Mechanism Of Ferroptosis In Mice Myocardium And In HeLa Cells Induced By Coxsackie Virus B3

Backgroud: Myocarditis is considered to be an important cause of sudden cardiac death in children,which is mainly caused by viral infection.In children,the most common virus detected is coxsackievirus B3(CVB3).Because cardiomyocytes are non-regenerable cells,the virus rapidly replicates in the cells and induces cell death after invading the myocardium,and then causes inflammatory cascade.There is still lack of effective treatment for viral myocarditis in clinic,so it is of great significance to explore a new mechanism to target CVB3 infection.Ferrop tosis is a new type of iron-dependent regulatory cell death.Recent studies have shown that iron,as an essential trace element,plays a crucial role in inhibiting viruses proliferation.However,it is not clear whether ferroptosis is involved in CVB3-induced myocarditis and the underlying mechanism of CVB3-induced cell death.Objective: By establishing the model of CVB3 myocarditis in vivo and model of CVB3 infection in vitro,we study whether ferroptosis was involved in the pathogenesis of CVB3 infection,and explore the potential molecular mechanism.Methods(1)48 Balb/c mice at 4 weeks were randomly divided into control group and CVB3 infection group.The mice were sacrificed on the 1st,4th,7th and 14 th day after intervention.The serum CK-MB and c Tn I of mice were detected by ELISA.The pathological sections of the myocardial tissue were stained with HE.The CVB3 virus particles and mitochondrial morphology were observed by electron microscope.The changes of myocardial iron,lipid peroxidation metabolite such as MDA and other markers of ferroptosis were detected by kits respectively.(2)The expression of ferroptosis genes ACSL4,GPX4,TFRC,NCOA4 were detected by quantitative Real-time PCR and Western blot.(3)The effects of the inhibitor Fer-1 on heart morphology,inflammation of myocardium,myocardial iron,lipid peroxidation metabolites by intraperitoneal injection Fer-1.(4)A stable model of CVB3 infected HeLa cells in vitro was establish,and cells at 6h,12 h,24h,36 h and 48 h after infection were harvested.The replication of the virus was observed by immunofluorescence,the cell survival rate was using CCK-8,the level of intracellular lipid ROS was detected by BODIPYTM 581/591 C11,the level of GSH and MDA in cells were using test kit.The expression of m RNA and protein of ACSL4,GPX4,TFRC,and NCOA4 by checked with quantitative Real-time PCR and Western blot.Cells were pretreated with Fer-1 and DFO respectively,and the CVB3 replication was observed by immunofluorescence,and cellular iron,MDA were measured with test kits.(5)The key gene of ferroptosis in CVB3 infection was screened by library and q-PCR detection.The cell survival rate,ferroptosis markers(iron,lipid ROS,GSH,MDA)and the expression of ferroptosis genes were measured by after knocking down TFRC.(6)Double luciferase reporter gene was used to detect the activity of the promoter of the gene,and chromatin immunoprecipitation(Ch IP)experiment was employed to verify the binding site of the transcription factor on the promoter of the gene.Results(1)In the myocardial tissue of viral myocarditis mice,ferroptosis mainly occurs on the 14 th day of CVB3 infection,manifested as myocardial iron ion overload,increased MDA,upregulation of m RNA and protein expression of promoting ferroptosis genes NCOA4,TFRC,ACSL4,and decreased inhibiting ferroptosis genes GPX4,Fth1,and SLC7A11.The inhibitor Fer-1 can reduce the levels of CK-MB and c Tn I,improve the pathological changes of myocarditis,reduce myocardial iron overload,and down regulate the expression of ferroptosis promoting genes,thus saving the myocardial injury of viral myocarditis.(2)In vitro HeLa cells,ferroptosis mainly occurs at 48 hours after CVB3 infection,due to an increase in intracellular iron,lipid ROS and MDA,decrease in GSH,and an increase in ACSL4 expression and inhibition of GPX4 m RNA and protein expression.After the intervention of two ferroptosis inhibitors,Fer-1 and DFO,the cell survival rate increased,the viral replication of CVB3 in cells decreased,ferroptosis markers such as free iron,MDA,and lipid ROS decreased,and reducing ACSL4 levels and increasing GPX4 levels.(3)The transferrin receptor(TFRC)is obviously increased induced by CVB3 infection in HeLa cell,and si TFRC can improve the cell survival rate in CVB3 infection and inhibit CVB3 virus replication.(4)TFRC expression is transferred from the cell membrane to the nucleus with CVB3 infection,and regulated by nuclear transcription factor Sp1.Sp1 can combine with specific position of TFRC promoter to regulate TFRC gene transcription.CVB3 regulates cell ferroptosis by Sp1/TFRC/Fe axis.Downregulation of Sp1 can cause a decrease in TFRC expression and intracellular iron concentration;Overexpression of Sp1 can increase the expression of TFRC,increase the intracellular iron,and aggravate ferroptosis.Conclusions(1)Ferroptosis is involved in the pathogenesis of CVB3 infected myocarditis.(2)Ferroptosis inhibitors Fer-1 and DFO can rescue the cell death of CVB3 infection and effectively inhibit the replication of intracellular virus in HeLa cells.(3)TFRC is the key molecule of ferroptosis in CVB3 infection and regulation the Sp1/TFRC/Fe axis can inhibit cell ferroptosis in CVB3 infection.