42 Nrf2 and nuclear factor kappa B cross-talk in bovine granulosa cells under lead challenge

Abstract

Lead (Pb²⁺), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb²⁺ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb²⁺ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5 mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO₂. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C₂H₃O₂)₂] (1, 2, or 3 µg mL⁻¹) for 2 h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H₂DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24 h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (^ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G₀/G₁ phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb⁺² driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.