基于线粒体损伤探讨双酚A诱导小鼠睾丸间质细胞凋亡的研究

doi:10.16431/j.cnki.1671-7236.2024.09.004

Abstract

Abstract: 【Objective】 The aim of this study was to investigate the mechanism of action of apoptosis induced by bisphenol A (BPA) exposure in mouse Leydig cells (TM3) from the perspective of mitochondrial damage. 【Method】 The TM3 cells were treated with different concentrations of BPA (0, 10, 30, 60, 90, 120 and 150 μmol/L) for 24 h, and the lowest BPA concentration causing apoptosis was screened by CCK-8 method.TM3 cells were randomly divided into control group (0 μmol/L BPA) and BPA group (60 μmol/L BPA) with three replicates in each group for 24 h.The state of cells was observed by inverted light microscope and apoptosis was observed by TUNEL staining.The TM3 cells were randomly divided into control group (0 μmol/L BPA), BPA group (60 μmol/L BPA), and BPA and N-acetyl-L-cysteine (NAC) co-treatment group (60 μmol/L BPA+5 mmol/L NAC), with three replicates in each group for 24 h.The content of malondialdehyde (MDA) and the activity of total superoxide dismutase (T-SOD) in the supernatant of cell culture were detected by ELISA method, the level of intracellular reactive oxygen species (ROS) was detected by kit, and the mitochondrial membrane potential was observed by JC-1 staining.The mRNA expression levels of glutathione peroxidase 1 (Gpx1), glutamate-cysteine ligase modifier subunit (Gclm), catalase (Cat), glutamate-cysteine ligase catalytic subunit (Gclc), mitochondrial fusion protein 2 (Mfn2), NAD-dependent protein deacetylase Sirtuin3 (Sirt3), caspase 3 (Casp3), cytochrome C (Cycs), B-cell lymphoma-2(BCL-2) associated X protein (Bax) in cells were detected by Real-time quantitative PCR.The relative expression of BAX and apoptosis regulatory factor BCL-2 proteins were detected by Western blotting, and the cell apoptosis rate was detected by flow cytometry. 【Result】 After treated with 60 μmol/L BPA for 24 h, the relative survival rate of TM3 cells began to decrease extremely significantly (P＜0.01), the number of apoptosis was extremely significantly increased (P＜0.01), and the number of cells were obviously decreased.Compared with control group, after 24 h of treatment with 60 μmol/L BPA, MDA content in TM3 cells was significantly increased (P＜0.01), T-SOD activity was decreased significantly (P＜0.01), the mRNA expression of antioxidation related genes (Gpx1, Cat, Gclm and Gclc genes) were significantly or extremely significantly reduced (P＜0.05 or P＜0.01), ROS production was extremely significantly increased (P＜0.01).The red/green fluorescence intensity ratio was extremely significantly decreased (P＜0.01).The mRNA expression of mitochondrial function related genes (Mfn2 and Sirt3 genes) were extremely significantly decreased (P＜0.01), while the mRNA expression of mitochondrial apoptosis pathway related genes (Casp3, Cycs and Bax genes) were extremely significantly increased (P＜0.01), the expression of BAX protein was extremely significantly increased (P＜0.01), the relative expression of BCL-2 protein was extremely significantly decreased and the apoptosis rate was extremely significantly increased (P＜0.01).Compared with the BPA-treated group, the MDA content in TM3 cells of the BPA+NAC treatment group was significantly reduced (P＜0.05), T-SOD activity was significantly increased (P＜0.05), the mRNA expression levels of antioxidant-related genes (Gpx1, Cat, Gclm and Gclc genes) were significantly increased (P＜0.05), the ROS production was extremely significantly decreased (P＜0.01).The red/green fluorescence intensity ratio was increased significantly (P＜0.05).The mRNA expression levels of mitochondrial function-related genes (Mfn2 and Sirt3 genes) were significantly or extremely significantly increased (P＜0.05 or P＜0.01), and the mRNA expression of mitochondrial apoptosis pathway related genes (Casp3, Cycs, Bax genes) were extremely significantly decreased (P＜0.01).The expression of BAX protein was extremely significantly decreased (P＜0.01), while the relative expression of BCL-2 extremely significantly increased (P＜0.01), and apoptosis rate was extremely significantly decreased (P＜0.01). 【Conclusion】 BPA exposure induced oxidative stress, resulting in mitochondrial dysfunction and subsequently triggering apoptosis in TM3 cells.

Key words: bisphenol A; oxidative stress; mitochondrial damage; apoptosis; mouse Leydig cells

CLC Number:

S852.21

YANG Wenzhe, LIU Kexiang, WANG Shirui, ZHAO Tong, PAN Feilong, ZHAO Shuchen, ZHAO Lijia. Study on Apoptosis of Mouse Leydig Cells Induced by BPA Based on Mitochondrial Damage[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3739-3751.

References

[1] XU A,LI X,LI K,et al.Linoleic acid promotes testosterone production by activating Leydig cell GPR120/ ERK pathway and restores BPA-impaired testicular toxicity[J].Steroids,2020,163:108677.
[2] KIM J Y,HAN E H,KIM H G,et al.Bisphenol A-induced aromatase activation is mediated by cyclooxygenase-2 up-regulation in rat testicular Leydig cells[J].Toxicology Letters,2010,193(2):200-208.
[3] 曹辉,余正,范一纯，等.双酚F暴露后对小鼠肝脏神经递质代谢的影响[J].南京医科大学学报(自然科学版),2023,43(9):1194-1200. CAO H,YU Z,FAN Y C，et al.Bisphenol F exposure disrupts metabolism of neurotransmitters in mouse liver[J].Journal of Nanjing Medical University (Natural Sciences),2023,43(9):1194-1200.(in Chinese)
[4] YUAN X,CHEN K,ZHENG F,et al.Low-dose BPA and its substitute BPS promote ovarian cancer cell stemness via a non-canonical PINK1/p53 mitophagic signaling[J].Journal of Hazardous Materials,2023,452:131288.
[5] GILL S,KUMARA V M R.Comparative neurodevelopment effects of bisphenol A and bisphenol F on rat fetal neural stem cell models[J].Cells,2021,10(4):793.
[6] MURATA M,KANG J H.Bisphenol A (BPA) and cell signaling pathways[J].Biotechnology Advances,2018,36(1):311-327.
[7] CASTELLINI C,TOTARO M,PARISI A,et al.Bisphenol A and male fertility:Myths and realities[J].Frontiers in Endocrinology,2020,11:353.
[8] ARCHANA M,SAIKANTH V,SATYAVANI M,et al.Prenatal exposure to bisphenol S and bisphenol A differentially affects male reproductive system in the adult offspring[J].Food and Chemical Toxicology,2022,167:113292.
[9] NGUYEN J L,EMILY A R,LIU T T,et al.Bisphenol-A analogs induce lower urinary tract dysfunction in male mice[J].Biochemical Pharmacology,2022,197:114889.
[10] RYU D Y,PANG W K,ADEGOKE E O,et al.Abnormal histone replacement following BPA exposure affects spermatogenesis and fertility sequentially[J].Environment International,2022,170:107617.
[11] SHARMA P,KAUSHAL N,SALETH L R,et al.Oxidative stress-induced apoptosis and autophagy:Balancing the contrary forces in spermatogenesis[J].Biochimica et Biophysica Acta-molecular Basis of Disease,2023,1869(6):166742.
[12] SIES H,BELOUSOV V V,CHANDEL N S,et al.Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology[J].Nature Reviews Molecular Cell Biology,2022,23(7):499-515.
[13] LIU L,LU O,LI D,et al.Sirtuin 3 mitigates oxidative-stress-induced apoptosis in bovine mammary epithelial cells[J].Journal of Dairy Science,2023,106(10):7266-7280.
[14] DIONÍSIO P A,AMARAL J D,RODRIGUES C M P.Oxidative stress and regulated cell death in Parkinson’s disease[J].Ageing Research Reviews,2021,67:101263.
[15] DENTON D,KUMAR S.Autophagy-dependent cell death[J].Cell Death & Disease,2019,26(4):605-616.
[16] CHUNCHAI T,APAIJAI N,BENJANUWATTRA J,et al.Erythropoietin exerted neuroprotection against cardiac ischemic/reperfusion injury by ameliorating oxidative stress,mitochondrial dysfunction,microglial activation,apoptosis and necroptosis[J].Alzheimers & Dementia,2021,17(3):e050179.
[17] SIES H,JONES D P.Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[J].Nature Reviews Molecular Cell Biology,2020,21(7):363-383.
[18] HURLEY J H,SCHULMAN B A.Atomistic autophagy:The structures of cellular self-digestion[J].Cell,2014,157(2):300-311.
[19] FILOMENI G,DE ZIO D,CECCONI F.Oxidative stress and autophagy:The clash between damage and metabolic needs[J].Cell Death & Disease,2015,22(3):377-388.
[20] ZHOU R,XIA M,ZHANG L,et al.Individual and combined effects of BPA,BPS and BPAF on the cardiomyocyte differentiation of embryonic stem cells[J].Ecotoxicology and Environmental Safety,2021,220:112366.
[21] ORNATOWSKI W,LU Q,YEGAMBARAM M,et al.Complex interplay between autophagy and oxidative stress in the development of pulmonary disease[J].Redox Biology,2020,36:101679.
[22] WANG C C,HE J Z,XU T F,et al.Bisphenol A(BPA),BPS and BPB-induced oxidative stress and apoptosis mediated by mitochondria in human neuroblastoma cell lines[J].Ecotoxicology and Environmental Safety,2021,207:111299.
[23] MURPHY M P.How mitochondria produce reactive oxygen species[J].Biochemical Journal,2009,417(1):1-13.
[24] 冯婉婷,李亚妮,夏国珺.双酚A对啮齿动物生殖毒性的研究进展[J].现代畜牧科技,2023，1:83-86. FENG W T,LI Y N,XIA G J.Research progress of BPA to reproductive toxicity in rodent[J].Modern Animal Husbandry Science & Technology,2023，1:83-86.(in Chinese)
[25] WANG X,NAG R,BRUNTON N P,et al.Hazard characterization of bisphenol A (BPA) based on rodent models-multilevel Meta-analysis and dose-response analysis for reproductive toxicity[J].Food and Chemical Toxicology,2023,172:113574.
[26] ZHANG M,MA B,YANG S,et al.Bisphenol A (BPA) induces apoptosis of mouse Leydig cells via oxidative stress[J].Environmental Toxicology,2023,38(2):312-321.
[27] ZHOU S M,YUAN W B,LI J Z,et al.TET1 involved in bisphenol A induced TM3 Leydig cell toxicity by regulating Cav3.3 hydroxymethylation[J].Chemosphere,2023,312(Pt 1):137171.
[28] MELI R,MONNOLO A,ANNUNZIATA C,et al.Oxidative stress and bpa toxicity:Antioxidant approach for male and female reproductive dysfunction[J].Antioxidants,2020,9(5):405.
[29] 熊忆茗,李红梅,秦占芬.双酚A对哺乳类实验动物雄性生殖系统发育的影响[J].生态毒理学报,2021,16(4):43-56. XIONG Y M,LI H M,QIN Z F.Effects of bisphenol A on development of male reproductive system in mammal laboratory animals[J].Asian Journal of Ecotoxicology,2021,16(4):43-56.(in Chinese)
[30] SHENG Z,WANG C,REN F,et al.Molecular mechanism of endocrine-disruptive effects induced by bisphenol A:The role of transmembrane G-protein estrogen receptor 1 and integrin αvβ3[J].Journal of Environmental Sciences,2019,75:1-13.
[31] CAMARCA A,GIANFRANI C,ARIEMMA F,et al.Human peripheral blood mononuclear cell function and dendritic cell differentiation are affected by bisphenol-A exposure[J].PLoS One,2016,11(8):e0161122.
[32] ELADAK S,MOISON D,GUERQUIN M J,et al.Effects of environmental bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis[J].PLoS One,2018,13(1):e0191934.
[33] WANG C,WANG C,WANG L,et al.Involvement of NLRP3/Caspase-1/GSDMD-dependent pyroptosis in BPA-Induced apoptosis of human neuroblastoma cells[J].Biochemical Pharmacology,2022,200:115042.
[34] SELÇUK Y,SEFA K,HASAN S,et al.Naringin protects against colistin-induced sciatic nerve damage by reducing oxidative stress,apoptosis and inflammation damage[J].Environmental Science and Pollution Research,2018,25(21):20968-20984.
[35] CHIANG Y W,SU C H,SUN H Y,et al.Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells[J].Environmental Toxicology,2021,37(1):131-141.
[36] HUANG M,LIU S,FU L,et al.Bisphenol A and its analogues bisphenol S,bisphenol F and bisphenol AF induce oxidative stress and biomacromolecular damage in human granulosa KGN cells[J].Chemosphere,2020,253:126707.
[37] MAO Y,CHEN Y,CAI W,et al.CypD-mediated mitochondrial dysfunction contributes to titanium ion-induced MC3T3-E1 cell injury[J].Biochemical and Biophysical Research Communications,2023,644:15-24.
[38] DING Y,ZHENG Y,HUANG J,et al.UCP2 ameliorates mitochondrial dysfunction,inflammation,and oxidative stress in lipopolysaccharide-induced acute kidney injury[J].International Immunopharmacology,2019,71:336-349.
[39] XU X,JIN K,BAIS A S,et al.Uncompensated mitochondrial oxidative stress underlies heart failure in an iPSC-derived model of congenital heart disease[J].Cell Stem Cell,2022,29(5):840-855.
[40] MOTAGHINEJAD M,MOTEVALIAN M,ULLOA L,et al.Minocycline protects against neuronal mitochondrial dysfunction and cognition impairment[J].Acta Neurobiologiae Experimentalis,2023,83(1):71-83.
[41] LI H,LEUNG J C K,YIU W H,et al.Tubular β-catenin alleviates mitochondrial dysfunction and cell death in acute kidney injury[J].Cell Death & Disease,2022,13(12):1061.
[42] LIU Y,YAO Y,TAO W,et al.Coenzyme Q10 ameliorates BPA-induced apoptosis by regulating autophagy-related lysosomal pathways[J].Ecotoxicology and Environmental Safety,2021,221:112450.
[43] JIANG W,ZHAO H,ZHANG L,et al.Maintenance of mitochondrial function by astaxanthin protects against bisphenol A-induced kidney toxicity in rats[J].Biomed Pharmacother,2020,121:109629.
[44] FADEL F,AL-KANDARI N,KHASHAB F,et al.JNK inhibition alleviates oxidative DNA damage,germ cell apoptosis,and mitochondrial dysfunction in testicular ischemia reperfusion injury[J].Acta Neurobiologiae Experimentalis,2020,52(8):891-900.
[45] KAMINSKYY V O,ZHIVOTOVSKY B.Free radicals in cross talk between autophagy and apoptosis[J].Antioxidants & Redox Signaling,2014,21(1):86-102.
[46] KOWALCZYK A.The role of the natural antioxidant mechanism in sperm cells[J].Reproductive Sciences,2022,29:1387-1394.
[47] JANSSEN O,QIAN J,LINKERMANN A,et al.CD95 ligand—Death factor and costimulatory molecule?[J].Cell Death & Disease,2003,10(11):1215-1225.
[48] WANG H W,LIU J,WEI S S,et al.Mitochondrial respiratory chain damage and mitochondrial fusion disorder are involved in liver dysfunction of fluoride-induced mice[J].Chemosphere,2020,241:125099.
[49] TANG W H,STITHAM J,JIN Y,et al.Aldose reductase-mediated phosphorylation of p53 leads to mitochondrial dysfunction and damage in diabetic platelets[J].Circulation,2014,129(15):1598-1609.

Study on Apoptosis of Mouse Leydig Cells Induced by BPA Based on Mitochondrial Damage

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