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组大鼠肝组织内MDA水平分别为（0.20±0.05）nmol/mg、（0.17±0.07）nmol/mg、（0.69±0.11）nmol/mg、（0.52±0.07）nmol/mg（ P<0.01），其中创伤组水平最高，创伤+B6组较创伤组降低；上述四组大鼠肝组织内氧化型谷胱甘肽水平分别为（11.75±2.09）μmol/g、（11.69±1.66）μmol/g、（19.75±3.40）μmol/g、（14.51±1.46）μmol/g（ P<0.01），其中创伤组水平最高，创伤+B6组较创伤组降低；创伤后肝组织内铁沉积显著增加，其中创伤+B6组的肝组织内铁沉积低于创伤组；电镜下创伤+B6组线粒体膜密度显著低于创伤组；创伤+B6组ACSL4蛋白表达量低于创伤组。（3）GO、KEGG、GSEA富集分析提示大剂量维生素B6可能通过增强肝细胞胆固醇合成代谢、减轻氧化应激，实现减轻创伤后肝细胞损伤，恢复肝细胞正常功能。 结论:大剂量维生素B6通过作用于坏死性凋亡、自噬、铁死亡来减轻大鼠严重创伤后应激性肝损伤，其分子机制可能与增强肝细胞胆固醇合成代谢、减轻氧化应激有关。

abstractsObjective: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 sequencing results of rats between the trauma group and the trauma+B6 group.Results:(1) In the liver tissues of rats, there were 344 significantly differentially expressed genes between the trauma group and trauma+B6 group, comprising 137 upregulated genes and 207 downregulated genes, of which 18 genes were associated with apoptosis, autophagy, necroptosis, ferroptosis, and pyroptosis. (2) No significant differences were found in TUNEL staining among the sham surgery group, sham surgery+B6 group, trauma group or trauma+B6 group; MLKL protein expression levels in the liver tissues after trauma were improved, of which the trauma+B6 group was lower than that of the trauma group; Electron microscopy showed that autophagic activity in the liver cells were significantly increased after trauma, which was significantly lower of the trauma+B6 group than that of the trauma group; MDA levels in the rat liver tissues were (0.20±0.05)nmol/mg, (0.17±0.07)nmol/mg, (0.69±0.11)nmol/mg and (0.52±0.07)nmol/mg in the sham surgery group, sham surgery+B6 group, trauma group, and trauma+B6 group respectively ( P<0.01), with the trauma group having the highest MDA levels and trauma+B6 group having lower MDA levels than the trauma group; Oxidized glutathione levels in the liver tissues of the four groups were (11.75±2.09)μmol/g, (11.69±1.66)μmol/g, (19.75±3.40)μmol/g, and (14.51±1.46)μmol/g respectively ( P<0.01), with the trauma group having the highest levels and trauma+B6 group having lower levels than the trauma group; Significantly increased iron deposition was observed in the liver tissues after trauma, with lower iron deposition in trauma+B6 group than the trauma group; Electron microscopy revealed significantly lower mitochondrial membrane density in the trauma+B6 group compared to the trauma group. ACSL4 protein expression level was lower in the trauma+B6 group compared to the trauma group; (3) GO, KEGG and GSEA enrichment analyses suggested that high-dose vitamin B6 may enhance cholesterol synthesis metabolism in the liver cells and alleviate oxidative stress to reduce liver cell damage and restore normal liver cell function after trauma. Conclusions:High-dose vitamin B6 attenuates stress-induced liver injury in rats with severe trauma by inhibiting the progression of necroptosis, autophagy and ferroptosis. Its molecular mechanism may be associated with enhanced hepatic cholesterol synthesis metabolism and alleviation of oxidative stress in the liver cells.