大黄-黄连调控AMPK/ULK1/Beclin1通路改善db/db小鼠肝脏胰岛素抵抗的机制  

Mechanism of Rhei Radix et Rhizoma-Coptidis Rhizoma Improving Liver Insulin Resistance in db/db Mice by Regulating AMPK/ULK1/Beclin1 Pathway

作　　者：赵泓彰 白敏 汪湛东 宋冰 郭超[1,2] 刘馨鸿 王琼 杨润泽[1] 汪永锋 张延英[1,2] ZHAO Hongzhang;BAI Min;WANG Zhandong;SONG Bing;GUO Chao;LIU Xinhong;WANG Qiong;YANG Runze;WANG Yongfeng;ZHANG Yanying(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Laboratory Animal Industry Technology Center of Gansu Province,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]甘肃省实验动物行业技术中心,兰州730000

出　　处：《中国实验方剂学杂志》2024年第8期9-16,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：甘肃省自然科学基金实验动物专项(22JR5RA581);甘肃省药品监督管理局项目(2022GSMPA001)。

摘　　要：目的:探讨大黄-黄连通过调控腺苷酸活化蛋白激酶(AMPK)/UNC-51样激酶1(ULK1)/自噬关键分子苄氯素1(Beclin1)通路减轻db/db小鼠胰岛素抵抗的作用及其机制。方法:采用db/db小鼠,雄性6周龄db/db小鼠60只,随机分为模型组、二甲双胍组(0.26 g·kg^(-1))、大黄-黄连低、中、高剂量组(2.25、4.5、9 g·kg^(-1));将同周龄db/m小鼠作为空白组,每组12只。连续灌胃给药干预8周,空白组和模型组每日给予等量蒸馏水灌胃,1次/d,观察小鼠生存状态并使用罗氏血糖设备检测空腹血糖(FBG);采用酶联免疫吸附测定法检测小鼠空腹血清胰岛素(FINS)并计算胰岛素抵抗指数(HOMA-IR);采用苏木素-伊红(HE)染色法观察小鼠肝脏病理变化;蛋白免疫印迹法(Western blot)检测肝脏组织中AMPK、Beclin1、自噬相关蛋白5(Atg5)及p62蛋白表达水平;免疫荧光检测肝脏组织中自噬相关蛋白1轻链3B(LC3B)、ULK1的蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测AMPK、Beclin1、Atg5、ULK1、p62 mRNA表达水平。结果:与空白组比较,模型组体质量显著升高(P<0.01);FBG、FINS、HOMA-IR水平均有所改变(P<0.01);肝细胞结构紊乱;肝脏组织中AMPK、Beclin1、Atg5蛋白表达水平显著降低(P<0.01);p62蛋白表达水平显著升高(P<0.01);mRNA与蛋白表达水平基本一致。与模型组比较,二甲双胍组、大黄-黄连高、中剂量组体质量明显降低(P<0.05);FBG、FINS、HOMA-IR均有所下降(P<0.05,P<0.01);治疗后各组小鼠的肝脏结构损伤均有不同程度减轻;肝脏组织中AMPK、Beclin1、Atg5、LC3B和ULK1蛋白表达升高(P<0.05,P<0.01),p62蛋白表达下降(P<0.01);且mRNA与蛋白表达水平大致相同。结论:大黄-黄连可以有效改善肝脏胰岛素抵抗,调控AMPK自噬信号通路,减轻db/db小鼠胰岛素抵抗从而有效防治2型糖尿病的发生发展。Objective:This study aims to examine the effect of Rhei Radix et Rhizoma-Coptidis Rhizoma on reducing insulin resistance in db/db mice by regulating the adenylate activated protein kinase(AMPK)/UNC-51-like kinase 1(ULK1)/key molecule of autophagy,benzyl chloride 1(Beclin1)pathway and elucidate the underlying mechanism.Method:Sixty 6-week-old male db/db mice were studied.They were randomly divided into the model group,metformin group(0.26 g·kg^(-1)),and low-,middle-,and high-dose groups(2.25,4.5,9 g·kg^(-1))of Rhei Radix et Rhizoma-Coptidis Rhizoma.A blank group of db/m mice of the same age was set,with 12 mice in each group.After eight weeks of continuous intragastric administration,the blank group and model group received distilled water intragastrically once a day.The survival status of the mice was observed,and fasting blood glucose(FBG)was measured using a Roche blood glucose device.Fasting serum insulin(FINS)was measured using an enzyme-linked immunosorbent assay,and the insulin resistance index(HOMA-IR)was calculated.Hematoxylin-eosin(HE)staining was performed to observe the pathological changes in the liver of the mice.The protein expression levels of AMPK,Beclin1,autophagy associated protein 5(Atg5),and p62 in liver tissue were determined by using Western blot.The protein expression levels of autophagy associated protein 1 light chain 3B(LC3B)and ULK1 in liver tissue were determined using immunofluorescence.Real-time fluorescence quantitative PCR(Real-time PCR)was used to measure mRNA expression levels of AMPK,Beclin1,Atg5,ULK1,and p62.Result:Compared with the blank group,the model group exhibited a significant increase in body mass(P<0.01).Additionally,the levels of FBG,FINS,and HOMA-IR significantly changed(P<0.01).The structure of liver cells was disordered.The protein expression levels of AMPK,Beclin1,and Atg5 in liver tissue were significantly decreased(P<0.01),while the expression level of p62 protein was significantly increased(P<0.01).The expression levels of mRNA and proteins were consistent.Co

关 键 词：大黄-黄连 肝损伤 胰岛素抵抗 肥胖型2型糖尿病 腺苷酸活化蛋白激酶(AMPK)/UNC-51样激酶1(ULK1)/自噬关键分子苄氯素1(Beclin1) 

分 类 号：R2-0[医药卫生—中医学] R33[文化科学—情报学] G353.11R246.82