大鼠体外循环后脑损伤与尿酸代谢的关系

Relationship of uric acid metabolism and brain injury post-cardiopulmonary bypass in rats

目的 探究大鼠体外循环(CPB)后脑损伤与尿酸代谢的关系。方法 将健康雄性SD大鼠分为假手术组与CPB组,每组12只。假手术组仅行血管穿刺,不进行CPB转流;CPB组建立CPB模型,转流时长110 min,结束后采集脑组织。CPB前及CPB开始后均收集微透析液1 h。采用TUNEL染色检测大鼠下丘脑室旁核(PVN)区域细胞凋亡情况,实时荧光定量PCR检测大脑皮质及下丘脑中Bax mRNA的表达,Western blotting检测凋亡相关蛋白的表达。采用RNA-seq分析两组脑组织中的差异表达基因(DEGs),基因本体论(GO)分析DEGs富集的通路,使用String在线软件和Cytoscape软件构建蛋白质-蛋白质相互作用(PPI)网络并筛选出关键基因;采用液相色谱串联质谱法(LC-MS/MS)分析CPB前后PVN处微透析液的差异代谢物并进行京都基因与基因组百科全书(KEGG)富集分析。采用尿酸测定试剂盒检测下丘脑中的尿酸浓度,采用实时荧光定量PCR检测下丘脑中尿酸代谢关键酶[黄嘌呤还原酶(XDH)、腺苷脱氨酶(ADA)]及尿酸转运体[有机阴离子转运体家族蛋白1(OAT1)、有机阴离子转运体家族蛋白3(OAT3)、ATP结合盒式转运体亚家族G成员2(ABCG2)、葡萄糖转运体9(GLUT9)]基因的表达。结果 实时荧光定量PCR检测结果显示,与假手术组比较,CPB组大鼠大脑皮质及下丘脑中Bax mRNA表达量明显增高(P<0.05)。TUNEL染色结果显示,CPB组大鼠PVN区域细胞凋亡率明显增高(19.0%±5.0%vs. 7.6%±0.8%,P=0.01)。Western blotting检测结果显示,与假手术组比较,CPB组大鼠下丘脑中Bcl-2/Bax比值明显增高(P<0.05)。假手术组与CPB组大鼠脑组织中的DEGs共2829个,其中上调基因1374个,下调基因1455个。GO富集分析显示,尿酸代谢相关通路主要富集于嘌呤核苷酸代谢及生物合成、嘌呤核苷单磷酸代谢、嘌呤核苷三磷酸代谢、嘌呤核糖核苷酸代谢及生物合成、嘌呤核糖核苷单磷酸盐代谢及生物合成、嘌呤核糖核苷三磷酸代谢及生物合成、对嘌呤化合物的反应等。两组微透析液差异代谢物共18种,其中上调代谢物13种,下调代谢物5种;KEGG富集分析得到7条显著富集的代谢通路,其中烟酸盐和烟酰胺代谢通路与尿酸代谢密切相关。RNA-seq与微透析液LC-MS/MS分析结果均提示CPB组尿酸代谢发生改变。CPB后下丘脑组织中尿酸浓度明显升高(P<0.01),XDH、ADA mRNA表达量明显增高(P<0.05),ABCG2、OAT1、OAT3、GLUT9 mRNA表达量明显降低(P<0.001)。结论 CPB过程中脑组织尿酸代谢发生了改变,而尿酸代谢改变可能是CPB后脑损伤的一个重要机制。

Objective To investigate the relationship between uric acid metabolism and brain injury following cardiopulmonary bypass(CPB)in rats.Methods Healthy male SD rats were randomly assigned to either a Sham group or a CPB group,each comprising 12 rats.The Sham group only underwent vascular puncture and did not perform CPB conversion,while the CPB group was subjected to a CPB procedure with a perfusion duration of 110 min,and the brain tissue was collected post-procedure.Microdialysate was collected 1 h before and after CPB initiation.Apoptosis in the paraventricular nucleus(PVN)was assessed using TUNEL staining,and the expression of Bax mRNA in cerebral cortex and hypothalamus was determined via real-time quantitative PCR.Apoptosis-related protein expression was analyzed by Western blotting.Differentially expressed genes(DEGs)were identified through RNA-sequencing between brain tissues of two groups,and Gene Ontology(GO)analysis was performed to identify enriched pathways among the DEGs.Protein-protein interaction(PPI)networks were constructed using String and Cytoscape softwares to identify key genes.Liquid chromatography tandem mass spectrometry(LC-MS/MS)was employed to analyze differential metabolites in the PVN before and after CPB,with Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis constructed subsequently.Uric acid levels in the hypothalamus was measured using a uric acid assay kit,and the expression of key enzymes of uric acid metabolism[xanthine reductase(XDH),adenosine deaminase(ADA)]and uric acid transporter[organic anion transporter family protein 1(OAT1),organic anion transporter family protein 3(OAT3),ATP-binding cassette transporter subfamily G member 2(ABCG2),glucose transporter 9(GLUT9)]genes in the hypothalamus was evaluated by real-time quantitative PCR.Results Real-time quantitative PCR revealed a significant upregulation of Bax mRNA in the cerebral cortex and hypothalamus of CPB group compared to Sham group(P<0.05).TUNEL staining indicated a significantly higher apoptosis rate of cells in PVN region in CPB group than that in Sham group(19.0%±5.0%vs.7.6%±0.8%,P=0.01).Western blotting showed a significantly increased Bcl-2/Bax ratio in the hypothalamus of CPB group compared to Sham group(P<0.05).A total of 2829 DEGs were identified between Sham group and CPB group,with 1374 upregulated genes and 1455 downregulated genes.Uric acid metabolism-related pathways were predominantly enriched in purine nucleoside metabolism and biosynthesis,purine nucleoside monophosphate metabolism,purine nucleoside triphosphate metabolism,purine ribonucleotide metabolism and biosynthesis,purine ribonucleoside monophosphate metabolism and biosynthesis,purine ribonucleoside triphosphate metabolism and biosynthesis,and reaction to purine compounds.Eighteen differential metabolites were identified in the microdialysate,with 13 upregulated and 5 downregulated metabolites.KEGG enrichment analysis identified 7 significantly enriched metabolic pathways,among which the nicotinate and nicotinamide metabolism pathways were closely related to uric acid metabolism.Both RNA-sequencing and LC-MS/MS analysis suggested alterations in uric acid metabolism in CPB groups.Post-CPB,uric acid concentration in the hypothalamic tissue significantly increased(P<0.01),and the expression of XDH and ADA mRNA in the hypothalamus were significantly increased(P<0.05),while the expression of ABCG2,OAT1,OAT3 and GLUT9 mRNA significantly decreased(P<0.001).Conclusion Uric acid metabolism in brain is altered during CPB,which may be an important mechanism for brain injury following CPB.