GDC-0449在CCl<sub>4</sub>联合二乙酰氨基芴诱导肝纤维化大鼠模型中的作用

胡永红; 肖准; 付亚东; 梁悦; 张霖璋; 刘伟; 慕永平; 刘成海; 刘平; 陈佳美

doi:10.3969/j.issn.1001-5256.2022.02.016

GDC-0449在CCl₄联合二乙酰氨基芴诱导肝纤维化大鼠模型中的作用

DOI: 10.3969/j.issn.1001-5256.2022.02.016

胡永红^{1, 2},
肖准^{1, 3},
付亚东^{1, 3},
梁悦^{1, 2},
张霖璋^{1, 3},
刘伟^{1, 2},
慕永平^{1, 2},
刘成海^{1, 2},
刘平^{1, 2, 3, ,},
陈佳美^{1, 2, ,}

1.
上海中医药大学附属曙光医院上海市中医药研究院肝病研究所，上海 201203
2.
肝肾疾病病证教育部重点实验室上海市中医临床重点实验室，上海 201203
3.
上海中医药大学交叉科学研究院，上海 201203

基金项目:

国家自然科学基金 (81973613);

国家自然科学基金 (81603465);

上海市科技启明星项目 (19QA1408900);

王宝恩肝纤维化基金 (CFHPC2020010);

上海中医药大学附属曙光医院四明青年基金 (SGKJ-202004)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：胡永红参与课题设计，实施课题，撰写论文；肖准、付亚东、梁悦、张霖璋参与课题实施；刘伟参与数据收集，统计分析；慕永平、刘成海参与课题设计，实验指导；刘平、陈佳美进行课题设计，拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
刘平，liuliver@vip.sina.com

陈佳美，cjm0102@126.com

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出版历程
- 收稿日期: 2021-10-07
- 录用日期: 2021-11-30
- 出版日期: 2022-02-20

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

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

摘要

摘要: 目的研究Hedgehog信号通路抑制剂维莫德吉(GDC-0449)对CCl₄联合二乙酰氨基芴(2-AAF)诱导的大鼠肝纤维化的干预作用。方法将18只Fisher344雌性大鼠随机分为正常组、CCl₄联合2-AAF模型组(CCl₄/2-AAF组)和GDC-0449组，每组6只。CCl₄/2-AAF组和GDC-0449组大鼠按2 mL/kg体质量皮下注射30% CCl₄-橄榄油溶液，每周2次，持续造模6周；自第7周始，在注射CCl₄-橄榄油溶液同时予2-AAF(100 mg·kg^-1·d^-1)灌胃，同时GDC-0449组大鼠按25 mg·kg^-1·d^-1灌胃干预，正常组大鼠予等体积橄榄油溶液注射及生理盐水灌胃，9周末处死取材。HE染色和天狼猩红(SR)染色观察各组大鼠肝组织病理及胶原沉积变化，SR阳性染色面积半定量分析和Ishak评分评估纤维化程度；碱水解法测定肝组织羟脯氨酸(Hyp)水平；免疫组化、Western Blot、实时荧光定量聚合酶链式反应(qRT-PCR)检测肝组织α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col-Ⅰ)、Ⅳ型胶原(Col-Ⅳ)、细胞角蛋白(CK)19、CK7、上皮细胞黏附分子(Epcam)及Hedgehog信号通路表达变化；免疫荧光共染观察CK19和卵圆细胞标志物6(OV6)共定位情况。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果与正常组相比，CCl₄/2-AAF组大鼠肝组织炎性细胞聚集、胶原纤维沉积明显，存在明显的假小叶结构；肝组织Hyp水平和胶原阳性面积比显著增加(P值均＜0.05)，Ishak评分显著上升(P＜0.05)；α-SMA、Col-Ⅰ、Col-Ⅳ、Epcam、CK19、CK7、跨膜转运蛋白Smoothened(Smo)、Hedgehog配体Desert Hedgehog(Dhh)、Indian Hedgehog结合膜受体Patched(Ptch2)、胶质瘤相关致癌基因(Gli1、Gli2、Gli3)的表达显著增多(P值均＜0.05)；免疫荧光共染显示CCl₄/2-AAF组大鼠肝组织内CK19阳性细胞均表达OV6，且较正常组显著增多。与CCl₄/2-AAF组相比，GDC-0449组肝组织炎性细胞聚集、胶原沉积明显减少；肝组织Hyp水平、胶原阳性面积比显著降低(P值均＜0.05)，Ishak评分显著下降(P＜0.05)；α-SMA、Epcam、CK19、CK7、Smo、Ptch2、Gli1、Gli2和Gli3的表达显著减少(P值均＜0.05)；免疫荧光共染显示肝组织内共表达OV6与CK19的阳性细胞数显著减少。结论 Hedgehog信号通路抑制剂GDC-0449显著抑制了CCl₄/2-AAF诱导的大鼠肝纤维化进展，且其作用机制与抑制肝星状细胞活化、胶原沉积，阻抑肝祖细胞活化增殖，并抑制其向胆管上皮细胞分化有关。
- 肝硬化 /
- Hedgehog信号通路 /
- 大鼠, 近交F344
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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图 1 各组大鼠肝组织HE、SR染色

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图 2 Col-Ⅳ和α-SMA免疫组化染色(×200)

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图 3 各组大鼠α-SMA蛋白水平

注：a，α-SMA的Western Blot结果；b，α-SMA蛋白的相对含量。

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图 4 Epcam、CK19和CK7免疫组化染色(×200)

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图 5 CK19(红色)和OV6(绿色)免疫荧光共染

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表 1 各组大鼠肝组织SR阳性染色面积比、Hyp水平及Ishak评分比较

组别	动物数(只)	SR阳性染色面积比(%)	Hyp(μg/mL)	Ishak评分(分)
正常组	6	1.05±0.37	335.63±51.75	0.00±0.00
CCl₄/2-AAF组	6	12.18±3.90¹⁾	1 003.18±297.33¹⁾	13.17±1.72¹⁾
GDC-0449组	6	3.99±1.43²⁾	645.54±69.60²⁾	8.50±1.38²⁾
F值		28.69	17.45	164.80
P值		＜0.001	＜0.001	＜0.001
注：与正常组比较，1)P＜0.05；与CCl₄/2-AAF组比较，2)P＜0.05。

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表 2 各组大鼠肝组织Col-Ⅰ、Col-Ⅳ、Acta2 mRNA表达量变化

组别	动物数(只)	Col-ⅠmRNA	Col-Ⅳ mRNA	Acta2 mRNA
正常组	6	0.59±0.29	0.78±0.23	1.82±0.66
CCl₄/2-AAF组	6	21.73±9.40¹⁾	6.56±2.02¹⁾	9.10±2.43¹⁾
GDC-0449组	6	17.34±2.19	4.80±1.43	5.18±0.97²⁾
F值		19.99	21.16	27.47
P值		＜0.001	＜0.001	＜0.001
注：与正常组比较，1)P＜0.05；与CCl₄/2-AAF组比较，2)P＜0.05。

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表 3 各组大鼠肝组织Epcam、CK19、CK7和Ccnd1 mRNA表达水平

组别	动物数(只)	Epcam mRNA	CK19 mRNA	CK7 mRNA	Ccnd1 mRNA
正常组	6	1.32±0.42	0.57±0.27	1.00±0.18	1.14±0.15
CCl₄/2-AAF组	6	7.59±1.95¹⁾	8.64±1.03¹⁾	1.71±0.27¹⁾	2.30±0.16¹⁾
GDC-0449组	6	3.07±1.49²⁾	5.47±1.81²⁾	1.18±0.24²⁾	1.91±0.30²⁾
F值		25.26	56.28	12.07	38.21
P值		＜0.001	＜0.001	0.002	＜0.001
注：与正常组比较，1)P＜0.05；与CCl₄/2-AAF组比较，2)P＜0.05。

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表 4 各组大鼠肝组织Hedgehog信号通路相关因子mRNA表达量变化

组别	动物数(只)	Smo mRNA	Dhh mRNA	Ptch2 mRNA	Gli1 mRNA	Gli2 mRNA	Gli3 mRNA
正常组	6	0.87±0.09	1.10±0.29	0.62±0.27	0.80±0.24	0.65±0.24	0.85±0.27
CCl₄/2-AAF组	6	4.31±1.30¹⁾	12.89±2.79¹⁾	3.20±0.86¹⁾	10.00±3.65¹⁾	1.62±0.39¹⁾	1.65±0.34¹⁾
GDC-0449组	6	2.60±0.35²⁾	10.14±4.53	0.82±0.22²⁾	0.48±0.26²⁾	0.96±0.03²⁾	0.98±0.13²⁾
F值		24.45	20.11	36.00	32.59	17.35	13.42
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与正常组比较，1)P＜0.05；与CCl₄/2-AAF组比较，2)P＜0.05。

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