Research On Toxicity And Apoptotic Mechanism Of Zearalenone On Porcine Granulosa Cells In Vitro

Zearalenone(ZEA) is a mycotoxin mainly produced by Fusarium.Its structural similarity with 17β-estradiol allow it to bind to estrogen receptors(ER) irreversibly,therefor,ZEA showes reproductive and developmental toxicity,in addition to,ZEA has immunotoxicity,carcinogenicity, genotoxicity, cytotoxicity and.Pigs are most sensitive to its toxicity.Objective:The aim of the study was to reveal the toxic effects of the porcine reproductive system through researching the toxic effects of ZEA on the porcine granulosa cells(GCs) in vitro,and to provide a theoretical basis for the detoxification method.Methods:GCs was extracted from follicles(1-5mm) by Mechanical separation. Morphology of GCs were observed by using Giemsa and crytal violet staining,cell viability was determined by 0.4% trypan blue, Immunocytochemistry was used to measure the FSHR expression to characterize GCs. Growth curve of GCs was detected by MTT assay; cell proliferation was determined using MTT method,SOD activity,GSH-Px activity and MDA content were measured by NBT chromogenic assay,TBA assay,and indirect method,respectively.TUNEL and Annexin-FITC/PI staining assay were used to detect apoposis. Measurement of ROS level and mitochondrial transmembrane potential by DCFA and Rh123 staining. Western blotting analysis of caspase-3 and caspase-9 expression, Immunofluorescence for caspase-3 expression and positioning and the inhibition of Z-VAD-FMK and Ac-LEHD-FMK on cell apoptosis.Results:GCs viability was about 65%,purity>97%, and cultured GCs grew well, logarithmic growth phase of GCs was from 24h to 96h. High doses of ZEA inhibits proliferation of porcine GCs, the IC50 was 90μM; 60μM,90μM and 120μM ZEA caused oxidative damage through significantly decreased antioxidant enzymes SOD, GSH-Px activity,increased MDA content in a dose-dependent manner,and induced cell apoptosis in a dose-dependent manner.Study reveals ZEA induced apoptosis in porcine ovarian granulosa cells by caspase-dependent mitochondrial apoptotic pathway, mainly in:a significant increase of intracellular ROS levels,significantly decrease of mitochondrial membrane potential, activation of Caspase-3,9 protein and an accumulation and translocation of caspase-3 from the cytoplasm into the nucleus, and the caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Ac-LEHD-FMK significantly inhibited ZEA induced apoptosis in porcine ovarian granulosa cells.Conclusion:ZEA inhibited proliferation of porcine GCs, caused cell oxidative damage and induced apoptosis via the mitochondrial caspase-dependent pathway induced apoptosis.