Effect and mechanism of epicardial adipose-derived exosomes on hippocampal microglia in canines with atrial fibrillation
Objective To investigate the effect of ex-osomes derived from epicardial adipose tissue (EAT) on hippocampal microglia and its mechanism. Meth-ods Eighteen male beagle canines were randomly di-vided into a sham group,pacing group,and GW4869 group,n=6 for each group.The sham group re-ceived pacemaker implantation without atrial pacing,an experimental model of AF was established by rapid atrial pacing (450 beats/min)for 2 weeks in the pacing group and the GW4869 group.The GW4869 group re-ceived intravenous GW4869 injection (an inhibitor of exosome biogenesis/release,0.3 mg/kg,once a day).All groups were locally injected with Ad-CD63-RFP in the EAT of the left atrial free wall to trace the exosomes.Exo-somes from EAT were extracted using ultracentrifugation,followed by RNA sequencing and quantitative real-time PCR (qRT-PCR)to detect RNA in exosomes and hippocampal tissues.The miRanda database was used to predict,and a luciferase reporter assay was employed to validate the target relationship between miRNA and mRNA.West-ern blotting was performed to identify EAT exosome markers (CD63,CD81,TSG101 ),and immunofluorescence was used to detect Ad-CD63-RFP in EAT and hippocampal tissues,as well as the microglial activation marker IBA-1 .In vitro,microglial cells (BV2 )were divided into control,mimics NC,cfa-miR-22 e mimics,and cfa-miR-22 e inhibitor groups.BV2 cells were treated with miRNA mimics and inhibitors,IBA-1 and RNA in BV2 cells were de-tected via immunofluorescence and qRT-PCR. Results Rapid atrial pacing increased AF susceptibility and the re-lease of EAT exosomes.The fluorescence of Ad-CD63-RFP transfected EAT in the pacing group was significantly higher than that in the sham group and GW4869 group (P<0.001 ),and the GW4869 group was higher than that in the sham group (P<0.05 );western blotting analysis showed that the EAT exosome markers (CD63,CD81,TSG101 )in the pacing group were significantly higher than those in the sham group.The fluorescence intensity of hippocampal Ad-CD63-RFP and microglial activation marker IBA-1 in the pacing group was significantly higher than in the sham group (P<0.001 ).Exosome inhibitors reversed exosome secretion and microglial cell activation.RNA sequencing showed that the differentially expressed gene of cfa-miR-22 e in EAT exosomes was upregulated,and its target gene IL-33 was downregulated in the hippocampus.The luciferase assay results showed that the fluorescence signal in the cfa-miR-22e-IL-33 WT group (0.41±0.07,n=5)in 293T cells was significantly lower than that in the miRNA-NC-IL-33 WT group (1.05±0.10,n=5,P<0.001)and the cfa-miR-22e-IL-33 MUT group (1.00±0.10,n=5,P<0.001),indicating a targeted interaction between cfa-miR-22e and IL-33.qRT-PCR results re-vealed that the levels of cfa-miR-22 e in EAT exosomes and hippocampal tissue were significantly higher in the pa-cing group canines compared to the sham group and GW4869 group (P<0.001 ),and the GW4869 group had higher levels than the sham group (P<0.001 ).In the hippocampal tissue of pacing group canines,IL-33 levels were lower than those in the sham group and GW4869 group (P<0.001 ),and the GW4869 group had lower lev-els than the sham group (P<0.001 ).In BV2 cells,the level of cfa-miR-22 e in the cfa-miR-22 e mimics group was elevated compared to the control group,mimics NC group,and the cfa-miR-22 e inhibitor group;conversely,the IL-33 level in the cfa-miR-22 e mimics group was lower than that in the control group,mimics NC group,and the cfa-miR-22e inhibitor group. Conclusion Exosomes containing cfa-miR-22e,secreted by the EAT of atrial fibril-lation canines,distribute in the hippocampus and activate hippocampal microglial cells through the cfa-miR-22 e/IL-33 signaling pathway.
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