Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis

FENG Wenmin; SU Anyu; HUANG Xiaoling; WANG Chunyi

doi:10.13422/j.cnki.syfjx.20230705

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Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis

更新时间：2023-04-13

- Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 10, Pages: 21-30(2023)
- 作者机构：
  
  广州中医药大学中药学院，广州 510006
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230705
  CLC： R2-0;R22;R285.5;R289;R33
- Published：20 May 2023，
  
  Published Online：23 February 2023，
  
  Received：21 December 2022，
- 稿件说明：
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冯雯敏,苏安宇,黄小玲等.古方黄芪散调控AMPK/mTOR自噬信号通路改善肝脏脂肪变性的机制[J].中国实验方剂学杂志,2023,29(10):21-30.

FENG Wenmin,SU Anyu,HUANG Xiaoling,et al.Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):21-30.
冯雯敏,苏安宇,黄小玲等.古方黄芪散调控AMPK/mTOR自噬信号通路改善肝脏脂肪变性的机制[J].中国实验方剂学杂志,2023,29(10):21-30. DOI： 10.13422/j.cnki.syfjx.20230705.

FENG Wenmin,SU Anyu,HUANG Xiaoling,et al.Mechanism of Huangqisan Regulating Autophagy by AMPK/mTOR Signaling Pathway Against Hepatic Steatosis[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(10):21-30. DOI： 10.13422/j.cnki.syfjx.20230705.

摘要

目的

基于腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路，探讨古方黄芪散调节自噬减轻小鼠肝脏脂肪变性改善非酒精性脂肪肝（NAFLD）的作用机制。

方法

通过数据库收集黄芪散主要化学成分及NAFLD相关靶点，对交集靶点进行基因本体（GO）分析和京都基因与基因组百科全书（KEGG）信号通路富集分析、构建蛋白质-蛋白质相互作用（PPI）网络，并进行体内实验验证。雄性C57BL/6J小鼠60只，适应性喂养1周后随机分为正常组、模型组、二甲双胍组（0.25 g·kg

-1

）、黄芪散低、高剂量组（0.5、1 g·kg

-1

），每组12只。采用高脂饮食（HFD）诱导小鼠NAFLD模型，造模的同时灌胃给药13周，每天1次。通过检测随机血糖、血清中总胆固醇（TC）、甘油三酯（TG）、游离脂肪酸（NEFA）、低密度脂蛋白胆固醇（LDL-C）水平和肝脏中TG含量；称量肝脏湿重并计算肝指数；采用苏木素-伊红（HE）染色、油红O染色、透射电子显微镜（TEM）、实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹法（Western blot）对C57BL/6J小鼠进行动物体内药效验证和机制探讨。

结果

通过网络药理学分析，结合前期研究基础，预测黄芪散可能通过AMPK/mTOR自噬信号通路改善NAFLD。体内实验结果表明，与正常组比较，模型组小鼠随机血糖、体质量、血清中TC、LDL-C和NEFA、肝脏湿重、肝指数和肝脏TG含量显著升高（

0.01），小鼠肝脏增大，HE染色可见小鼠肝脏脂滴（LDs）大量空泡化，油红O染色显示肝细胞脂肪堆积，透射电镜未见明显自噬体和自噬溶酶体，微管相关蛋白1轻链3（LC3）A、LC3B、AMPK

1 mRNA表达和AMPK、磷酸化（p）-AMPK、p-AMPK/AMPK蛋白表达显著下调（

0.01），LC3Ⅱ/Ⅰ、p-mTOR蛋白水平显著上调（

0.01）。与模型组比较，黄芪散低、高剂量组小鼠体质量、肝脏湿重、血清TG、NEFA水平明显降低（

0.05，

0.01），HE染色和油红O染色显示肝脏病理改变得到改善，肝脏透射电镜可见少量自噬溶酶体和自噬小体；黄芪散低剂量组肝指数显著降低（

0.01），黄芪散高剂量组随机血糖、血清TC水平、肝脏TG含量明显降低（

0.05）；Western blot和Real-time PCR结果显示，黄芪散低、高剂量组LC3A、LC3B mRNA和LC3Ⅱ/Ⅰ、p-AMPK、p-AMPK/AMPK蛋白表达显著上调（

0.01），p62

mRNA和p62、p-mTOR蛋白表达明显下调（

0.05，

0.01）。

结论

黄芪散可能通过AMPK/mTOR信号通路促进自噬和恢复自噬通量，减轻肝脏脂肪变性，从而改善NAFLD。

Abstract

Objective

To explore the mechanism of Huangqisan （HQS） in regulating autophagy to alleviate hepatic steatosis and improve non-alcoholic fatty liver disease （NAFLD） based on adenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway.

Method

The main chemical components and targets of HQS and NAFLD-related targets were collected from database and the intersection targets were used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network was constructed， and

in vivo

experimental verification was conducted. Sixty C57BL/6J male mice were randomly divided into normal control group （NCD）， model group high-fat diet （HFD）， metformin group （MET， 0.25 g·kg

-1

）， low-dose Huangqisan group （HQS-L， 0.5 g·kg

-1

）， and the high-dose Huangqisan group （HQS-H， 1 g·kg

-1

）， with 12 mice in each group after a one-week acclimatization period. NAFLD model was induced by HFD， and intragastric administration was performed at the same time， once a day for 13 weeks. Random blood glucose， serum total cholesterol （TC）， triglyceride （TG）， non-esterified fatty acid （NEFA）， low density lipoprotein-chdesterol （LDL-C） levels， and liver TG content were determined. The liver weight was weighed， and liver index was calculated. Hematoxylin-eosin （HE） staining， oil red O staining， transmission electron microscope （TEM）， real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot were used to verify the effect and reveal the potential mechanism of C57BL/6J mice

in vivo

Result

Through network pharmacology analysis， combined with previous studies， it was predicted that HQS may improve NAFLD by regulating autophagy via the AMPK/mTOR signaling pathway. The result of

in vivo

experiment showed that， as compared with NCD group， random blood glucose， body weight， serum TC， LDL-C， NEFA， liver weight， liver index， and liver TG content of mice in the HFD groups were significantly increased （

0.01）. HE staining showed massive lipid droplets （LDs） vacuolated， oil red O staining showed lipid accumulation in liver cells， and no obvious autophagosomes and autolysosome were observed under TEM. The relative mRNA expression of LC3A、LC3B、AMPK

1 and protein expression of AMPK， phosphory phosphorylated（p）-AMPK， and p-AMPK/AMPK were significantly down-regulated （

0.01）， while the protein expression of microtubule-associated protein 1 light chain 3 （LC3）Ⅱ/Ⅰ and p-mTOR was significantly up-regulated （

0.01）. As compared with HFD groups， liver weight， serum TG， and NEFA levels in HQS-L and HQS-H groups were significantly deceased （

0.05，

0.01）. HE staining and oil red O staining showed the improvement of liver pathological changes after HQS administration. Under TEM， a small amount of autophagosome and autolysosome were observed. Besides， liver index was significantly decreased in the HQS-L group （

0.01）， and random blood glucose， serum TC level and liver TG content were significantly decreased in the HQS-H group （

0.05）. The results of Western blot and Real-time PCR showed that the mRNA expression of LC3A and LC3B and the protein expression of LC3Ⅱ/Ⅰ， p-AMPK， and p-AMPK/AMPK were significantly up-regulated （

0.01）， while the mRNA expressions of p62 and protein expression of p62 and p-mTOR were significantly down-regulated （

0.05，

0.01）.

Conclusion

HQS may promote autophagy and restore autophagy flux via the AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.

关键词

黄芪散非酒精性脂肪肝网络药理学腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）自噬

Keywords

Huangqisannon-alcoholic fatty liver diseasenetwork pharmacologyadenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）autophagy

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