| Objective: Stroke is one of the leading causes of adult morbidity and mortality in most developing and developed countries.According to the World Health Organisation(WHO),15 million people worldwide are affected by stroke each year,most of whom suffer from neurological and physical disabilities or die.This is despite increasing awareness and better management of overall cardiovascular disease risk,which has led to a reduction in stroke mortality.However,there is no effective treatment for the cell death caused by stroke.There is therefore an urgent need to explore the mechanisms that lead to CNS tissue damage and death after ischemic stroke at the molecular,cellular,tissue and systemic levels.This study established ischemic stroke models of neurons and investigated the mechanisms of key genes to provide new therapeutic targets for the very early stage of ischemic stroke and improve patient prognosis.Methods: To model the very early stage and optimal time window for clinical treatment of ischemic stroke,we used HT22 cell lines to establish 3 hours(3H)and 6 hours(6H)models of oxygen and glucose deprivation(OGD),followed by extraction of total RNA from the cells for library building and sequencing.The sequencing data were subjected to differential expression genes analysis and gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signalling pathway enrichment analysis to screen for key gene.Next,we detected changes in c-fos at the gene and protein levels by real-time fluorescence quantitative PCR(RT-qPCR)as well as western blot(WB)experiments.To further investigate the mechanism of c-fos in ischemic stroke,we constructed c-fos knockdown strains of HT22 cell lines using lentivirus and examined oxidative stress,mitochondrial biogenesis,mitochondrial function and apoptosis in OGD-stimulated knockdown strains(KD)and controls(NC).Results: Analysis of the sequencing results showed that the most significantly up-regulated gene was c-fos at 3H of OGD treatment and then largely returned to normal levels at 6H.In combination with our previous study analysis of peripheral blood microarray data from ischemic stroke patients and brain tissue microarray data from rats with ischemic stroke(not yet published),c-fos was selected as the key gene.GO analysis resolved the results suggesting that c-fos mainly functions as a transcriptional regulator and is involved in the oxidative stress response.Then,by knocking down the c-fos,we found that the levels of Sod2 and GPx decreased while the levels of MDA increased,suggesting that c-fos knockdown in OGD-treated neurons leads to an increase in oxidative stress.In addition,WB results also showed that the expression of Pcg-1α and Tfam decreased after c-fos knockdown,reflecting the results that c-fos knockdown leads to impaired mitochondrial biogenesis;on the other hand,immunofluorescence results showed an increase in cytochrome c content in the cytoplasm after c-fos knockdown,suggesting that c-fos knockdown leads to increased impairment of mitochondrial function in HT22 cells.The MTT results showed a significant decrease in cell survival rate after c-fos knockdown,the WB results showed a significant increase in the Bax/Bcl-2 ratio after c-fos knockdown,and the ATP content also increased after c-fos knockdown,all three results suggested that apoptosis was significantly increased after c-fos knockdown..Conclusion: c-fos is involved in anti-oxidative stress and anti-apoptotic responses in the ultra-early stages of ischemic stroke,exerting a neuroprotective effect. |
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