Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis

LIANG Jianqing; ZHANG Yuanyuan; ZHU Xiangdong; SI Xiaoli

doi:10.13422/j.cnki.syfjx.20231002

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Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis

更新时间：2023-08-22

- Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 18, Pages: 1-7(2023)
- 作者机构：
  
  1.甘肃中医药大学基础医学院，甘肃省中医方药挖掘与创新转化重点实验室，糖尿病实验室，兰州 730000
  2.宁夏医科大学，银川 750004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20231002
  CLC： R2-0;R22;R285.5;R289;R33
- Published：20 September 2023，
  
  Published Online：24 April 2023，
  
  Received：27 January 2023，
- 稿件说明：
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梁建庆,张媛媛,朱向东等.基于AMPK-FoxO3a自噬轴探讨葛根芩连汤改善2型糖尿病db/db小鼠肝脏脂质异位蓄积的机制[J].中国实验方剂学杂志,2023,29(18):1-7.

LIANG Jianqing,ZHANG Yuanyuan,ZHU Xiangdong,et al.Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(18):1-7.
梁建庆,张媛媛,朱向东等.基于AMPK-FoxO3a自噬轴探讨葛根芩连汤改善2型糖尿病db/db小鼠肝脏脂质异位蓄积的机制[J].中国实验方剂学杂志,2023,29(18):1-7. DOI： 10.13422/j.cnki.syfjx.20231002.

LIANG Jianqing,ZHANG Yuanyuan,ZHU Xiangdong,et al.Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(18):1-7. DOI： 10.13422/j.cnki.syfjx.20231002.

摘要

目的

探讨葛根芩连汤调控腺苷酸活化蛋白激酶（AMPK）-叉头框蛋白O3a（FoxO3a）自噬轴改善2型糖尿病db/db小鼠肝脏脂质异位蓄积的机制研究，为阐明葛根芩连汤的降糖机制及推广应用提供科学依据。

方法

选取SPF级自发性2型糖尿病动物模型db/db小鼠75只及正常db/m小鼠15只予维持饲料喂养1周后检测血糖，随机分为6组，每组15只。正常组（生理盐水0.2 g·kg

-1

）、二甲双胍组（0.2 g·kg

-1

）、葛根芩连汤高、中、低剂量组（31.9、19.1、6.9 g·kg

-1

）及模型组（生理盐水0.2 g·kg

-1

），连续灌胃给药12周，1次/d，检测各组小鼠空腹血糖（FBG）、糖化血红蛋白（HbA1c），酶联免疫吸附测定法（ELISA）检测血清空腹胰岛素（FINS）、游离脂肪酸（FFA）水平，苏木素-伊红（HE）染色观察肝组织病理改变，使用蛋白免疫印迹法（Western blot）检测肝脏组织中磷酸化（p）-AMPK、p-FoxO3a及自噬相关蛋白微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）、p62蛋白的表达；免疫荧光检测肝脏组织中缺氧诱导因子-1

（HIF-1

）蛋白表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测肝组织AMPK、FoxO3a、LC3 mRNA表达。

结果

与正常组比较，模型组小鼠肝脏组织有病理学改变；模型组小鼠FBG、HbA1c、FINS和FFA水平显著升高（

0.01）；肝脏组织中p-AMPK、p62、HIF-1

蛋白表达水平显著升高，p-FoxO3a、LC3Ⅱ的蛋白表达显著降低（

0.01）；模型组小鼠肝脏组织AMPK mRNA表达显著降低，FoxO3a表达显著升高（

0.01）。与模型组比较，各给药组肝脏组织病变程度减轻；各给药组FBG、HbA1c、FINS和FFA水平有所降低（

0.01）；葛根芩连汤中、高剂量组p-AMPK、p62、HIF-1

蛋白表达有所增加，p-FoxO3a的表达呈剂量依赖性降低，二甲双胍组、葛根芩连汤高剂量组LC3Ⅱ的表达有所升高（

0.01）；给药组AMPK mRNA的表达呈剂量依赖性增加，FoxO3a的表达呈剂量依赖性降低（

0.01）。

结论

葛根芩连汤可改善2型糖尿病db/db小鼠肝脏脂质异位蓄积，可能与调控AMPK-FoxO3a自噬轴相关。

Abstract

Objective

To explore the mechanism of Gegen Qinliantang （GQT） in improving ectopic lipid accumulation in the liver of db/db mice with type 2 diabetes mellitus （T2DM） by regulating the adenosine monophosphate-activated protein kinase （AMPK）-forkhead box O3a （FoxO3a） autophagy axis， to provide a scientific basis for clarifying the hypoglycemic mechanism of GQT and its clinical application.

Method

Seventy-five spontaneous T2DM db/db mice and 15 normal db/m mice were selected and maintained on a regular diet for one week， followed by the measurement of blood glucose. They were then randomly divided into six groups， with 15 mice in each group， including normal group （0.2 g·kg

-1

saline）， metformin group （0.2 g·kg

-1

）， high-， medium， and low-dose GQT group （31.9， 19.1， 6.9 g·kg

-1

）， and model group （0.2 g·kg

-1

saline）. The mice were orally administered the corresponding drugs once daily for 12 weeks. Fasting blood glucose （FBG） and glycated hemoglobin （HbA1c） were detected. Fasting insulin （FINS） and free fatty acid （FFA） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Pathological changes in liver tissues were observed by hematoxylin-eosin （HE） staining. The protein expression levels of phosphorylated （p）-AMPK， p-FoxO3a， and autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ） and p62 were detected using Western blot. Immunofluorescence was used to detect the expression of hypoxia-inducible factor-1

（HIF-1

） in liver tissues. Real-time polymerase chain reaction （Real-time PCR） was performed to detect the mRNA expression of AMPK， FoxO3a， and LC3 in liver tissues.

Result

Compared with the normal group， the model group showed pathological changes in liver tissues， increased FBG， HbA1c， FINS， and FFA levels （

0.01）， increased protein expression levels of p-AMPK， p62， and HIF-1

， decreased protein expression levels of p-FoxO3a and LC3Ⅱ in liver tissues （

0.01）， decreased mRNA expression of AMPK， and increased expression of FoxO3a （

0.01）. Compared with the model group， the treatment groups showed relieved liver tissue lesions and decreased FBG， HbA1c， FINS， and FFA levels （

0.01）. The expression of p-AMPK， p62， and HIF-1α increased， while the expression of p-FoxO3a showed a dose-dependent decrease in the high-dose GQT group. The expression of LC3Ⅱ increased in the metformin group and the high-dose GQT group （

0.01）. The mRNA expression of AMPK showed a dose-dependent increase， and the expression of FoxO3a showed a dose-dependent decrease in the treatment groups （

0.01）.

Conclusion

GQT can improve ectopic lipid accumulation in the liver of T2DM db/db mice， which may be related to the regulation of the AMPK-FoxO3a autophagy axis.

关键词

葛根芩连汤2型糖尿病腺苷酸活化蛋白激酶（AMPK）-叉头框蛋白O3a（FoxO3a）自噬轴脂质的异位蓄积

Keywords

Gegen Qinliantangtype 2 diabetes mellitusadenosine monophosphate-activated protein kinase （AMPK）-forkhead box O3a （FoxO3a） autophagy axisectopic lipid accumulation

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