PEDV infection induces ferroptosis in Vero cells via an ACSL-mediated lipid peroxidation pathway

Abstract Porcine epidemic diarrhea virus (PEDV) is a highly contagious viral pathogen causing severe economic losses in the swine industry. However, the underlying mechanisms of PEDV-induced host cell death largely unknown. In this study, we investigated the role of ferroptosis, a non-apoptotic form of programmed cell death, in PEDV pathogenesis. The experiments were divided into four groups: a control group, a PEDV (MOI = 1.0) group, an Erastin (5µM) positive control group and a Liprostatin (0.5µM) negative control group. Levels of GSH, ROS, Fe3+ and cell viability were evaluated using ELISA test kits. Fluorescence microscopy was employed to assess Fe2+ aggregation, while flow cytometry was utilizeed to measure lipid peroxide levels. The mRNA transcript levels of key gene involved the ferroptosis pathway-ACSL4, GPX4, ALOX15 and LPCAT3 - were determined by quantitative reverse transcription PCR. Compared to the control group, the PEDV group exhibited a significant decrease in GSH levels (P < 0.01) and a gradual reduction in Fe3+ levels (P < 0.05) over time. Furthermore, the PEDV group showed a substantial increase in ROS release (P < 0.05) and a corresponding decrease in cell viability (P < 0.05) relative to the control group. The results of the qRT-PCR revealed that the expression levels of ACSL4, ALOX15 and LPCAT3 mRNA were significantly elevated in the PEDV group (P<0.01). Additionally, Western blot analysis confirmed that the protein expression levels of ACSL4, ALOX15 and LPCAT3 also increased progressively (P < 0.01). In conclusion, our findings demonstrated that PEDV can induce ferroptosis in Vero cells through the lipid peroxidation pathway mediated by ACSL 4.