-1 RSL3 for 24 h), RSL3+ferroptosis inhibitor (Ferrostatin-1, Fer-1) group (treated with 10 nmol·L-1 RSL3 for 24 h+2 μmol·L-1 Fer-1 for 12 h), RSL3+low dose of AZA group (treated with 10 nmol·L-1 RSL3 for 24 h +5 μmol·L-1 AZA for 12 h), RSL3+medium dose of AZA group (treated with 10 nmol·L-1 RSL3 for 24 h+10 μmol·L?1 AZA for 12 h), and RSL3+high dose of AZA group (treated with 10 nmol·L-1 RSL3 for 24 h+20 μmol·L-1 AZA for 12 h). The MTT method was used to detect the activities of the GC-2 cells in various groups after treated with different concentrations of AZA and RSL3; the GSH and GSSG levels in the GC-2 cells were detected by GSH and oxidized glutathione(GSSG) detection kits; the malondialdehyde(MDA) levels in the GC-2 cells in various groups were detected by MDA detection kit; Western blotting method was used to detect the expression levels of long-chain acyl-CoA synthetase 4 (ACSL4), heme oxygenase-1(HO-1), and glutathione peroxidase 4(GPX4) proteins in the cells in various groups; immunofluorescence staining was used to detect the expressions of ACSL4 protein in the cells in various groups. Results Compared with control group, the differences in activities of the GC-2 cells in 5, 10, and 20 μmol·L?1 AZA groups had no significant differences(P>0.05), while the activities of the GC-2 cells in 30 and 40 μmol·L?1 AZA groups were significantly decreased(P<0.01); therefore, the AZA concentration was selected to be within 20 μmol·L?1. Compared with control group, the differences of the activities of the GC-2 cells in 1, 5, and 10 nmol·L?1 RSL3 groups had no significant differences(P>0.05), while the activities of the GC-2 cells in 50, 100, 500, and 1 000 nmol·L?1 RSL3 groups were significantly decreased(P<0.05 or P<0.01); therefore, the RSL3 concentration was set to be within 10 nmol·L?1. The GSH and MDA detection kits results showed that compared with control group, the levels of GSSG and MDA in the GC-2 cells in RSL3 group were significantly increased(P<0.05), while the GSH levels were significantly decreased(P<0.05); compared with RSL3 group, the levels of GSSG and MDA in the GC-2 cells in RSL3+Fer-1 group and RSL3+AZA group were significantly decreased (P<0.01), while the GSH levels were significantly increased(P<0.01). The Western blotting results showed that compared with control group, the expression levels of ACSL4 and HO-1 proteins in the GC-2 cells in RSL3 group were significantly increased(P<0.05), while the expression level of GPX4 protein was significantly decreased(P<0.01); compared with RSL3 group, the expression levels of GPX4 protein in the GC-2 cells in RSL3+Fer-1 group and RSL3+AZA group were significantly increased(P<0.05), while the expression levels of ACSL4 and HO-1 proteins were significantly decreased(P<0.01). The immunofluorescence staining results showed that compared with control group, the expression amount of ACSL4 protein in the GC-2 cells in RSL3 group was significantly increased, and compared with RSL3 group, the expression amounts of ACSL4 protein in the cells in RSL3+Fer-1 group and RSL3+AZA group were significantly decreased. Conclusion AZA can alleviate the ferroptosis-induced by RSL3 in spermatocytes of the mice.

Key words: Azathioprine, Ferroptosis, Spermatocyte cells, Glutathione peroxidase 4 inhibitor, Acyl CoA synthetase long-chain family member 4

中图分类号: 

  • R332