Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene

Yonghong HU; Zhun XIAO; Yadong FU; Yue LIANG; Linzhang ZHANG; Wei LIU; Yongping MU; Chenghai LIU; Ping LIU; Jiamei CHEN

doi:10.3969/j.issn.1001-5256.2022.02.016

Volume 38 Issue 2

Feb. 2022

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Article Navigation > Journal of Clinical Hepatology > 2022 > 38(2): 334-341

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Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene

DOI: 10.3969/j.issn.1001-5256.2022.02.016

Yonghong HU^{1, 2},
Zhun XIAO^{1, 3},
Yadong FU^{1, 3},
Yue LIANG^{1, 2},
Linzhang ZHANG^{1, 3},
Wei LIU^{1, 2},
Yongping MU^{1, 2},
Chenghai LIU^{1, 2},
Ping LIU^{1, 2, 3
,
,},
Jiamei CHEN^{1, 2
,
,}

1.
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine; Institute of Liver Diseases, Shanghai Institute of Traditional Chinese Medicine, Shanghai 201203, China
2.
Key Laboratory of Liver and Kidney Diseases, Ministry of Education; Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
3.
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Research funding:

National Natural Science Foundation of China (81973613);

National Natural Science Foundation of China (81603465);

Morning Star Project of Shanghai Sci & Tech (19QA1408900);

Wang Baoen Liver Fibrosis Foundation (CFHPC2020010);

Siming Youth Fund Project of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (SGKJ-202004)

More Information

Corresponding author: LIU Ping, liuliver@vip.sina.com; CHEN Jiamei, cjm0102@126.com
Received Date: 2021-10-07
Accepted Date: 2021-11-30
Published Date: 2022-02-20

Abstract

Abstract

Objective To investigate the intervention effect of GDC-0449, a hedgehog signaling pathway inhibitor, on rats with liver fibrosis induced by carbon tetrachloride (CCl₄) combined with 2-acetylaminofluorene (2-AAF). Methods A total of 18 female Fisher344 rats were randomly divided into normal group, CCl₄/2-AAF group, and GDC-0449 group, with 6 rats in each group. The rats in the CCl₄/2-AAF group and the GDC-0449 group were given subcutaneously injected 30% CCl₄-olive oil solution at a dose of 2 mL/kg twice a week for 6 weeks to induce liver fibrosis; since week 7, in addition to the injection of CCl₄-olive oil solution, the rats in these two groups were given 2-AAF (100 mg/kg/d) by gavage, and the rats in the GDC-0449 group were given GDC-0449 (25 mg/kg/d) by gavage, while those in the normal group were given an equal volume of olive oil solution by injection and normal saline by gavage. All rats were sacrificed at the end of week 9, and related samples were collected. HE staining and sirius red (SR) staining were used to observe the changes in liver histopathology and collagen deposition, and the semi-quantitative analysis of SR-positive area and Ishak score were used to evaluate fibrosis degree; the alkaline hydrolysis method was used to measure the level of hydroxyproline (Hyp) in liver tissue; immunohistochemistry, Western blot, and qRT-PCR were used to measure the expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-Ⅰ), type Ⅳ collagen (Col-Ⅳ), cytokeratin 19 (CK19), cytokeratin 7 (CK7), the epithelial cell adhesion molecule Epcam, and the hedgehog signaling pathway in liver tissue; double immunofluorescence staining was used to observe the colocalization of CK19 and the oval cell marker OV6. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Compared with the normal group, the CCl₄/2-AAF group had marked inflammatory cell aggregation and collagen deposition in liver tissue, with the formation of a pseudolobular structure, as well as significant increases in Hyp level and collagen positive area ratio in liver tissue (P < 0.05), Ishak score (P < 0.05), and the expression of α-SMA, Col-Ⅰ, Col-Ⅳ, Epcam, CK19, CK7, the transmembrane transporter Smoothened (Smo), Hedgehog ligand Desert Hedgehog (Dhh), the Indian Hedgehog membrane-binding receptor Patched (Ptch2), and glioma-related oncogenes Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed that CK19-positive cells also expressed OV6 in the liver tissue of rats in the CCl₄/2-AAF group, with a significant increase compared with the normal group. Compared with the CCl₄/2-AAF group, the GDC-0449 group had significant reductions in inflammatory cell aggregation and collagen deposition in liver tissue, Hyp level and collagen positive area ratio in liver tissue (P < 0.05), Ishak score (P < 0.05), and the expression of α-SMA, Epcam, CK19, CK7, Smo, Ptch2, Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed a significant reduction in the number of cells with co-expression of OV6 and CK19 in liver tissue. Conclusion The Hedgehog signaling pathway inhibitor GDC-0449 can significantly inhibit the progression of liver fibrosis induced by CCl₄/2-AAF in rats, possibly by inhibiting hepatic stellate cell activation, collagen deposition, activation and proliferation of hepatic progenitor cells, and differentiation of hepatic progenitor cells into biliary epithelial cells.
- Liver Cirrhosis,
- Hedgehog Signaling Pathway,
- Rats, Inbred F344

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Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene

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Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene

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