ATPR Alleviates Lipopolysaccharide-induced Acute Myocardial Injury In Mice By Inhibiting Ferroptosis

Objective To investigate the effect and mechanism of ATPR on lipopolysaccharide-induced acute myocardial injury in C57BL/6 mice.Methods Male mice of C57BL/6 strain were randomly divided into control group,LPS group,LPS+ATPR group and LPS+ Ferrostatin-1(Fer-1)group.The mice in the LPS+ATPR group and LPS+ Fer-1 group were injected intraperitoneally with ATPR(15mg/kg-d)and Fer-1(2 mg/kg-d),respectively,and control group and LPS group were given solvent,and after one week of continuous administration,LPS group,LPS+ATPR group and LPS+ Fer-1 group were injected intraperitoneally with LPS(6 mg/kg),and 6h later All mice were executed.The m RNA levels of IL-6 and TNF-α in the heart tissues were detected by q PCR;the changes of heart tissues were observed by H&E staining;the changes of myocardial mitochondrial structure were observed by transmission electron microscope(TEM);Western Blot(WB)to detect the expression of Ferroptosis-related proteins GPX4,FTH1,SLC7A11,ACSL4 and related regulatory proteins(NRF2,Keap1,P62).H9C2 was cultured,and cell proliferation was detected by MTT assay,6 concentrations of ATPR pretreatment groups(2,4,6,8,10,16 μM)were set,and the changes of proliferation in the no-pretreatment group and ATPR pretreatment group were compared after LPS stimulation.After 24 h of pretreatment,LPS was added to the LPS group,LPS+ATPR group and LPS+ Fer-1 group at a concentration of 400ng/ml.WB was performed to detect relevant proteins in ferroptosis pathway and immunoprecipitation was performed to detect the effect of LPS on the interaction between P62 and Keap1.Results Compared with normal mice,the serum levels of MDA increased,GSH decreased,and the ratio of reduced glutathione to oxidized glutathione(GSH/GSSG)decreased in the LPS group.Compared with LPS group,MDA content decreased,GSH content increased and GSH/GSSG increased in LPS+ATPR group and LPS+Fer-1 group;m RNA levels of IL-6 and TNF-α in cardiac tissue increased after mice received LPS stimulation,decrease in LPS+ ATPR group and LPS+Fer-1 group.There was no significant difference in H&E staining of myocardial tissues of mice after LPS stimulation.Observation under TEM showed that myocardial mitochondria in the LPS group showed a decrease or disappearance of cristae compared with the control group,and the number of mitochondrial cristae in ATPR and Fer-1 pretreated mice was increased and intact.The results of MTT experiments showed that the concentration of ATPR pretreatment at 10 μM effectively attenuated the effect of LPS.Compared with the control group,the expression of GPX4,FTH1,SLC7A11,NRF2,and P62 proteins were reduced in mouse myocardial tissue or H9C2 cells after LPS stimulation,and the expression of ACSL4 and Keap1 proteins were basically not different.The expression levels of GPX4,FTH1,SLC7A11,NRF2,and P62 proteins increased in LPS+ATPR group and LPS+Fer-1 group of mice or H9C2.Immunoprecipitation results showed that LPS attenuated the interaction between P62 and Keap1,and ATPR pretreatment of cells increased the interaction between P62 and Keap1.Conclusion ATPR alleviates lipopolysaccharide-induced acute myocardial injury in mice by inhibiting Ferroptosis...