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

Abstract

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

CLC Number:

R966

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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References 32

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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^△△

Inhibitory effect of diosmetin on ferroptosis of GC-2 spermatocytes induced by RSL3 in mice and its mechanism

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