Preliminary Study On The Pathway Of Ferroptosis Induced By Porcine Epidemic Diarrhea Virus In Vero Cells

Porcine epidemic diarrhea virus（PEDV）is a member of the coronavirus family,its infection leads to severe watery diarrhea in newborn piglets.PEDV is widespread all over the world,causing high morbidity and mortality in pigs,which poses a huge threat to the pig industry.Ferroptosis is a non-apoptotic cell death pathway which exhibits a specific iron-dependent expression pattern and is associated with lipid peroxidation.Glutathione peroxidase 4（GPX4）is a key regulator of ferroptosis.Whether PEDV infection can activate cell ferroptosis remains unexplored.In this study,we analyzed the programmed cell death pathway of Vero cells caused by PEDV infection and explored its molecular mechanism.The experiment was divided into,and ELISA detection kit was used to detect the We analyzed the levels of GSH,ROS and Fe³⁺in the media via ELISA as well as cell viability in cells incubated under different treatments,including blank,PEDV（MOI=1.0）,Erastin（5μM）and PEDV+Liprostatin（0.5μM）.Fe²⁺aggregation was analyzed using fluorescence microscopy imaging.Lipid peroxidation level was demonstrated by flow cytometry;m RNA expression levels of core genes of ferroptosis pathway,including ACSL4,TFRC,ATF4,HMOX1,SLC3A2,GPX4,FTH1,P53,ALOX15 and LPCAT3,were detected by q RT-PCR.Protein expression levels of GPX4,ALOX15,LPCAT3,and ACSL4 were examined by Western Blot.The experimental results indicated that compared with the blank group,the GSH activity level decreased over time（P<0.01）,while the Fe³⁺level gradually decreased（P<0.05）in the virus treated group.ROS release level of the virus infected group was also gradually increased than that of the blank group（P<0.05）.Compared with the blank group,fluorescence caused by Fe²⁺aggregation at 36 hours post infection was significantly brighter,while pretreatment with the ferroptosis inhibitor Liprostatin（0.5μM）significantly reduced the fluorescence intensity.The viability of the cells in the virus infected group gradually decreased over time（P<0.05）compared with blank control,and the reduce of viability is attenuated with Liprostatin pretreatment（P<0.01）.Lipid peroxidation level were also increased by PEDV infection,while Liprostatin significantly reduced the increased generation of lipid peroxides（P<0.05）.m RNA and protein expression levels of ACSL4,ALOX15 and LPCAT3 were all gradually increased overtime after PEDV infection compared to blank group,indicating the activation of ferroptosis pathway.In conclusion,we found that PEDV infection activated ferroptosis in Vero cells through ACSL4-mediated lipid peroxidation pathway.