基于生物信息学分析探讨羟基红花黄色素A通过JAK2/STAT3信号通路抑制缺血性脑卒中后神经元凋亡的机制

Mechanism of Hydroxysafflor Yellow A Inhibiting Neuronal Apoptosis after Ischemic Stroke through JAK2/STAT3 Signaling Pathway Based on Bioinformatics Analysis

目的:利用生物信息学技术筛选并鉴定缺血性脑卒中(IS)的关键基因,基于基因富集分析结合相关实验探讨羟基红花黄色素A(HSYA)对脑缺血损伤后神经元凋亡的影响及机制。方法:从GEO数据库获得IS相关的样本数据,进行差异表达基因(DEGs)分析及基因富集分析,获得关键基因与关键信号通路,并通过体内外实验进行相关验证。建立Sprague-Dawley大鼠大脑中动脉闭塞再灌注模型(MCAO/R),采用蛋白免疫印迹法(Western Blot)和免疫荧光检测Janus激酶2(JAK2)/信号传导转录激活因子3(STAT3)通路的磷酸化水平及凋亡相关蛋白的表达;线粒体膜电位探针检测线粒体膜电位情况进而明确细胞凋亡水平。利用HT-22海马神经元细胞建立糖氧剥夺/复糖氧模型(OGD/R),使用JAK2/STAT3信号通路抑制剂进一步验证HSYA对神经元凋亡的作用机制。结果:通过生物信息学分析研究GSE22255数据集发现23个显著上调的DEGs和2个显著下调的DEGs。富集分析发现其与脂多糖的反应、细胞凋亡的调控、炎症反应、急性炎症反应调节、分子干预信号通路相关。生物过程方面,通过建立特征基因功能网络发现,特征基因与急性炎症反应、凋亡信号通路的调节、脂多糖介导的反应、稳态分子的反应等功能相关。基因集富集分析显示,肿瘤坏死因子α(TNF-α)介导的核因子κB(NF-κB)信号通路、血红素代谢信号通路、细胞凋亡相关信号通路、JAK/STAT3信号通路、P53信号通路发挥着关键作用。筛选出JAK2/STAT3信号通路和凋亡相关通路为研究重点。与假手术组比较,MCAO/R组大鼠磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)和促凋亡相关蛋白表达升高(P<0.001),抑凋亡相关蛋白表达降低(P<0.001)。HSYA干预后可抑制JAK2/STAT3信号通路磷酸化激活和神经元凋亡(P<0.01)。体外实验显示,OGD/R组JAK2/STAT3通路被磷酸化激活,促凋亡相关蛋白表达较Normal组升高(P<0.001),抑凋亡相关蛋白表达低于Normal组(P<0.001);加入抑制剂AG490后JAK2、STAT3磷酸化程度降低(P<0.01)。与Normal组比较,OGD/R组凋亡相关蛋白半胱氨酸蛋白酶3(Cleaved Caspase-3)、Bcl-2关联X蛋白(Bax)表达水平升高(P<0.001),Bcl-2表达降低(P<0.001)。HSYA抑制了神经元的凋亡(P<0.01)。结论:JAK2/STAT3信号通路和凋亡相关信号通路在IS后发挥着关键作用,HSYA可能通过调控JAK2/STAT3信号通路,抑制缺血缺氧后神经元凋亡,从而减轻脑损伤。

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 factorκB(NF-κB)signaling pathway,heme metabolism signaling pathway,apoptosis-related signaling pathway,JAK/STAT3 signaling pathway and P53 signaling pathway mediated by tumor necrosis factorα(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.