American Journal of Clinical and Experimental Medicine

Submit a Manuscript

Publishing with us to make your research visible to the widest possible audience.

Propose a Special Issue

Building a community of authors and readers to discuss the latest research and develop new ideas.

Research Article |

Mechanism Study of GanDouLing Mitigating Liver Fibrosis in Wilson’s Disease via Regulating the PI3K/AKT/mTOR Pathway to Activate Autophagy

The hepatic copper accumulation characteristic of Wilson's disease (WD) leads to activation of hepatic stellate cells, extracellular matrix deposition, and the progression to liver fibrosis (LF), with specific therapeutic interventions for this condition notably deficient. Increasing evidence suggests the potential utility of GanDouLing (GDL) in addressing WD-related LF. In this study, the pharmacological effects of GDL on WD and LF were assessed by establishing a copper-induced human hepatic stellate cell (LX-2) model and elucidating potential mechanisms underlying GDL treatment. The findings indicate that GDL treatment significantly attenuates the viability of copper-induced LX-2 cells and reduces the expression of fibrotic markers. This inhibitory effect is mediated through the PAM pathway, as it can be reversed by activators such as IGF-1 or MHY1485. Furthermore, GDL treatment downregulates critical components of the PAM pathway at both mRNA and protein levels. Molecular docking simulations further confirm the strong affinity and stable binding between active constituents of GDL, such as luteolin and vestitol, and their target proteins (AKT1, PIK3CA, mTOR). In copper-induced LX-2 cells, GDL treatment upregulates autophagic indicators, including LC3, Beclin-1, and AO, while downregulating p62. TEM experiments reveal that GDL treatment increases the presence of autophagosomes in copper-induced LX-2 cells, thereby ameliorating mitochondrial damage. In conclusion, GDL exerts its inhibitory effects on copper-induced LX-2 cell activation by downregulating the PAM pathway. These findings provide a novel approach for addressing LF related to WD and support the utilization of herbal-based supplements and alternative therapies.

GanDouLing, Wilson Disease-Associated Liver Fibrosis, Autophagy, PI3K/AKT/mTOR Pathway

APA Style

Shao-Peng Huang, Sen Chen, Huan Wu, An Zhou, Huai-Zhou Jiang, et al. (2023). Mechanism Study of GanDouLing Mitigating Liver Fibrosis in Wilson’s Disease via Regulating the PI3K/AKT/mTOR Pathway to Activate Autophagy. American Journal of Clinical and Experimental Medicine, 11(4), 73-85.

ACS Style

Shao-Peng Huang; Sen Chen; Huan Wu; An Zhou; Huai-Zhou Jiang, et al. Mechanism Study of GanDouLing Mitigating Liver Fibrosis in Wilson’s Disease via Regulating the PI3K/AKT/mTOR Pathway to Activate Autophagy. Am. J. Clin. Exp. Med. 2023, 11(4), 73-85. doi: 10.11648/j.ajcem.20231104.12

AMA Style

Shao-Peng Huang, Sen Chen, Huan Wu, An Zhou, Huai-Zhou Jiang, et al. Mechanism Study of GanDouLing Mitigating Liver Fibrosis in Wilson’s Disease via Regulating the PI3K/AKT/mTOR Pathway to Activate Autophagy. Am J Clin Exp Med. 2023;11(4):73-85. doi: 10.11648/j.ajcem.20231104.12

Copyright © 2023 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. GOYAL R, CHACHAM S, GOYAL J P, et al. Wilson's Disease Masquerading as Juvenile Idiopathic Arthritis in an Adolescent [J]. Cureus, 2020, 12(7): e9160.
2. CHOE E J, CHOI J W, KANG M, et al. A population-based epidemiology of Wilson's disease in South Korea between 2010 and 2016 [J]. Sci Rep, 2020, 10(1): 14041.
3. LEMOINNE S, FRIEDMAN S L. New and emerging anti-fibrotic therapeutics entering or already in clinical trials in chronic liver diseases [J]. Curr Opin Pharmacol, 2019, 49: 60-70.
4. HUANG C, GAN D, LUO F, et al. Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells [J]. Front Pharmacol, 2019, 10: 431.
5. ZHANG X W, ZHOU J C, PENG D, et al. Disrupting the TRIB3-SQSTM1 interaction reduces liver fibrosis by restoring autophagy and suppressing exosome-mediated HSC activation [J]. Autophagy, 2020, 16(5): 782-96.
6. WEISKIRCHEN R, TACKE F. Relevance of Autophagy in Parenchymal and Non-Parenchymal Liver Cells for Health and Disease [J]. Cells, 2019, 8(1).
7. ZHANG Z, GUO M, LI Y, et al. RNA-binding protein ZFP36/TTP protects against ferroptosis by regulating autophagy signaling pathway in hepatic stellate cells [J]. Autophagy, 2020, 16(8): 1482-505.
8. GAO J, WEI B, DE ASSUNCAO T M, et al. Hepatic stellate cell autophagy inhibits extracellular vesicle release to attenuate liver fibrosis [J]. J Hepatol, 2020, 73(5): 1144-54.
9. LI M Y, ZHU X L, ZHAO B X, et al. Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis [J]. Cell Death Dis, 2019, 10(7): 489.
10. XIU A Y, DING Q, LI Z, et al. Doxazosin Attenuates Liver Fibrosis by Inhibiting Autophagy in Hepatic Stellate Cells via Activation of the PI3K/Akt/mTOR Signaling Pathway [J]. Drug Des Devel Ther, 2021, 15: 3643-59.
11. LIU C, LIU Q, NIAN M, et al. Identification and quantitative analysis of the chemical constituents of Gandouling tablets using ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry [J]. J Sep Sci, 2023, 46(16): e2300060.
12. YANG Y, HAO W, YANG Y, et al. Chemical Profiling and Quantification of Potential Bioactive Components in Gandouling Pill by Ultra-High Performance Liquid Chromatography Coupled with Diode Array Detector/Quadruple-Qrbitrap Mass Spectrometry [J]. Molecules, 2022, 27(23).
13. ZHANG J, XIE D, LI Y, et al. Evaluation of efficacy and safety of gandouling plus sodium dimercaptosulphonate in treatment of patients with neurological Wilson's disease from China [J]. J Tradit Chin Med, 2018, 38(5): 781-6.
14. ZHANG J, CHEN H, BAO Y, et al. System Pharmacology-Based Strategy to Decode the Synergistic Mechanism of GanDouLing for Wilson's Disease [J]. Evid Based Complement Alternat Med, 2021, 2021: 1248920.
15. ZHANG J, LI L, CHEN H, et al. Clinical efficacy and safety of Gandouling plus low-dose D-penicillamine for treatment of Wilson's disease with neurological symptoms [J]. J Tradit Chin Med, 2018, 38(1): 89-94.
16. ROSE P W, PRLIC A, ALTUNKAYA A, et al. The RCSB protein data bank: integrative view of protein, gene and 3D structural information [J]. Nucleic Acids Res, 2017, 45(D1): D271-D81.
17. ZHANG J, XIAO L, YANG W. Combined sodium Dimercaptopropanesulfonate and zinc versus D-penicillamine as first-line therapy for neurological Wilson's disease [J]. BMC Neurol, 2020, 20(1): 255.
18. HAMILTON J P, KOGANTI L, MUCHENDITSI A, et al. Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice [J]. Hepatology, 2016, 63(6): 1828-41.
19. LI T Y, YANG Y, ZHOU G, et al. Immune suppression in chronic hepatitis B infection associated liver disease: A review [J]. World J Gastroenterol, 2019, 25(27): 3527-37.
20. LIU J, KONG D, QIU J, et al. Praziquantel ameliorates CCl4 -induced liver fibrosis in mice by inhibiting TGF-beta/Smad signalling via up-regulating Smad7 in hepatic stellate cells [J]. Br J Pharmacol, 2019, 176(24): 4666-80.
21. KONG L, HUANG H, LUAN S, et al. Inhibition of ASIC1a-Mediated ERS Improves the Activation of HSCs and Copper Transport Under Copper Load [J]. Front Pharmacol, 2021, 12: 653272.
22. ZHOU J, JIANG Y Y, CHEN H, et al. Tanshinone I attenuates the malignant biological properties of ovarian cancer by inducing apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway [J]. Cell Prolif, 2020, 53(2): e12739.
23. SARAVIA J, RAYNOR J L, CHAPMAN N M, et al. Signaling networks in immunometabolism [J]. Cell Res, 2020, 30(4): 328-42.
24. KUANG H, TAN C Y, TIAN H Z, et al. Exploring the bi-directional relationship between autophagy and Alzheimer's disease [J]. CNS Neurosci Ther, 2020, 26(2): 155-66.
25. LI J, LI X, XU W, et al. Antifibrotic effects of luteolin on hepatic stellate cells and liver fibrosis by targeting AKT/mTOR/p70S6K and TGFbeta/Smad signalling pathways [J]. Liver Int, 2015, 35(4): 1222-33.
26. BUENO-SILVA B, ROSALEN P L, ALENCAR S M, et al. Vestitol drives LPS-activated macrophages into M2 phenotype through modulation of NF-kappaB pathway [J]. Int Immunopharmacol, 2020, 82: 106329.
27. CHEN X, WANG H, ZHOU M, et al. Valproic Acid Attenuates Traumatic Brain Injury-Induced Inflammation in Vivo: Involvement of Autophagy and the Nrf2/ARE Signaling Pathway [J]. Front Mol Neurosci, 2018, 11: 117.
28. PARKER K H, HORN L A, OSTRAND-ROSENBERG S. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy [J]. J Leukoc Biol, 2016, 100(3): 463-70.
29. HE W, PENG H, MA J, et al. Autophagy changes in lung tissues of mice at 30 days after carbon black-metal ion co-exposure [J]. Cell Prolif, 2020, 53(7): e12813.
30. NIU Q, ZHAO W, WANG J, et al. LicA induces autophagy through ULK1/Atg13 and ROS pathway in human hepatocellular carcinoma cells [J]. Int J Mol Med, 2018, 41(5): 2601-8.
31. DURGAN J, LYSTAD A H, SLOAN K, et al. Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine [J]. Mol Cell, 2021, 81(9): 2031-40 e8.
32. ZENG Z, LIANG J, WU L, et al. Exercise-Induced Autophagy Suppresses Sarcopenia Through Akt/mTOR and Akt/FoxO3a Signal Pathways and AMPK-Mediated Mitochondrial Quality Control [J]. Front Physiol, 2020, 11: 583478.
33. LV H, ZHEN C, LIU J, et al. beta-Phenethyl Isothiocyanate Induces Cell Death in Human Osteosarcoma through Altering Iron Metabolism, Disturbing the Redox Balance, and Activating the MAPK Signaling Pathway [J]. Oxid Med Cell Longev, 2020, 2020: 5021983.
34. LIU Y, PENG W, QU K, et al. TET2: A Novel Epigenetic Regulator and Potential Intervention Target for Atherosclerosis [J]. DNA Cell Biol, 2018, 37(6): 517-23.
35. HINZE L, LABROSSE R, DEGAR J, et al. Exploiting the Therapeutic Interaction of WNT Pathway Activation and Asparaginase for Colorectal Cancer Therapy [J]. Cancer Discov, 2020, 10(11): 1690-705.
36. MWANGI S M, LI G, YE L, et al. Glial Cell Line-Derived Neurotrophic Factor Enhances Autophagic Flux in Mouse and Rat Hepatocytes and Protects Against Palmitate Lipotoxicity [J]. Hepatology, 2019, 69(6): 2455-70.
37. TO U, SCHILSKY M L. A Case for Not Going Global: "Americanization" of Diet Accelerates Hepatic Mitochondrial Injury in a Model of Wilson Disease [J]. Cell Mol Gastroenterol Hepatol, 2019, 7(3): 684-5.
38. ORNATOWSKI W, LU Q, YEGAMBARAM M, et al. Complex interplay between autophagy and oxidative stress in the development of pulmonary disease [J]. Redox Biol, 2020, 36: 101679.