山奈黄酮醇抑制HK2/LDHA依赖的糖酵解通路减轻大鼠肝缺血-再灌注损伤  

Kaempferol inhibits HK2/LDHA-dependent glycolysis pathway and alleviates hepatic ischemia-reperfusion injury in rats

作　　者：殷娟[1] 李依玲 孟杰 沈威 Yin Juan;Li Yiling;Meng Jie;Shen Wei(Department of Critical Care Medicine,Xiaogan Hospital Affiliated to Wuhan University of Science and Technology,Xiaogan 432000,China)

机构地区：[1]武汉科技大学附属孝感医院重症医学科,湖北孝感432000 [2]武汉科技大学附属孝感医院麻醉科,湖北孝感432000

出　　处：《中国急救医学》2023年第11期905-911,共7页Chinese Journal of Critical Care Medicine

基　　金：湖北省公共卫生青年拔尖人才项目;孝感市自然科学计划项目(XGKJ2021010038)。

摘　　要：目的 探讨山奈黄酮醇(KM)对大鼠肝缺血-再灌注损伤(HIRI)的影响和与己糖激酶2(HK2)依赖的糖代谢重编程的关系。方法 采用随机数表法将60只SPF级雄性SD大鼠分为五组:假手术组(Sham组)、肝缺血-再灌注损伤组(HIRI组)、HIRI+山奈黄酮醇预处理组(KM组)、HIRI+山奈黄酮醇+己糖激酶激活剂Hexokinase预处理组(KM+Hex组)和HIRI+Hexokinase预处理组(Hex组)。通过选择性肝门阻断法制备大鼠HIRI模型,KM组、KM+Hex和Hex组于HIRI建模前3 d每日分别腹腔注射KM 50 mg/kg、Hexokinase 20 mg/kg。HIRI模型建立6 h后麻醉处死大鼠,并采集肝脏和下腔静脉血液样本,ELISA法检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBil)、白细胞介素-6(IL-6)和肝组织核因子p65亚基(p65)活性,化学比色法检测肝组织超氧化物歧化酶(SOD)、丙二醛(MDA)和丙酮酸含量,蛋白质凝胶电泳实验检测HK2、乳酸脱氢酶A(LDHA)、磷酸化AMP依赖的蛋白激酶α(p-AMPKα)蛋白表达量,HE染色观察肝脏组织病理改变,免疫组织化学染色后评估肝脏凋亡蛋白裂解的半胱氨酸-天冬氨酸蛋白酶3(cleaved caspase-3)表达水平。结果 与Sham组比较,HIRI组血清ALT、AST、TBil、IL-6含量和肝组织MDA含量、乳酸/丙酮酸比值、活性升高[ALT(μg/L):46.3±7.2 vs.106.4±18.3;AST(μg/L):61.8±9.7 vs.190.8±26.1;TBil(μg/L):4.62±0.68 vs.21.23±2.85;IL-6(ng/L):39.8±6.9 vs.209.3±30.1;MDA(U/gprot):10.61±2.12 vs.28.40±4.21;乳酸/丙酮酸比值:11.5±3.1 vs.61.7±8.9;p65(%):0.97±0.11 vs.3.85±0.54],肝组织SOD含量、p-AMPKα表达降低[SOD(U/g prot):68.9±19.7 vs.161.8±20.4;p-AMPKα:0.56±0.09 vs.1.14±0.15],肝组织HK2、LDHA和cleaved caspase-3表达明显升高(HK2:0.61±0.09 vs.0.16±0.03;LDHA:0.85±0.11 vs.0.38±0.05;cleaved caspase-3:4.74±0.63 vs.1.09±0.13)(P<0.05),肝细胞损伤明显减轻;与HIRI组比较,KM组血清ALT、AST、TBil、IL-6含量和肝组织MDA含量、乳酸/丙酮酸比值、p65活性降低[ALT(μg/Objective To evaluate the effect of kaempferol(KM) on hepatic ischemia-reperfusion injury(HIRI) in rats and the relaitonship with hexokinase 2(HK2)-dependent glycolysis reprogramming.Methods 60 SPF male SD rats were randomly divided into 5 groups:sham group(Sham group),hepatic ischemia-reperfusion injury group(HIRI group),HIRI+KM pretreatment group(KM group),HIRI+KM+HK pretreatment group(KM+Hex group) and HIRI+HK pretreatment group(Hex group).The rat model of HIRI was established by selective hepatic portal occlusion,KM group,KM+Hex group and Hex group were intraperitoneally injected with 50 mg/kg KM and 20 mg/kg HK daily 3 days before HIRI modeling.At 6 h after the establishment of model,the rats were anesthetized and sacrificed,and liver and blood samples were collected.The serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBil),interleukin-6(IL-6) content and p65 activity in liver tissue were detected by ELISA.The contents of superoxide dismutase(SOD),malondialdehyde(MDA) and pyruvate in liver tissue were detected by chemistry colorimetric method.The protein expression of HK2,lactate dehydrogenase A(LDHA) and phosphorylated AMP-activated protein kinase α(p-AMPKα) were detected by Western blotting.The pathological changes of liver tissue were evaluated after hematoxylin-eosin staining.The expression level of hepatic apoptosis protein cleaved caspase-3 was evaluated after immunohistochemistry staining.Results Compared with Sham group,the level of serum ALT,AST,TBil and IL-6,the content of MDA,lactate/pyruvate ratio and the activity of p65 in liver tissue increased[ALT(μg/L):46.3±7.2 vs.106.4±18.3;AST(μg/L):61.8±9.7 vs.190.8±26.1;TBil(μg/L):4.62±0.68 vs.21.23±2.85;IL-6(ng/L):39.8±6.9 vs.209.3±30.1;MDA(U/g prot):10.61±2.12 vs.28.40±4.21;lactate/pyruvate ratio:11.5±3.1 vs.61.7±8.9;p65(%):0.97±0.11 vs.3.85±0.54];the tissue content of SOD and the expression of p-AMPKα were decreased [SOD(U/g prot):68.9±19.7 vs.161.8±20.4;p-AMPKα:0.56±0.09 vs.1.14±0.15],the ex

关 键 词：山奈黄酮醇 肝缺血-再灌注损伤 糖酵解 己糖激酶2 

分 类 号：R285.5[医药卫生—中药学]