跨膜转运蛋白zgc:158423功能缺失导致氧化磷酸化功能紊乱诱发脑出血OA

Objective To explore the mechanism by which the SLC3A2 homologous gene zgc:158423 influences intracerebral hemorrhage in adult zebrafish.Methods Clustered regularly interspaced short palindromic repeats associated protein 9(CRISPR/Cas9)technology was employed to generate zgc:158423 zebrafish mutants.Vascular endothelial cell-labeled transgenic fish Tg(kdrl:eGFP)were crossed with zgc:158423 mutants.zgc:158423-/-;Tg(kdrl:eGFP)mutants were obtained through genotyping and screening.Confocal laser scanning microscopy was used to observe cerebral vascular development phenotypes in 4 and 10 d post fertilization(dpf)larvae.Brain tissue clearing and light-sheet imaging were employed to examine cerebral vascular development in adult homozygous mutants,with cerebral hemorrhage rates quantified across different age groups.Transcriptome sequencing and bioinformatics analysis were performed on whole-brain tissue from adult mutants,with reverse transcription quantitative polymerase chain reaction(RT-qPCR)used to detect related gene expression.Results The zebrafish gene zgc:158423 is a direct homolog of human.SLC3A2,exhibiting high sequence conservation across species.Bioinformatics analysis and in situ hybridization experiments confirmed that zgc:158423 is specifically highly expressed in brain vascular endothelial cells during wild-type zebrafish development.zgc:158423 homozygous mutants exhibited a brain hemorrhage phenotype at 5,7 and 9 months post fertilization(mpf)with significantly increased hemorrhage rates;leakage was significantly increased in the brain tissue of the 9 mpf mutant,indicating impaired blood-brain barrier function.Ribonucleic acid sequencing(RNA-Seq)result identified 2,148 differentially expressed genes,comprising 950 upregulated and 1,198 downregulated genes.After knocking out zgc:158423,the blood-brain barrier(BBB)-related gene set and wingless/integrated(WNT)signaling pathway-related genes were significantly affected.kyoto encyclopedia of genes and genomes(KEGG)metabolic pathway analysis indicated enrichment of differentially expressed genes primarily involved in oxidative phosphorylation signaling.RT-qPCR detection revealed significant downregulation of key pathway genes atp5l,atp5fld,cox8a,cox 17,and ndufc2,while cox 15 expression was upregulated,indicating disrupted oxidative phosphorylation signaling.There was a reduction in ATP,NAD+,and the NAD+/NADH ratio in mutant brain tissue,while NADH significantly increased,further indicating impaired oxidative phosphorylation.Conclusions Deletion of the zgc:158423 gene in zebrafish disrupts oxidative phosphorylation signaling,inducing cerebral hemorrhage in adult zebrafish.