百克敏对小鼠精母细胞GC-2线粒体功能和融合分裂动态的影响

doi:10.13481/j.1671-587X.20260506

摘要/Abstract

摘要：

目的探讨百克敏对小鼠精母细胞系GC-2细胞线粒体功能及动力学平衡的影响，并阐明其可能的作用机制。方法采用噻唑蓝（MTT）法检测不同浓度百克敏作用下各组GC-2细胞活性，确定合适的作用浓度。将小鼠精母细胞GC-2分为对照组、低浓度百克敏（0.8 μmol·L^-1）组、中浓度百克敏（1.6 μmol·L^-1）组和高浓度百克敏（2.4 μmol·L^-1）组。采用线粒体膜电位JC-1检测试剂盒检测各组GC-2细胞的线粒体内膜跨膜电位差（ΔΨ_m），2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光探针法检测各组GC-2细胞中活性氧（ROS）水平，三磷酸腺苷（ATP）检测试剂盒测定各组GC-2细胞中ATP水平，Mito-Tracker荧光探针法检测各组GC-2细胞中线粒体融合分裂情况，Western blotting法检测各组GC-2细胞中线粒体融合分裂相关蛋白视神经萎缩蛋白1（OPA1）和动力相关蛋白1（DRP1）蛋白表达水平。结果与对照组比较，百克敏浓度≥1.0 μmol·L^-1时各组GC-2细胞活性明显降低（P<0.001），百克敏对GC-2细胞的半数抑制浓度（IC??）为4.8 μmol·L^-1。与对照组比较，低、中和高浓度百克敏组GC-2细胞中ΔΨ_m明显降低（P<0.05或P<0.01），细胞中ROS水平明显升高（P<0.05或P<0.01），细胞中ATP水平明显降低（P<0.001）。Mito-Tracker染色，对照组GC-2细胞线粒体呈连续网络状分布，融合状态良好；低、中和高浓度百克敏组GC-2细胞线粒体呈碎片化形态，网络结构断裂明显，融合减少、分裂增加。Western blotting法，与对照组比较，中和高浓度百克敏组GC-2细胞中OPA1蛋白表达水平明显降低（P<0.05或P<0.01），低、中和高浓度百克敏组GC-2细胞中DRP1蛋白表达水平明显升高（P<0.001）。结论百克敏可诱导GC-2细胞线粒体融合与分裂失衡，导致线粒体功能紊乱，其毒性作用可能与线粒体动力学调控异常有关。

关键词: 百克敏, 小鼠精母细胞, 线粒体功能, 线粒体融合分裂, 动力学平衡

Abstract:

Objective To investigate the effect of pyraclostrobin on mitochondrial function and dynamic balance in mouse spermatocyte GC-2 cells， and to elucidate its possible mechanism. Methods Methylthiazolyldiphenyl-tetrazolium bromide（MTT） method was used to detect the cell activity of GC-2 cells in various groups after treatment with different concentrations of pyraclostrobin， so as to determine the appropriate treatment concentration. Mouse spermatocyte cell line GC-2 was divided into control group， low dose of pyraclostrobin （0.8 μmol·L^-1） group， medium dose of pyraclostrobin （1.6 μmol·L^-1） group， and high dose of pyraclostrobin （2.4 μmol·L^-1） group. JC-1 mitochondrial membrane potential assay kit was used to detect the mitochondrial transmembrane potential difference （ΔΨ_m）across the inner membrane of mitochondrial in GC-2 cells in various groups. 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescent probe method was used to detect the levels of reactive oxygen species （ROS） in GC-2 cells in various groups. Adenosine triphosphate （ATP） assay kit was used to measure the levels of ATP in GC-2 cells in various groups. Mito-tracker fluorescence probe method was used to detect the fusion and fission of mitochondria in GC-2 cells in various groups. Western blotting method was used to detect the expression levels of mitochondrial fusion and fission related proteins， optic atrophy 1 （OPA1） and dynamin-related protein 1 （DRP1）， in GC-2 cells in various groups. Results Compared with control group， the cell activities of GC-2 cells were significantly decreased in groups with pyraclostrobin concentration≥1 μmol·L^-1 （P<0.001）. The half maximal inhibitory concentration （IC₅₀） of pyraclostrobin against GC-2 cells was 4.8 μmol·L^-1. Compared with the control group， the ΔΨ_m in the GC-2 cells were significantly decreased in low dose of pyraclostrobin group， medium dose of pyraclostrobin group， and high dose of pyraclostrobin group （P<0.05 or P<0.01），the levels of ROS in the GC-2 cells were significantly increased（P<0.05 or P<0.01），and the ATP levels in the GC-2 cells were significantly decreased （P<0.001）. The Mito-Tracker staining results showed that the mitochondria in GC-2 cells in control group were distributed in a continuous network pattern with good fusion， while those in low dose of pyraclostrobin group， medium dose of pyraclostrobin group， and high dose of pyraclostrobin group showed fragmented morphology with obvious network structure breakage， decreased fusion， and increased fission. The Western blotting results showed that compared with control group， the expression levels of OPA1 protein in the GC-2 cells were decreased in medium dose of pyraclostrobin group and high dose of pyraclostrobin group （P<0.05 or P<0.01）， and the expression levels of DRP1 protein in the GC-2 cells were significantly increased in low dose of pyraclostrobin group，medium dose of pyraclostrobin group， and high dose of pyraclostrobin group （P<0.001）. Conclusion Pyraclostrobin induced an imbalance between mitochondrial fusion and fission in GC-2 cells， leading to mitochondrial dysfunction. Its toxic effect may be associated with abnormal regulation of mitochondrial dynamics.

Key words: Pyraclostrobin, Mouse spermatocyte, Mitochondrial function, Mitochondrial fusion and fission, Dynamic balance

中图分类号:

R994.6

阳诗盈,刘闻强,冯敏,李青青,蔡自胜,王春柔,尹俐. 百克敏对小鼠精母细胞GC-2线粒体功能和融合分裂动态的影响[J]. 吉林大学学报(医学版), 2026, 52(5): 1244-1251.

Shiying YANG,Wenqiang LIU,Min FENG,Qingqing LI,Zisheng CAI,Chunrou WANG,Li YIN. Effects of pyraclostrobin on mitochondrial function and fusion-fission dynamics in mouse spermatocyte GC-2 cells[J]. Journal of Jilin University(Medicine Edition), 2026, 52(5): 1244-1251.

图/表 4

参考文献 33

[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]	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.
[3]	SILVA V， MOL H G J， ZOMER P， et al. Pesticide residues in European agricultural soils-A hidden reality unfolded［J］. Sci Total Environ， 2019， 653： 1532-1545.
[4]	EUROPEAN Food Safety Authority. Peer review of the pesticide risk assessment of the active substance pyraclostrobin［J］. EFSA J， 2025， 23（3）： e9257.
[5]	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.
[6]	KIM C， CHOE H， PARK J， et al. Molecular mechanisms of developmental toxicities of azoxystrobin and pyraclostrobin toward zebrafish （Danio rerio） embryos： Visualization of abnormal development using two transgenic lines［J］. Environ Pollut， 2021， 270： 116087.
[7]	LI H， JING T F， LI T B， et al. Ecotoxicological effects of pyraclostrobin on tilapia （Oreochromis niloticus） via various exposure routes［J］. Environ Pollut， 2021， 285： 117188.
[8]	HUANG X P， YANG S， LI B X， et al. Comparative toxicity of multiple exposure routes of pyraclostrobin in adult zebrafish （Danio rerio）［J］. Sci Total Environ， 2021， 777： 145957.
[9]	JIANG J H， ZHOU W J， FANG N， et al. Chronic exposure to pyraclostrobin induced hepatic toxicity and glucolipid metabolism dysfunction in adult zebrafish［J］. Aquat Toxicol， 2025， 286： 107440.
[10]	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.
[11]	ZEMHERI-NAVRUZ F， INCE S， ARSLAN-ACAROZ D， et al. Resveratrol alleviates pyraclostrobin-induced lipid peroxidation， oxidative stress， and DNA damage in rats［J］. Environ Sci Pollut Res， 2023， 30（3）： 6414-6423.
[12]	WOLFE J， MARSIT C. Pyrethroid pesticide exposure and placental effects［J］. Mol Cell Endocrinol， 2023， 578： 112070.
[13]	MARTYNIUK C J， MEHINTO A C， DENSLOW N D. Organochlorine pesticides： Agrochemicals with potent endocrine-disrupting properties in fish［J］. Mol Cell Endocrinol， 2020， 507： 110764.
[14]	BRETVELD R， BROUWERS M， EBISCH I， et al. Influence of pesticides on male fertility［J］. Scand J Work Environ Health， 2007， 33（1）： 13-28.
[15]	ZHANG H L， LI Y， ZHU N， et al. Chronic exposure to polystyrene micro/nanoplastics triggers testicular dysfunction through PI3K/AKT/mTOR signaling-mediated spermatocyte senescence in mice［J］. Food Chem Toxicol， 2025， 205： 115703.
[16]	WANG X L， LV C Y， GUO Y， et al. Mitochondria associated germinal structures in spermatogenesis： piRNA pathway regulation and beyond［J］. Cells， 2020， 9（2）： 399.
[17]	GUO X， WANG L Y， XUAN J Y， et al. Fluoride induces spermatocyte apoptosis by IP3R1/MCU-mediated mitochondrial calcium overload through MAMs［J］. J Hazard Mater， 2025， 489： 137514.
[18]	ZHAO J T， CAO M S， YI H S， et al. Triptolide causes spermatogenic disorders by inducing apoptosis in the mitochondrial pathway of mouse testicular spermatocytes［J］. Toxics， 2024， 12（12）： 896.
[19]	ADEBAYO M， SINGH S， SINGH A P， et al. Mitochondrial fusion and fission： The fine-tune balance for cellular homeostasis［J］. FASEB J， 2021， 35（6）： e21620.
[20]	YAPA N M B， LISNYAK V， RELJIC B， et al. Mitochondrial dynamics in health and disease［J］. FEBS Lett， 2021， 595（8）： 1184-1204.
[21]	GREL H， WOZNICA D， RATAJCZAK K， et al. Mitochondrial dynamics in neurodegenerative diseases： unraveling the role of fusion and fission processes［J］. Int J Mol Sci， 2023， 24（17）： 13033.
[22]	YOU Y H， NI H B， MA Q， et al. Ketogenic diet ameliorates MASLD via balancing mitochondrial dynamics and improving mitochondrial dysfunction［J］. Nutr Diabetes， 2025， 15（1）： 37.
[23]	SULE R O， CONDON L， GOMES A V. A common feature of pesticides： oxidative stress-the role of oxidative stress in pesticide-induced toxicity［J］. Oxid Med Cell Longev， 2022， 2022： 5563759.
[24]	LIU X L， TANG D C， YIN F H， et al. Mitochondrion-targeted triphenylphosphonium-based kresoxim-methyl analogues： synthesis， fungicidal activity， and action mechanism approach［J］. J Agric Food Chem， 2022， 70（42）： 13563-13573.
[25]	SHI X K， BIAN X B， HUANG T， et al. Azoxystrobin induces apoptosis of human esophageal squamous cell carcinoma KYSE-150 cells through triggering of the mitochondrial pathway［J］. Front Pharmacol， 2017， 8： 277.
[26]	KINOSHITA M， SAITO Y， OTANI K， et al. Mitochondrial dynamics as a potential therapeutic target in acute myeloid leukemia［J］. Int J Hematol， 2024， 120（5）： 601-612.
[27]	ZHOU X Y， CHEN H M， WANG L， et al. Mitochondrial dynamics： a potential therapeutic target for ischemic stroke［J］. Front Aging Neurosci， 2021， 13： 721428.
[28]	陈敏，朱慧艳，陶静，等. 甜菜碱对大鼠脑微血管内皮细胞氧糖剥夺损伤的改善作用及对PI3K/AKT通路的影响［J］. 吉林大学学报（医学版）， 2025， 51（1）： 96-104.
[29]	REN L， CHEN X D， CHEN X B， et al. Mitochondrial dynamics： fission and fusion in fate determination of mesenchymal stem cells［J］. Front Cell Dev Biol， 2020， 8： 580070.
[30]	OJAIMI MAL， SALAH A， EL-HATTAB A W. Mitochondrial fission and fusion： molecular mechanisms， biological functions， and related disorders［J］. Membranes， 2022， 12（9）： 893.
[31]	田广，覃施媛，宋成文，等. 基于网络药理学定锚分析探究大黄素治疗肝损伤的作用机制［J］. 中国兽医学报， 2025， 45（9）： 1965-1976.
[32]	LEE Y H， KUK M U， SO M K， et al. Targeting mitochondrial oxidative stress as a strategy to treat aging and age-related diseases［J］. Antioxidants， 2023， 12（4）： 934.
[33]	杨露，傅家财，李凤金，等. 五味子乙素对胰腺癌细胞迁移和侵袭的抑制作用及其机制［J］. 吉林大学学报（医学版）， 2025， 51（1）： 44-50.