肝细胞铁死亡对糖尿病大鼠肝脏缺血再灌注损伤的影响

Effect of ferroptosis on hepatic ischemia reperfusion injury in diabetic rats model

目的探讨肝细胞铁死亡对糖尿病(DM)大鼠肝脏缺血再灌注损伤(HIRI)的影响。方法采用随机数表法将40只健康雄性SD大鼠分为4组:假手术(Sham)组、HIRI组、DM组、DM+HIRI组。通过连续4周喂养高脂高糖饲料复合腹腔注射链脲佐菌素的方法建立DM模型,并在此基础上采用部分肝血流阻断法建立HIRI损伤模型。采用酶联免疫吸附测定法检测大鼠胰岛素、肝功能及血脂代谢指标,化学发光法检测肝脏超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、多不饱和脂肪酸(PUFA)及脂氧合酶-1(LOX-1)含量,HE染色法评估肝组织病理学变化,蛋白质印迹法检测肝细胞铁死亡相关蛋白长链脂酰辅酶A合成酶4(ACSL4)和谷胱甘肽过氧化物酶4(GPX4)的表达水平。结果与Sham组相比,DM组大鼠空腹血糖、胰岛素、胰岛素抵抗指数、血清总胆固醇、甘油三酯及肝脏PUFA含量均显著升高,差异具有统计学意义(P<0.05)。HE染色提示DM组大鼠肝细胞呈脂肪变性,DM+HIRI组大鼠肝细胞广泛气球样变及坏死。与HIRI组相比,DM+HIRI组大鼠血清总胆固醇[(5.87±0.76)比(1.34±0.2)mmol/L]、甘油三酯[(2.93±0.47)比(0.71±0.34)mmol/L]、丙氨酸氨基转移酶[(339.5±40.09)比(155.17±18.53)U/L]、天冬氨酸氨基转移酶[(325.50±37.52)比(102.39±22.68)U/L]、PUFA[(21.58±3.01)比(8.12±0.94)mg/g]、LOX-1[(200.81±26.03)比(73.34±10.66)U/mg]含量均显著升高,差异具有统计学意义(P<0.05)。与HIRI组相比,DM+HIRI组ACSL4蛋白表达上调[(0.46±0.06)比(1.02±0.11)],GPX4蛋白表达下调[(0.43±0.07)比(0.14±0.02)],差异均具有统计学意义(P<0.05)。结论DM大鼠对HIRI的耐受性明显降低,其机制可能与肝细胞脂质代谢异常及铁死亡有关。

Objective To investigate the effect of ferroptosis on hepatic ischemia reperfusion injury in diabetic rats model.Methods Forty Sprague Dawley rats were randomly divided into four groups:sham operation group(Sham),hepatic ischemia reperfusion group(HIRI),diabetes mellitus group(DM),diabetes mellitus+hepatic ischemia reperfusion group(DM+HIRI).The diabetic rat model was established by feeding the rats with high-fat and high-sugar feed for four consecutive weeks combined with intraperitoneal injection of 50 mg/kg 1%streptozotocin,and on this basis,the hepatic ischemia reperfusion injury model was established by local hepatic blood flow occlusion.ELISA assay was used to detect insulin content,liver function and serum lipid metabolism biomarkers.Chemiluminescence method was used to detect liver superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),polyunsaturated fatty acids(PUFA)and lipoxygenase(LOX-1)contents.HE staining was used to evaluate the pathological changes of liver tissue structure,and Western blotting was used to detect the ferroptosis-related protein expression of ACSL4 and GPX4.Results Compared with Sham group,the level of fasting blood glucose,insulin,insulin resistance index,serum TC,TG and liver PUFA content of DM group were increased significantly(P<0.05),HE staining showed there were a large number of fatty degeneration of hepatocytes in DM group,and extensive ballooning and necrosis of hepatocytes in DM+HIRI group.Compared with HIRI group,level of serum TC,TG,ALT,AST and the liver PUFA and LOX-1 in DM+HIRI group were significantly increased[TC(5.87±0.76)vs(1.34±0.2)mmol/L,TG(2.93±0.47)vs(0.71±0.34)mmol/L,ALT(339.5±40.09)vs(155.17±18.53)U/L,AST(325.50±37.52)vs(102.39±22.68)U/L,PUFA(21.58±3.01)vs(8.12±0.94)mg/g,LOX-1(200.81±26.03)vs(73.34±10.66)U/m](P<0.05).Compared with HIRI group,the protein expression of ACSL4 in DM+HIRI group was up-regulated[(0.46±0.06)vs(1.02±0.11)],while the expression of GPX4 was down-regulated[(0.43±0.07)vs(0.14±0.02)]significantly(P<0.05).Conclusion The tolerance of DM rats to hepatic ischemia reperfusion injury was significantly reduced,which may be related to hepatic abnormal lipid metabolism and ferroptosis.