大剂量维生素B6对大鼠严重创伤后应激性肝细胞死亡方式的影响及可能机制  

Effect of high-dose vitamin B6 on stress-induced liver cell death in rats with severe trauma and the possible mechanism

作　　者：张寅杰 王之怀 唐雪琳 周海洋 高鹏 朱春富 贾中芝 岳茂兴 秦锡虎[1,2] Zhang Yinjie;Wang Zhihuai;Tang Xuelin;Zhou Haiyang;Gao Peng;Zhu Chunfu;Jia Zhongzhi;Yue Maoxing;Qin Xihu(Graduate School,Nanjing Medical University,Nanjing 210000,China;Department of Hepatobiliary Surgery,Changzhou No.2 People′s Hospital Affiliated to Nanjing Medical University,Changzhou 213000,China;Department of Intensive Care Unit,Changzhou No.2 People′s Hospital Affiliated to Nanjing Medical University,Changzhou 213000,China;Trauma Center,Changzhou No.2 People′s Hospital Affiliated to Nanjing Medical University,Changzhou 213000,China;Department of Vascular Intervention,Changzhou No.2 People′s Hospital Affiliated to Nanjing Medical University,Changzhou 213000,China)

机构地区：[1]南京医科大学研究生院,南京210000 [2]南京医科大学附属常州第二人民医院肝胆外科,常州213000 [3]南京医科大学附属常州第二人民医院ICU,常州213000 [4]南京医科大学附属常州第二人民医院创伤中心,常州213000 [5]南京医科大学附属常州第二人民医院介入血管科,常州213000

出　　处：《中华创伤杂志》2024年第6期558-568,共11页Chinese Journal of Trauma

基　　金：常州市社会发展项目(CE20215040);常州市2022卫生健康卓越人才项目(2022CZZY005)。

摘　　要：目的探讨大剂量维生素B6对大鼠严重创伤后应激性肝细胞死亡方式的影响及可能机制。方法选取32只雄性SD大鼠,按随机数字表法分为假手术组、假手术+B6组、创伤组、创伤+B6组,每组8只。采用腹壁创伤、双侧股骨骨折、单侧颅脑损伤和股动脉抽血4 ml的方法构建大鼠严重创伤模型。假手术+B6组、创伤+B6组予生理盐水+大剂量维生素B6治疗;假手术组、创伤组仅滴注生理盐水治疗。在伤后36 h收集大鼠肝脏组织:(1)二代测序筛选创伤组与创伤+B6组大鼠肝组织的差异基因,并分析可能参与的细胞死亡方式。(2)验证大剂量维生素B6能否影响假手术组、假手术+B6组、创伤组、创伤+B6组大鼠肝细胞的各种细胞死亡方式,包括通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色验证凋亡;通过混合谱系激酶结构域样蛋白(MLKL)免疫组化染色验证坏死性凋亡;通过透射电镜验证自噬;通过检测各组大鼠肝组织内丙二醛(MDA)与氧化型谷胱甘肽水平、二氨基联苯氨(DAB)加强法普鲁士蓝染色、透射电镜、酰基辅酶A合成酶长链家族成员4(ACSL4)免疫组化染色验证铁死亡。(3)生物信息学分析[基因本体(GO)、京都基因与基因组百科全书(KEGG)、基因富集分析(GSEA)]创伤组与创伤+B6组大鼠肝组织测序结果代表的生物学过程及信号通路。结果(1)创伤组与创伤+B6组大鼠肝组织存在显著差异表达的基因有344个(上调137个,下调207个),涉及与凋亡、自噬、坏死性凋亡、铁死亡和焦亡相关的18个基因。(2)假手术组、假手术+B6组、创伤组、创伤+B6组TUNEL染色未体现明显凋亡差异;创伤后肝组织MLKL蛋白表达量增加,其中创伤+B6组MLKL蛋白表达量低于创伤组;电镜下可见创伤后大鼠肝细胞的自噬活动显著增加,其中创伤+B6组的细胞自噬活动低于创伤组;假手术组、假手术+B6组、创伤组、创伤+B6组大鼠肝�Objective To investigate the effect of high-dose vitamin B6 on stress-induced liver cell death in rats with severe trauma and its possible mechanism.Methods Thirty-two male SD rats were selected and divided into sham surgery group,sham surgery+B6 group,trauma group,and trauma+B6 group by using a random number table,with 8 rats in each group.Rat models of severe trauma were established by inducing abdominal wall injury,bilateral femoral fractures,unilateral cranial injury,and withdrawal of 4 ml blood from the femoral artery.The sham surgery+B6 group and trauma+B6 group were treated with saline solution plus high-dose vitamin B6,while the sham surgery group and trauma group with infusion of saline solution only.At 36 hours after injury,rat liver tissues were collected for the following experiments:(1)the genes differentially expressed in the liver tissues of the rats of the trauma group and the trauma+B6 group were screened with next-generation sequencing,followed by an analysis of the possible involvement of cell death pathways;(2)validation was conducted to ascertain whether high-dose vitamin B6 could influence various cell death pathways in the liver cells in the sham surgery group,sham surgery+B6 group,trauma group,and trauma+B6 group:apoptosis was confirmed through terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining;necroptosis was verified by mixed lineage kinase domain-like protein(MLKL)immunohistochemical staining;autophagy was examined via transmission electron microscopy;ferroptosis was confirmed by detecting oxidative malondialdehyde(MDA)levels,oxidized glutathione levels,Prussian blue staining with diaminobenzidine(DAB)enhancement,transmission electron microscopy,and immunohistochemical staining for acyl-CoA synthetase long-chain family member 4(ACSL4);(3)Biological information analyses[Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Gene Enrichment analysis(GSEA)]were performed for biological processes and signaling pathways represented by liver tissue se

关 键 词：维生素B6 应激障碍 创伤性 肝功能不全 细胞死亡 

分 类 号：R575[医药卫生—消化系统]