花生四烯酸12-脂氧化酶-12-氢基二十碳四烯酸-GPR31轴在肝脏再灌注肝缺血损伤中的作用  

Role of ALOX12-12-HETE-GPR31 axis in hepatic ischemia-reperfusion injury after liver reperfusion

作　　者：高杨[1] 张升宁[1] 李来邦[1] 冉江华[1] GAO Yang;ZHANG Shengning;LI Laibang;RAN Jianghua(Department of Hepatobiliary and Pancreatic Vascular Surgery,Kunming First People's Hospital,Kunming,Yunnan 650051,China)

机构地区：[1]昆明市第一人民医院肝胆胰血管外科,云南昆明650051

出　　处：《安徽医药》2022年第1期29-34,共6页Anhui Medical and Pharmaceutical Journal

基　　金：国家自然科学基金资助项目(81660112)。

摘　　要：目的探究花生四烯酸12-脂氧化酶(ALOX12)–12-氢基二十碳四烯酸(HETE)–GPR31轴在肝脏再灌注肝缺血损伤中的作用机制。方法采用8周龄雄性B6.Cg-Tg(MX1-cre)Cgn/J小鼠为研究对象,采用随机数字表法分为三组:空白对照组(n=12)、实验对照组(n=12)和实验组(n=12)。实验组小鼠进行基于胚胎干细胞的基因打靶技术制备基因敲除手术;进行肝血流阻断。肝血流阻断45 min后发送移走血管夹,以恢复血液供应。实验对照组进行肝血流阻断。空白对照组同样进行开腹但并不进行肝血流阻断。采用蛋白质印迹法(Western blotting)检测ALOX12–12-HETE–GPR31轴中基因表达水平。采用酶联免疫吸附测定(ELISA)法分别检测肝脏血清中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)三项炎性因子水平和12-HETE含量。采用原位细胞凋亡检测试剂盒检测细胞凋亡情况。采用日立7180全自动生化分析仪检测小鼠肝脏血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、AST/ALT。结果肝脏再灌注肝缺血损伤小鼠的ALOX12–12-HETE-GPR31轴中基因转录水平均高于空白对照组(P<0.05),且随着再灌注时间的延长而逐渐增大(P<0.05)。空白对照组和实验组小鼠,在肝血流阻断并再灌注后,随着再灌注时间延长ALOX12–12-HETE–GPR31轴中基因转录水平间差异无统计学意义(P>0.05)。实验对照组小鼠肝脏中IL-1β[(20.53±1.32)ng/L比(10.61±0.83)ng/L]、IL-6[(322.1±11.41)ng/L比(107.34±9.02)ng/L]、TNF-α水平[(31.78±2.42)ng/L比(11.41±0.98)ng/L]、12-HETE含量、肝脏组织细胞凋亡率、ALT[(47.94±1.48)U/L比(24.85±1.50)U/L]、AST[(54.45±3.17)U/L比(30.69±2.08)U/L]和AST/ALT[(1.23±0.04)比(0.69±0.03)]均高于空白对照组(P<0.05)。实验组小鼠各项指标低于实验对照组(P<0.05),且与空白对照组差异无统计学意义(P>0.05)。结论ALOX12表达量的上调会促进12-HETE的积累,特异性敲除ALOObjective To explore the mechanism of ALOX12–12-HETE–GPR31 axis in hepatic ischemia-reperfusion injury.Methods Eight-week-old male B6.Cg-Tg(MX1-cre)Cg n/J mice were selected as the research objects and randomly assigned into three groups:blank control group(n=12),experimental control group(n=12)and experimental group(n=12).The experimental group:mice were subjected to gene knockout operation based on embryonic stem cell gene targeting technology,and hepatic blood flow was blocked.After 45 minutes of hepatic blood flow occlusion,the removal clamp was sent to restore blood supply.The experimental control group:hepatic blood flow was blocked.Blank control group:the same laparotomy without hepatic blood flow occlusion.Western blot was used to detect gene expression in the ALOX12–12-HETE–GPR31 axis.The level of three inflammatory factors and 12-HETE content of IL-1 beta,IL-6 and TNF-αin liver serum were detected by ELISA.In situ apoptosis assay kit was used to detect the apoptosis of cells.Hitachi 7180 automatic biochemical analyzer was used to detect alanine aminotransferase(ALT),aspartate aminotransferase(AST)and AST/ALT in the liver serum of mice.Results The gene expression level of ALOX12–12-HETE–GPR31 axis in mice with hepatic reperfusion ischemia injury was higher than that in blank control group(P<0.05),and increased gradually with the prolongation of reperfusion time(P<0.05).There was no significant difference in gene expression level in ALOX12-12-HETE-GPR31 axis between blank control group and experimental group after hepatic blood flow occlusion and reperfusion(P>0.05).The levels of three inflammatory factors,IL-1beta[(20.53±1.32)ng/L vs.(10.61±0.83)ng/L],IL-6[(322.1±11.41)ng/L vs.(107.34±9.02)ng/L],TNF-α[(31.78±2.42)ng/L vs.(11.41±0.98)ng/L],12-HETE content,apoptotic rate of liver tissue,ALT[(47.94±1.48)U/L vs.(24.85±1.50)U/L],AST[(54.45±3.17)U/L vs.(30.69±2.08)U/L]and AST/ALT[(1.23±0.04)vs.(0.69±0.03)]in the experimental control group were higher than those in the blank control gr

关 键 词：再灌注损伤 肝细胞 花生四烯酸12-脂氧化酶基因 12-氢基二十碳四烯酸积累 GPR31 肝缺血 

分 类 号：R575[医药卫生—消化系统]