Mechanism of Hydroxysafflor Yellow A Inhibiting Neuronal Apoptosis after Ischemic Stroke through JAK2/STAT3 Signaling Pathway Based on Bioinformatics Analysis
Objective:To screen and identify key genes related to ischemic stroke(IS)by bioinformatics technology,and to explored the effects and its mechanism of hydroxysafflor yellow A(HSYA)on neuronal apoptosis after cerebral ischemic injury.Methods:Is-related sample data were obtained from GEO database,and differential expression gene(DEGs)analysis and gene enrichment analysis were performed to obtain key genes and key signaling pathways,and relevant verification was carried out through in vivo and in vitro experiments.The Sprague-Dawley rat middle cerebral artery occlusion and reperfusion model(MCAO/R)was established.The phosphorylation level of Janus kinase 2(JAK2)/signal transduction transcriptional activator 3(STAT3)pathway and the expression of apoptosis-related proteins were detected by Western Blot and immunofluorescence.Mitochondrial membrane potential probes were used to detect mitochondrial membrane potential to determine the level of apoptosis.The glucose-oxygen deprivation/reoxygen model(OGD/R)was established in HT-22 hippocampal neurons,and JAK2/STAT3 signaling pathway inhibitors were used to verify the mechanism of HSYA on neuronal apoptosis.Results:Bioinformatics analysis of the GSE22255 dataset revealed 23 significantly up-regulated DEGs and 2 significantly down-regulated DEGs.Enrichment analysis showed that it was related to lipopolysaccharide response,apoptosis regulation,inflammatory response,acute inflammatory response regulation,and molecular intervention signal pathway.In terms of biological processes,by establishing functional networks of feature genes,it was found that feature genes were related to acute inflammatory response,regulation of apoptosis signaling pathway,lipopolysaccharids-mediated response,and homeostasis molecular response.Gene set enrichment analysis showed that nuclear factor κB(NF-κB)signaling pathway,heme metabolism signaling pathway,apoptosis-related signaling pathway,JAK/STAT3 signaling pathway and P53 signaling pathway mediated by tumor necrosis factor α(TNF-α)played some key role.JAK2/STAT3 signaling pathway and apoptosis-related pathways were screen out as the research focus.Compared with the sham group,the expressions of phosphorylated JAK2(p-JAK2),phosphorylated STAT3(p-STAT3)and pro-apoptosis-related proteins in the MCAO/R group increased(P＜0.001),while the expressions of anti-apoptosis-related proteins decreased(P＜0.001).HSYA inhibited phosphorylation activation of JAK2/STAT3 signaling pathway and neuronal apoptosis(P＜0.01).Vitro experiments showed that JAK2/STAT3 pathway was activated by phosphorylation in the OGD/R group,the expression of pro-apoptosis-related proteins was more than that in the normal group(P＜0.001),and the expression of anti-apoptosis-related proteins was less than that in the normal group(P＜0.001).The phosphorylation of JAK2 and STAT3 decreased after the addition of inhibitor AG490(P＜0.01).Compared with the normal group,the expression levels of apoptosis-associated protein cysteine protease 3(Cleaved Caspase-3)and Bcl-2 associated X protein(Bax)in the OGD/R group increased(P＜0.001),and the expression of Bcl-2 decreased(P＜0.001).HSYA inhibited neuronal apoptosis(P＜0.01).Conclusion:JAK2/STAT3 signaling pathway and apoptosis-related signaling pathway played key roles after IS,and HSYA might inhibit neuronal apoptosis after ischemia and hypoxia by regulating JAK2/STAT3 signaling pathway,thereby alleviating brain injury.
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维普
