GSH/GPX4参与双酚A诱导小尾寒羊颗粒细胞铁死亡的研究OA

Bisphenol A(BPA),a common environmental endocrine disruptor,can induce apoptosis of cells,affect the function of ovarian granulosa cells,and pose a threat to the development of animal husbandry.This study aimed to preliminarily investigate the mechanism by which BPA induced ferroptosis in granulosa cells of Small-tailed Han sheep(Ovis aries)via the glutathione(GSH)/glutathione peroxidase 4(GPX4)pathway.The viability of ovarian granulosa cells was assessed using the CCK-8 assay.The cells were treated with varying concentrations of BPA(0,50,100,200,and 400 μmol/L)for 24,48,and 72 h.Based on the results,the optimal conditions were determined to be a BPA concentration of 200 μmol/L and a treatment duration of 24 h.To validate the effect of BPA on ferroptosis in these cells,the experiment was divided into 3 groups:Control group,BPA-treated group(200 μmol/L BPA),and ferroptosis inhibition group(200 μmol/L BPA+2 μmol/L Ferrostatin-1).To evaluate the key markers of ferroptosis,the expression of ferroptosis related genes and proteins were quantified by qRT-PCR and Western blot.Intracellular reactive oxygen species(ROS)levels were detected using the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)method,while Fe2+,malondialdehyde(MDA),and glutathione(GSH)levels were quantified using commercial assay kits.The results showed that treatment with 200 μmol/L BPA extremely significantly increased the contents of Fe2+,ROS,and MDA(P＜0.01),and extremely significantly decreased the level of GSH(P＜0.01).mRNA level of solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),and ferritin heavy chain 1(FTH1)was significantly upregulated(P＜0.05),whereas GPX4 and SLC7A11 protein abundance significantly decreased(P＜0.05)and FTH1 protein extremely significantly increased(P＜0.01).Treatment with 2 μmol/L Ferrostatin-1 extremely significantly decreased Fe2+,ROS,and MDA contents(P＜0.01),extremely significantly elevated GSH levels(P＜0.01).The protein levels of GPX4 and SLC7A11 in the ferroptosis inhibition group were significantly higher than those in the BPA treatment group(P＜0.05),while the FTH1 protein level was extremely significantly decreased(P＜0.01).These results indicated that Ferrostatin-1 alleviated BPA-induced ferroptosis.This research demonstrated that BPA exposure triggered oxidative stress and disrupted iron metabolism in granulosa cells of Small Tail Han sheep by impairing the GSH/GPX4 axis,ultimately leading to ferroptosis.The addition of Ferrostatin-1 in vitro exhibited a significant antagonistic effect on this process.This study adopted a targeted ferroptosis strategy to mitigate the damage of environmental pollutants to sheep reproductive performance,providing a critical theoretical foundation for high fecundity and stress resistance breeding of sheep.