二甲双胍在小鼠肠缺血再灌注致肝损伤中的作用及其与Akt的关系

Effect of metformin on liver injury induced by intestinal ischemia-reperfusion in mice and its relationship with Akt

目的:评价二甲双胍在小鼠肠缺血再灌注致肝损伤中的作用及其与蛋白激酶B(Akt)的关系。方法:SPF级健康雄性C57BL/6小鼠24只,采用随机数字表法分为4组(n=6):假手术组(S组)、缺血再灌注组(I/R组)、缺血再灌注+二甲双胍组(M组)和缺血再灌注+二甲双胍+Akt抑制剂Triciribine组(MT组)。采用夹闭肠系膜上动脉45 min后再灌注120 min的方法建立小鼠肠I/R模型。再灌注120 min时采集眶静脉血样,检测血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平,随后处死小鼠取肝组织,HE染色观察肝组织病理学变化,Eckhoff评分法评估肝损伤程度,检测肝组织谷胱甘肽(GSH)活性、丙二醛(MDA)含量,荧光定量PCR法测定IL-6mRNA表达,Western Blot法检测Akt及磷酸化Akt(p-AKT)、谷胱甘肽过氧化物酶4(GPX4)和酶酰基辅酶A合成酶长链4(ACSL4)的表达。结果:与S组比较,I/R组肝组织Eckhoff评分、血清ALT和AST水平升高,肝组织GSH活性降低,MDA含量升高,IL-6 mRNA表达上调(P<0.05),GPX4表达下调,p-Akt和ACSL4表达上调(P<0.05);与I/R组比较,M组肝组织Eckhoff评分、血清ALT和AST水平降低,肝组织GSH活性升高,MDA含量降低,IL-6 mRNA表达下调(P<0.05),p-Akt和GPX4表达上调,ACSL4表达下调(P<0.05);与M组比较,MT组肝组织Eckhoff评分、血清ALT和AST水平升高,肝组织GSH活性降低,MDA含量升高,IL-6 mRNA表达上调(P<0.05),p-Akt和GPX4表达下调,ACSL4表达上调(P<0.05)。结论:二甲双胍通过抑制铁死亡对小鼠肠缺血再灌注所致的肝损伤产生保护作用,其机制可能与激活Akt有关。

Objective:To evaluate the effect of metformin on liver injury induced by intestinal ischemia-re-perfusion in mice and its relationship with protein kinase B(Akt).Methods:Twenty-four SPF-grade healthy male C57BL/6 mice were randomly divided into four groups(n=6):sham operation group(S group),intestinal ischemia-reperfusion group(I/R group),metformin+ischemia-reperfusion group(M group),and metformin+ischemia-reperfusion+protein kinase B inhibitor Triciribine(TCN)group(MT group).The mouse model of intestinal ischemia-reperfusion injury was established by clamping the superior mesenteric artery for 45 minutes followed by reperfusion for 120 minutes.Orbital venous blood samples were collected at the end of reperfusion for determination of serum alanine aminotrans-ferase(ALT)and aspartate aminotransferase(AST)levels,then the mice were sacrificed,and liver tissue samples were obtained for microscope examination of the pathological changes and determina-tion of glutathione(GSH)activity,malondialdehyde(MDA)content,the expression of interleukin-6(IL-6)mRNA(by fluorescence quantitative polymerase chain reaction),the expression of protein ki-nase B(Akt)and its phosphorylated protein(p-Akt),glutathione peroxidase 4(GPX4),and Acly-CoA synthetase long-chain family 4(ACSL4)(by Western Blot).The degree of liver injury was assessed using the Eckhoff scoring method.Results:Compared with those in the S group,the Eckhoff score,serum ALT and AST levels increased,GSH activity decreased,MDA content increased,the expres-sion of IL-6 mRNA was up-regulated(P<0.05),the expression of GPX4 was down-regulated,and the expression of p-Akt and ACSL4 were up-regulated in the I/R group(P<0.05).Compared with those in the I/R group,the Eckhoff score,serum ALT and AST levels decreased,GSH activity in-creased,MDA content decreased,the expression of IL-6 mRNA was down-regulated(P<0.05),the expression of GPX4 and p-Akt were up-regulated,and the expression of ACSL4 was down-regulated in M group(P<0.05).Compared with those in the M group,the Eckhoff score,serum ALT and AST levels increased,GSH activity decreased,MDA content increased,the expression of IL-6 mRNA was up-regulated,the expression of GPX4 was down-regulated,and the expression of p-Akt and ACSL4 were up-regulated in the MT group(P<0.05).Conclusion:Metformin exerts a protec-tive effect on liver injury induced by intestinal ischemia-reperfusion in mice by inhibiting ferroptosis,and the mechanism may be related to the activation of Akt.