二甲双胍对肝脏缺血再灌注损伤的影响及其机制

Effect of metformin on hepatic ischemia-reperfusion injury and mechanism

目的探讨二甲双胍对肝脏缺血再灌注损伤的影响及其机制。方法30只C57BL/6小鼠随机分为假手术组(Sham组)、缺血再灌注组(I/R组)、缺血再灌注组+二甲双胍组(I/R+MET组)。I/R组和I/R+MET组小鼠建立肝脏缺血再灌注损伤模型,Sham组小鼠不建模。I/R+MET组小鼠术前每日二甲双胍200 mg/kg腹腔注射,术前24 h停止给药。Sham组和I/R组小鼠术前每日腹腔注射等体积生理盐水。3组小鼠在术后6 h,取静脉血,检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)浓度。原位缺口末端标记法(TUNEL)染色试剂盒分析3组肝脏组织细胞凋亡。放射免疫法检测3组小鼠氧化应激反应。酶联免疫吸附试验(ELISA)分析3组小鼠肝脏炎性因子水平变化。蛋白质免疫印迹分析3组小鼠肝脏组织腺苷酸活化蛋白激酶(AMPK)、UNC-51样激酶(ULK1)、天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)、自噬底物p62蛋白表达水平。组间计量数据比较采用单因素方差分析。结果I/R+MET组小鼠血清ALT和AST水平[(62.37±8.07)、(121.51±20.45)U/L]明显低于I/R组[(140.53±13.07)、(187.19±18.73)U/L],差异有统计学意义(t=18.150、7.489,P<0.05)。I/R+MET组小鼠肝脏组织炎性因子[(55.70±6.36)、(39.62±6.66)、(64.12±5.33)pg/g]低于I/R组[(99.15±7.45)、(82.73±8.90)、(102.40±13.17)pg/g],差异有统计学意义(t=18.580、14.030、9.630,P<0.05)。I/R+MET组小鼠血清MDA水平[(1.03±0.05)μmol/ml]明显低于I/R组[(1.73±0.14)μmol/ml],差异有统计学意义(t=14.970,P<0.05)。I/R+MET组小鼠血清SOD水平[(129.70±7.12)U/ml]明显高于I/R组[(99.50±10.66)U/ml],差异有统计学意义(t=14.970,P<0.05)。I/R+MET组小鼠肝脏组织TUNEL染色阳性率[(13.57±2.01)%]明显低于I/R组[(26.40±4.05)%],差异有统计学意义(t=8.967,P<0.05)。I/R+MET组小鼠肝脏组织AMPK磷酸化和ULK1磷酸化水平(1.25±0.06、1.37±0.09)明显高于I/R组(0.65±0.07、0.96±0.06),差异有统计学意义(t=19.480、11.670,P<0.05)。I/R+MET组小鼠肝脏组织自噬底物蛋白p62水平(0.67±0.08)明显低于I/R组(0.98±0.05),差异有统计学意义(t=10.050,P<0.05)。结论二甲双胍通过激活AMPK,促进细胞自噬,缓解肝脏缺氧缺血引起的氧化应激和炎性反应,对缺血再灌注损伤肝脏起保护作用。

Objective To investigate the effects of metformin on hepatic ischemia-reperfusion injury(IRI)and its molecular mechanism.Methods Totally,30 C57BL/6 mice were randomly divided into Sham group,ischemia reperfusion group(I/R group),ischemia reperfusion group+metformin group(I/R+MET group).Liver IRI model was established in I/R group and I/R+MET group,but not in Sham group.Mice in I/R+MET group were intraperitoneally injected with 200 mg/kg metformin daily before surgery,and the administration was stopped 24 h before surgery.Rats in Sham group and I/R group were intraperitoneally injected with equal volume of normal saline daily before surgery.The serum glutamic pyruvic transaminase(ALT)and glutamic oxaloacetic transaminase(AST)concentrations in the three groups were determined by venous blood samples 6 h after operation.The apoptosis of liver cells in the three groups was analyzed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining kit.The oxidative stress reaction of the three groups of rats was detected by radioimmunoassay.The changes of liver inflammatory factors in three groups of mice were analyzed by enzyme linked immunosorbent assay(ELISA).AMP-activated protein kinase(AMPK)and UNC-51 like kinase 1(ULK1)and autophagy substrate p62 protein expression levels in the liver tissues of the three groups of mice were analyzed by Western blotting.One-way analysis of variance was used to compare the measurement data between groups.Results The levels of serum ALT and AST in I/R+MET group[(62.37±8.07),(121.51±20.45)U/L]were significantly lower than those in I/R group[(140.53±13.07),(187.19±18.73)U/L,t=18.150,7.489,P<0.05].Inflammatory factors of mice liver tissue in the I/R+MET group[(55.70±6.36),(39.62±6.66),(64.12±5.33)pg/g]were lower than in the I/R group[(99.15±7.45),(82.73±8.90),(102.40±13.17)pg/g,t=18.580,14.030,9.630,P<0.05].The serum MDA level in the I/R+MET group[(1.03±0.05)μmol/ml]was significantly lower than that in the I/R group[(1.73±0.14)μmol/ml,t=14.970,P<0.05].The serum SOD level of mice in the I/R+MET group[(129.70±7.12)U/ml]was significantly higher than that in the I/R group[(99.50±10.66)U/ml,t=14.970,P<0.05].The positive rate of TUNEL staining in liver tissue of mice in the I/R+MET group[(13.57±2.01)%]was significantly lower than that in the I/R group[(26.40±4.05)%,t=8.967,P<0.05].The phosphorylation levels of AMPK and ULK1 in liver tissue of mice in the I/R+MET group(1.25±0.06,1.37±0.09)were significantly higher than those in the I/R group(0.65±0.07,0.96±0.06,t=19.480,11.670,P<0.05).The level of autophagy substrate protein p62 in liver tissue of mice in the I/R+MET group(0.67±0.08)was significantly lower than that of mice in the I/R group(0.98±0.05,t=10.050,P<0.05).Conclusion Metformin can promote autophagy by activating AMPK.It can relieve oxidative stress and inflammation caused by liver hypoxia and ischemia,and protect liver from IRI.