香叶木素对RSL3诱导小鼠精母细胞GC-2铁死亡的抑制作用及其机制

doi:10.13481/j.1671-587X.20240601

摘要/Abstract

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^-1Fer-1）。分别采用0、1、5、10、50、100、500和1 000 nmol·L^-1 RSL3溶液及0、0.1、0.5、1.0、5.0、10.0和50.0 μmol·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^-1DIO组及RSL3+0.8 nmol·L^-1DIO组、RSL3+4.0 nmol·L^-1DIO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1 000 nmol·L^-1 RSL3组GC-2细胞存活率均明显降低（P<0.01）；与0 μmol·L^-1 DIO组比较，0.5、1.0、5.0、10.0和50.0 μmol·L^-1DIO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<0.1 μmol·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^-1DIO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<0.05或P<0.01），RSL3作用36和48 h时GC-2细胞中HO-1蛋白表达水平均明显降低（P<0.01）；将100 nmol·L^-1RSL3作用GC-2细胞12 h作为后续实验条件。与对照组比较，RSL3组GC-2细胞中MDA水平明显升高（P<0.01），SOD活性和GSH/GSSG比值均明显降低（P<0.05）；与RSL3组比较，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+Fer-1组GC-2细胞中SOD活性均明显升高（P<0.05或P<0.01），RSL3+20.0 nmol·L^-1 DIO组和RSL3+Fer-1组GC-2细胞中MDA水平均明显降低（P<0.05或P<0.01），RSL3+4.0 nmol·L^-1 DIO组、RSL3+20.0 nmol·L^-1 DIO组和RSL3+Fer-1组GC-2细胞中GSH/GSSG比值均明显升高（P<0.05或P<0.01）。免疫荧光法观察，与对照组比较，RSL3组GC-2细胞中ACSL4蛋白荧光强度明显增强；与RSL3组比较，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+Fer-1组GC-2细胞中ACSL4蛋白荧光强度均明显减弱。Western blotting法检测，与对照组比较，RSL3组GC-2细胞中HO-1蛋白表达水平升高（P<0.05），GPX4和FTH1蛋白表达水平均明显降低（P<0.05或P<0.01）；与RSL3组比较，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+Fer-1组GC-2细胞中HO-1蛋白表达水平均明显降低（P<0.05或P<0.01），GPX4和FTH1蛋白表达水平均明显升高（P<0.05或P<0.01）。结论 DOI能够减轻RSL3诱导的小鼠精母细胞GC-2铁死亡，其机制可能与抑制HO-1蛋白表达，上调GPX4和FTH1蛋白表达有关。

关键词: 铁死亡, 香叶木素, 小鼠精母细胞GC-2, 谷胱甘肽过氧化酶4, 铁蛋白重链1, 酰基辅酶A合成酶长链家族成员4

Abstract:

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?1 DIO group， RSL3+4.0 nmol·L?1 DIO group， RSL3+20.0 nmol·L?1 DIO group， and RSL3+ferroptosis inhibitor Ferrostain-1（Fer-1） group （200 nmol·L?1 Fer-1）. The cells were treated with 0， 1， 5， 10， 50， 100， 500， and 1 000 nmol·L?1 RSL3 solutions， and 0， 0.5， 0.1， 1.0， 5.0， 10.0， and 50.0 μmol·L?1 DIO 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?1 DIO groups， as well as RSL3+0.8 nmol·L?1 DIO group， RSL3+4.0 nmol·L?1 DIO group， and RSL3+20.0 nmol·L?1 DIO 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?1 RSL3 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 1 000 nmol·L^-1 RSL3 groups were significantly decreased （P<0.01）； compared with 0 μmol·L^-1 DIO group， the survival rates of the GC-2 cells in 0.5， 1.0， 5.0， 10.0， and 50.0 μmol·L^-1 DIO groups were significantly decreased （P<0.01）， and 100 nmol·L^-1 RSL3 with DIO concentration< 0.1 μmol·L^-1 were selected for the subsequent experiments. Compared with blank group， the survival rates of the GC-2 cells in model group was significantly decreased （P<0.01）； compared with model group， the survival rates of the GC-2 cells in RSL3 + 20.0 nmol·L^-1 DIO group was significantly increased （P<0.01）. The Western blotting results showed that compared with 0 h， the expression level of GPX4 protein in the GC-2 cells was significantly decreased after treated with RSL3 for 6 h （P<0.01）， and the expression level of HO-1 protein was significantly increased after treated with RSL3 for 12 h （P<0.05）； after treated with RSL3 for 12 h， the expression levels of GPX4 and FTH1 proteins were significantly decreased （P<0.05 or P<0.01）； after treated with RSL3 for 24 h， the expression levels of GPX4 and HO-1 proteins were significantly decreased （P<0.05 or P<0.01）； after treated with RSL3 for 36 and 48 h， the expression levels of HO-1 protein were significantly decreased （P<0.01）. Therefore， 100 nmol·L^-1 RSL3 and for 12 h were selected as the experimental condition for the subsequent experiments.Compared with control group， the MDA level in the GC-2 cells in RSL3 group was significantly increased （P<0.01）， and the SOD activity and GSH/GSSG ratio were significantly decreased （P<0.05）. Compared with RSL3 group， the SOD activities in the cells in RSL3+0.8 nmol·L^-1 DIO group， RSL3+4.0 nmol·L^-1 DIO group， RSL3+20.0 nmol·L?1 DIO group， and RSL3+Fer-1 group were significantly increased （P<0.05 or P<0.01）. The MDA levels in the cells in RSL3+20.0 nmol·L^-1 DIO group and RSL3+Fer-1 group were significantly decreased （P<0.05 or P<0.01）， and the GSH/GSSG ratio in the cells in RSL3+4.0 nmol·L^-1 DIO group， RSL3+20.0 nmol·L^-1 DIO group， and RSL3+Fer-1 group were significantly increased （P<0.05 or P<0.01）.The immunofluorescence observation results showed that compared with control group， the fluorescence intensity of ACSL4 protein in the GC-2 cells in RSL3 group was significantly increased； compared with RSL3 group， the fluorescence intensities of ACSL4 protein in the cells in RSL3+0.8 nmol·L^-1 DIO group， RSL3+4.0 nmol·L^-1 DIO group， RSL3+20.0 nmol·L^-1 DIO group， and RSL3+Fer-1 group were significantly decreased.The Western blotting results showed that compared with control group， the expression level of HO-1 protein in the cells in RSL3 group was increased （P<0.05）， and the expression levels of GPX4 and FTH1 proteins were significantly decreased （P<0.05 or P<0.01）； compared with RSL3 group， the expression levels of HO-1 protein in the cells in RSL3+0.8 nmol·L^-1 DIO group， RSL3+4.0 nmol·L?1 DIO group， RSL3+20.0 nmol·L^-1 DIO group， and RSL3+Fer-1 group were significantly decreased （P<0.05 or P<0.01）， and the expression levels of GPX4 and FTH1 proteins were significantly increased （P<0.05 or P<0.01）. Conclusion DIO can alleviate the RSL3-induced ferroptosis in the GC-2 spermatocytes of the mice， and its mechanism may be related to the inhibition of HO-1 protein expression and the upregulation of expressions of GPX4 and FTH1 proteins.

Key words: Ferroptosis, Diosmetin, Mouse spermatocyte GC-2, Glutathione peroxidase 4, Ferritin heavy chain 1, Acyl-CoA synthetase long-chain family member 4

中图分类号:

R966

马宝莲,胡晓雪,艾霄文,张永兰. 香叶木素对RSL3诱导小鼠精母细胞GC-2铁死亡的抑制作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(6): 1481-1490.

Baolian MA,Xiaoxue HU,Xiaowen AI,Yonglan ZHANG. Inhibitory effect of diosmetin on ferroptosis of GC-2 spermatocytes induced by RSL3 in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(6): 1481-1490.

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Group	Survival rate
Concentration of RSL3 （nmol·L^－1）
0	100.00±5.12
1	96.00±2.65
5	97.28±4.64
10	91.09±1.32
50	73.54±7.51^*
100	59.61±0.35^*
500	15.21±0.61^*
1 000	13.66±0.07^*

Group	Survival rate
Concentration of DOI （μmol·L^-1）
0	100.00±1.97
0.1	94.70±3.96
0.5	85.57±0.55^*
1.0	84.30±1.47^*
5.0	76.50±2.30^*
10.0	64.41±8.46^*
50.0	18.74±0.22^*

Group	Survival rate
Blank	100.00±4.47
Model	67.94±0.38^*
0.8 nmol·L^－1 DIO	99.41±2.65
4.0 nmol·L^－1 DIO	95.46±0.74
20.0 nmol·L^－1 DIO	95.25±0.87
RSL3+0.8 nmol·L^－1 DIO	70.81±0.74
RSL3+4.0 nmol·L^－1 DIO	71.33±2.32
RSL3+20.0 nmol·L^－1 DIO	76.96±5.13^△

Group	FTH1	GPX4	HO-1
Time after treated with RSL3
0 h	1.00±0.00	1.00±0.00	1.00±0.00
6 h	0.84±0.12	0.63±0.16^**	1.09±0.06
12 h	0.72±0.01^*	0.68±0.05^**	1.23±0.03^*
24 h	0.84±0.15	0.65±0.11^**	0.56±0.32^*
36 h	0.78±0.02	0.83±0.05	0.47±0.18^**
48 h	0.85±0.11	1.10±0.03	0.42±0.23^**

Group	SOD [λ_B/（U·mg^－1）]	MDA[m_B/（mol·g^－1）]	Ratio of GSH/GSSG
Control	355.69±24.05	1.00±0.00	1.00±0.00
RSL3	269.64±39.82^*	1.85±0.35^**	0.36±0.04^*
RSL3+0.8 nmol·L^－1 DIO	373.40±28.41^△	1.42±0.48	0.79±0.31
RSL3+4.0 nmol·L^－1 DIO	402.08±39.52^△△	1.26±0.22	0.96±0.18^△
RSL3+20.0 nmol·L^－1 DIO	425.60±25.37^△△	1.08±0.21^△	1.20±0.23^△△
RSL3+Fer-1	377.03±22.71^△	1.04±0.20^△△	1.28±0.32^△△