农药百克敏对小鼠精母细胞GC-2铁死亡的诱导作用

doi:10.13481/j.1671-587X.20260103

摘要/Abstract

摘要：

目的探讨百克敏对小鼠精母细胞GC-2铁死亡的影响，阐明百克敏是否能引起雄性生殖毒性。方法小鼠精母细胞GC-2分为对照组（普通培养基）、低剂量百克敏组（0.8 μmol·L^-1百克敏）、中剂量百克敏组（1.6 μmol·L^-1百克敏）和高剂量百克敏组（2.4 μmol·L^-1百克敏）。给予百克敏处理24 h后，采用噻唑蓝（MTT）法检测不同剂量百克敏作用下GC-2细胞活性，JC-1线粒体膜电位检测试剂盒检测各组GC-2细胞中线粒体膜电位，超氧化物歧化酶（SOD）活性检测试剂盒检测各组GC-2细胞中SOD活性，丙二醛（MDA）检测试剂盒检测各组GC-2细胞中MDA水平，还原型谷胱甘肽（GSH）和氧化型GSH（GSSG）检测试剂盒检测各组GC-2细胞中GSH和GSSG水平，并计算GSH/GSSG比值；Western blotting法检测各组GC-2细胞中血红素氧合酶1（HO-1）、铁蛋白重链1（FTH1）和GSH过氧化物酶4（GPX4）蛋白表达水平。结果与对照组比较，百克敏剂量≥1.0 μg·L^-1各组GC-2细胞活性均降低（P<0.01），设置其低、中和高剂量分别为0.8、1.6和2.4 μmol·L^-1百克敏。JC-1法，与对照组比较，中和高剂量百克敏组GC-2细胞线粒体膜电位均降低。与对照组比较，低、中和高剂量百克敏组GC-2细胞中SOD活性均降低（P<0.01），MDA水平升高（P<0.01）。与对照组比较，低、中和高剂量百克敏组GC-2细胞中GSH水平均降低（P<0.01）、GSSG水平均升高（P<0.01），且GSH/GSSG比值均降低（P<0.01）。与对照组比较，低、中和高剂量百克敏组GC-2细胞中HO-1蛋白表达水平明显升高（P<0.05或P<0.01），FTH1和GPX4蛋白表达水平明显降低（P<0.05或P<0.01）。结论百克敏能够诱导小鼠精母细胞GC-2铁死亡。

关键词: 百克敏, 小鼠精母细胞, 铁死亡, 谷胱甘肽过氧化物酶4, 氧化应激

Abstract:

Objective To discuss the effect of pyraclostrobin on ferroptosis in the spermatocytes GC-2 of the mice， and to clarify whether pyraclostrobin can cause male reproductive toxicity. Methods The mouse spermatocytes GC-2 were divided into control group， low dose of pyraclostrobin group （0.8 μmol·L^-1 pyraclostrobin）， medium dose of pyraclostrobin group （1.6 μmol·L^-1 pyraclostrobin） and high dose of pyraclostrobin group （2.4 μmol·L^-1 pyraclostrobin）. After being treated with pyraclostrobin for 24 h，methylthiazolyldiphenyl-tetrazolium bromide （MTT） method was used to detect the proliferation activities of the GC-2 cells after treated with different doses of pyraclostrobin； mitochondrial membrane potential detection kit JC-1 was used to detect the mitochondrial membrane potential in the GC-2 cells in various groups； superoxide dismutase （SOD） activity detection kit was used to detect the activities of SOD in the GC-2 cells in various groups； malondialdehyde （MDA） assay kit was used to determine the levels of MDA in the GC-2 cells in various groups； reduced glutathione （GSH） and oxidized glutathione （GSSG） detection kits were used to detect the levels of GSH and GSSG in the GC-2 cells， and the ratios of GSH/GSSG in various groups were calculated； Western blotting method was used to detect the expression levels of heme oxygenase-1 （HO-1）， ferritin heavy chain 1 （FTH1） and glutathione peroxidase 4 （GPX4） in the GC-2 cells in various groups. Results Compared with control group， the proliferation activities of the GC-2 cells in various pyaclostrobin dose ≥1.0 μg·L^-1 groups were decreased （P<0.01）. The low， medium， and high doses of pyraclostrobin were 0.8， 1.6， and 2.4 μmol·L^-1. The results of JC-1 method showed that compared with control group， the mitochondrial membrane potential of the GC-2 cells in medium， and high doses of pyraclostrobin groups were decreased. Compared with control group， the SOD activity in the GC-2 the cells in low， medium， and high doses of pyraclostrobin groups were decreased （P<0.01）， and the MDA levels in the GC-2 cells were increased （P<0.01）. Compared with control group， the GSH levels in the GC-2 cells in low， medium， and high doses of pyraclostrobin groups were significantly decreased （P<0.01）， the GSSG levels were increased （P<0.01）， and the GSH/GSSG ratios were decreased （P<0.01）. Compared with control group， the expression levels of HO-1 protein in the GC-2 cells in low， medium， and high doses of pyraclostrobin groups were significantly increased （P<0.05 or P<0.01）， while the expression levels of FTH1 and GPX4 proteins were significantly decreased （P<0.05 or P<0.01）. Conclusion Pyraclostrobin can induce the ferroptosis of the spermatocytes GC-2.

Key words: Pyraclostrobin, Mouse spermatocytes, Ferroptosis, Glutathione peroxidase 4, Oxidative stress

中图分类号:

R966

汤子怡,阳诗盈,杨天珍,刘闻强,钟江雪,尹俐. 农药百克敏对小鼠精母细胞GC-2铁死亡的诱导作用[J]. 吉林大学学报(医学版), 2026, 52(1): 18-25.

Ziyi TANG,Shiying YANG,Tianzhen YANG,Wenqiang LIU,Jiangxue ZHONG,Li YIN. Induction effect of pesticide pyraclostrobin on ferroptosis of spermatocytes GC-2 of mice[J]. Journal of Jilin University(Medicine Edition), 2026, 52(1): 18-25.

图/表 6

表1

图1

表2

表3

图2

表4

参考文献 30

[1]	张明浩，康珊珊，王曦，等. 基于多作物中吡唑醚菌酯的环境行为评估长期膳食风险［J］. 农业工程学报， 2023， 39（11）： 286-294.
[2]	JOSHI J， SHARMA S， GURUPRASAD K N. Foliar application of pyraclostrobin fungicide enhances the growth， rhizobial-nodule formation and nitrogenase activity in soybean （var. JS-335）［J］. Pestic Biochem Physiol， 2014， 114： 61-66.
[3]	刘冰卿，陈霞，凌金锋，等. 氯氟醚菌唑·吡唑醚菌酯对柑橘炭疽病的田间防治效果［J］. 热带农业科学， 2023， 43（7）： 53-56.
[4]	罗跃，吴小毛，胡贤锋，等. 吡唑醚菌酯的降解代谢及毒理研究进展［J］. 农业资源与环境学报， 2022， 39（4）： 651-663.
[5]	李烨青，张昌朋，赵学平，等. 吡唑醚菌酯对斑马鱼早期生命阶段的亚致死效应研究［J］. 生态毒理学报， 2024， 19（4）： 338-349.
[6]	LI H， ZHAO F， CAO F J， et al. Mitochondrial dysfunction-based cardiotoxicity and neurotoxicity induced by pyraclostrobin in zebrafish larvae［J］. Environ Pollut， 2019， 251： 203-211.
[7]	WU Y Z， WANG Y J， TONG Z， et al. Pyraclostrobin induces developmental toxicity and cardiotoxicity through oxidative stress and inflammation in zebrafish embryos［J］. Environ Pollut， 2024， 358： 124490.
[8]	WU M Q， BIAN J H， HAN S， et al. Characterization of hepatotoxic effects induced by pyraclostrobin in human HepG2 cells and zebrafish larvae［J］. Chemosphere， 2023， 340： 139732.
[9]	MAO L G， JIA W， ZHANG L， et al. Embryonic development and oxidative stress effects in the larvae and adult fish livers of zebrafish （Danio rerio） exposed to the strobilurin fungicides， kresoxim-methyl and pyraclostrobin［J］. Sci Total Environ， 2020， 729： 139031.
[10]	KOVAČEVIĆ M， HACKENBERGER D K， HACKENBERGER B K. Effects of strobilurin fungicides （azoxystrobin， pyraclostrobin， and trifloxystrobin） on survival， reproduction and hatching success of Enchytraeus crypticus［J］. Sci Total Environ， 2021， 790： 148143.
[11]	WANG Q Y， LUO N N， LEI M， et al. Facial irritant contact dermatitis caused by pyraclostrobin［J］. Clin Cosmet Investig Dermatol， 2022， 15： 1643-1647.
[12]	TANG D L， KANG R， VANDEN BERGHE T， et al. The molecular machinery of regulated cell death［J］. Cell Res， 2019， 29（5）： 347-364.
[13]	STOCKWELL B R， FRIEDMANN ANGELI J P， BAYIR H， et al. Ferroptosis： a regulated cell death nexus linking metabolism， redox biology， and disease［J］. Cell， 2017， 171（2）： 273-285.
[14]	ZHANG S P， XIN W， ANDERSON G J， et al. Double-edge sword roles of iron in driving energy production versus instigating ferroptosis［J］. Cell Death Dis， 2022， 13（1）： 40.
[15]	WU S F， GA Y， MA D Y， et al. The role of ferroptosis in environmental pollution-induced male reproductive system toxicity［J］. Environ Pollut， 2024， 363（Pt 1）： 125118.
[16]	NAKAMURA T， NAGURO I， ICHIJO H. Iron homeostasis and iron-regulated ROS in cell death， senescence and human diseases［J］. Biochim Biophys Acta Gen Subj， 2019， 1863（9）： 1398-1409.
[17]	YANG L L， CAI X M， LI R B. Ferroptosis induced by pollutants： an emerging mechanism in environmental toxicology［J］. Environ Sci Technol， 2024， 58（5）： 2166-2184.
[18]	HAN L X， WANG Y R， WANG Y J， et al. Pyraclostrobin repeated treatment altered the degradation behavior in soil and negatively affected soil bacterial communities and functions［J］. J Hazard Mater， 2025， 485： 136876.
[19]	YUAN W Z， SUN Z B， JI G J， et al. Emerging roles of ferroptosis in male reproductive diseases［J］. Cell Death Discov， 2023， 9（1）： 358.
[20]	焦丹丽，马秉哲，赵琛，等. 铁死亡在睾丸生精功能障碍中的机制研究进展［J］. 中华男科学杂志， 2022， 28（11）： 1044-1048.
[21]	LIU Y， CAO X H， HE C， et al. Effects of ferroptosis on male reproduction［J］. Int J Mol Sci， 2022， 23（13）： 7139.
[22]	ZHANG W， LIU Y， LIAO Y， et al. GPX4， ferroptosis， and diseases［J］. Biomed Pharmacother， 2024， 174： 116512.
[23]	IMAI H， HAKKAKU N， IWAMOTO R， et al. Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice［J］. J Biol Chem， 2009， 284（47）： 32522-32532.
[24]	CHEN Y， MA Q， ZHANG J， et al. Endogenous iron（Ⅱ） self-enriched fenton nanocatalyst via FTH1 activity inhibition and iron（Ⅲ） reduction for amplified cancer ferroptosis therapy［J］. Mol Pharm， 2025， 22（3）： 1568-1583.
[25]	ZHOU S M， SHI Y， LI J Y， et al. Bisphenol F induces spermatogenic cell ferroptosis via FTO-mediated m6A regulation of FTH1［J］. Free Radic Biol Med， 2025， 229： 364-373.
[26]	TIAN Y， LU J， HAO X Q， et al. FTH1 inhibits ferroptosis through ferritinophagy in the 6-OHDA model of parkinson’s disease［J］. Neurotherapeutics， 2020， 17（4）： 1796-1812.
[27]	TANG Z M， JU Y H， DAI X C， et al. HO-1-mediated ferroptosis as a target for protection against retinal pigment epithelium degeneration［J］. Redox Biol， 2021， 43： 101971.
[28]	CARBONE M， MATHIEU B， VANDENSANDE Y， et al. Impact of exposure to pyraclostrobin and to a pyraclostrobin/boscalid mixture on the mitochondrial function of human hepatocytes［J］. Molecules， 2023， 28（20）： 7013.
[29]	CHANG H C， WU R X， SHANG M， et al. Reduction in mitochondrial iron alleviates cardiac damage during injury［J］. EMBO Mol Med， 2016， 8（3）： 247-267.
[30]	AHOLA S， LANGER T. Ferroptosis in mitochondrial cardiomyopathy［J］. Trends Cell Biol， 2024， 34（2）： 150-160.

Group	Cell activity
Control	1.00±0.02
Concentration of pyraclostrobin (μmol·L^-1)
0.5	0.99±0.00
1.0	0.85±0.01^*
1.5	0.80±0.04^*
2.0	0.72±0.00^*
2.5	0.62±0.04^*
3.0	0.60±0.03^*
3.5	0.56±0.01^*
4.0	0.57±0.01^*
4.5	0.54±0.01^*

Group	SOD [λ_B/（U·mg^-1)]	MDA [w_B/（μmol·g^-1)]
Control	70.13±5.11	1.00±0.07
Pyraclostrobin
Low dose	35.69±6.08^*	1.56±0.11^*
Medium dose	18.36±0.58^*	1.66±0.02^*
High dose	14.98±1.69^*	2.67±0.91^*

Group	Level of GSH [w_B/（μmol·g^-1）]	Level of GSSG [w_B/（μmol·g^-1）]	Ratio of GSH/GSSG
Control	23.49±2.12	4.70±0.05	4.99±0.41
Pyraclostrobin
Low dose	14.72±0.76^*	5.33±0.18	2.76±0.05^*
Medium dose	9.00±1.14^*	7.36±0.39^*	1.22±0.17^*
High dose	8.72±2.64^*	6.91±0.32^*	1.25±0.32^*

Group	HO-1	FTH1	GPX4
Control	1.00±0.21	1.00±0.15	1.00±0.15
Pyraclostrobin
Low dose	1.86±0.33^*	0.31±0.07^**	0.45±0.17^*
Medium dose	2.76±0.21^**	0.28±0.07^**	0.45±0.19^*
High dose	3.81±0.41^**	0.17±0.02^**	0.46±0.08^*