During the hyperacute phase of intracerebral hemorrhage (ICH), the mass effect and blood components mechanically lead to brain damage and neurotoxicity. Our findings revealed that the mass effect and transferrin precipitate neuronal oxidative stress and iron uptake, culminating in ferroptosis in neurons. M6A (N6-methyladenosine) modification, the most prevalent mRNA modification, plays a critical role in various cell death pathways. The Fto (fat mass and obesity-associated protein) demethylase has been implicated in numerous signaling pathways of neurological diseases by modulating m6A mRNA levels. Regulation of Fto protein levels in neurons effectively mitigated mass effect-induced neuronal ferroptosis. Applying nanopore direct RNA sequencing, we identified voltage-dependent anion channel 3 (Vdac3) as a potential target associated with ferroptosis. Fto influenced neuronal ferroptosis by regulating the m6A methylation of Vdac3 mRNA. These findings elucidate the intricate interplay between Fto, Vdac3, m6A methylation, and ferroptosis in neurons during the hyperacute phase post-ICH and suggest novel therapeutic strategies for ICH.
Ferroptosis/physiology* ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics* ; Animals ; Neurons/metabolism* ; Transferrin/pharmacology* ; Mice ; Methylation ; Mice, Inbred C57BL ; Adenosine/metabolism* ; RNA, Messenger/metabolism* ; Male ; Oxidative Stress/physiology*

Objective To explore the protective effect and related mechanism of microRNA(miR)-375-5p on heart failure(HF)rats.Methods Fifty male Sprague Dawley rats were randomly divided into the sham operation group,HF group,miR-NC group,miR-375-5p group and miR-375-5p+Compound C(CC)group,with 10 rats in each group.The rats in the HF group,miR-NC group,miR-375-5p group and miR-375-5p+CC group were intraperitoneally injected with adriamycin solution to prepare HF models,and the rats in the sham operation group were intraperitoneally injected with an equal amount of NaCl solution.On the next day after modeling,the rats in the miR-375-5p group were intravenously injected with 100 μL of miR-375-5p mimics via tail vein;the rats in the miR-NC group were intravenously injected with 100 μL of miR-NC mimics via tail vein;the rats in the sham operation group and HF group were intravenously injected with an equal volume of normal saline via tail vein;the rats in the miR-375-5p+CC group were intravenously injected with 100 μL of miR-375-5p mimics and adenosine monophosphate-activated protein kinase(AMPK)inhibitor CC(0.2 mg·kg-1)via tail vein;and the rats in each group were administered once daily for 4 consecutive weeks.The cardiac function parameters of rats in each group were detected by color Doppler ultrasound;the expression levels of miR-375-5p in myocardial tissues of rats in each group were detected by reverse transcription polymerase chain reaction;the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,malondial-dehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in serum of rats in each group were measured by enzyme-linked immunosorbent assay;the pathological changes in myocardial tissues of rats in each group were observed by hematoxylin-eosin staining;the myocardial cell apoptosis of rats in each group was detected by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay;and the relative expression levels of phosphorylated-AMPK(p-AMPK),AMPK and sirtuin 3(SIRT3)proteins in myocardial tissues of rats in each group were measured by Western blot.Results Compared with the sham operation group,the left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5 p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats in the HF group,miR-NC group and miR-375-5p group significantly decreased,while the left ventricular end-diastolic dimension(LVEDD),left ventricular end-systolic dimension(LVESD),myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1β and MDA significantly increased(P＜0.05).There was no significant differences in LVEF,LVFS,LVEDD,LVESD,myocardial cell apoptosis rate,serum levels of TNF-α,IL-6,IL-1β,MDA and GSH,SOD activity,p-AMPK/AMPK,miR-375-5p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats between the miR-NC group and the HF group(P＞0.05).Compared with the HF group,LVEF,LVFS,serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats in the miR-375-5p group significantly increased,while the LVEDD,LVESD,myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1β and MDA significantly decreased(P＜0.05).Compared with the miR-375-5p group,LVEF,LVFS,serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5p expression levels,and relative expression level of SIRT3 protein in myocardial tissues of rats in the miR-375-5p+CC group significantly decreased,while the LVEDD,LVESD,myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1β and MDA significantly increased(P＜0.05).Myocardial cells of rats in the sham operation group were regularly arranged,with obvious nuclei and no inflammatory cell infiltration;myocardial cells of rats in the HF group were altered morphologically and arranged irregularly,with enlarged cell gaps,lighter staining,myocardial fibrosis,and a large number of inflammatory cell infiltration.There was no significant difference in the pathological changes of myocardial tissues between the HF group and the miR-NC group.Compared with the HF group,myocardial cells of rats in the miR-375-5p group were arranged orderly,and the degree and extent of necrocytosis were reduced.The pathological changes in myocardial cells of rats were similar between the miR-375-5p+CC group and the HF group.Conclusion miR-375-5p can inhibit the apoptosis of myocardial cells in rats with HF,which may be related to its activation of the AMPK/SIRT3 signaling pathway.