鳖甲煎丸对肝癌Huh-7细胞皮下移植裸鼠模型的影响及作用机制

卢露; 陈焕灵; 徐健; 杜沅沁; 刘晓丽; 吴颖升; 吴成挺; 班惟; 黄晶晶; 黄鸿娜

doi:10.12449/JCH260115

鳖甲煎丸对肝癌Huh-7细胞皮下移植裸鼠模型的影响及作用机制

DOI: 10.12449/JCH260115

卢露¹,
陈焕灵¹,
徐健¹,
杜沅沁¹,
刘晓丽¹,
吴颖升¹,
吴成挺¹,
班惟¹,
黄晶晶^{2, 3, , ,},
黄鸿娜^{4, 5}

1.
广西中医药大学研究生院，南宁 530022
2.
广西中医药大学第一附属医院仙葫院区脾胃肝病科，南宁 530022
3.
广西高发传染病中西医结合转化医学重点实验室，南宁 530022
4.
广西中医药大学第一附属医院中医内科教研室，南宁 530022
5.
广西中医药大学第一附属医院肝病科，南宁 530022

基金项目:

国家自然科学基金 (8246150205);

广西自然科学基金 (2022GXNSFAA035460);

广西自然科学基金 (2024GXNSFDA010005)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：卢露、陈焕灵负责课题设计，起草论文；卢露、吴颖升、徐健、班惟负责实验操作，研究过程的实施；卢露、吴颖升、陈焕灵、刘晓丽负责数据收集，统计学分析，绘制图表；杜沅沁、卢露、吴成挺负责拟定写作思路；黄晶晶、黄鸿娜负责指导撰写文章并最后定稿。

详细信息

通信作者:
黄晶晶， 55869563@qq.com （ORCID： 0000-0002-4932-0838）

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出版历程
- 收稿日期: 2025-07-12
- 录用日期: 2025-10-15
- 出版日期: 2026-01-25

Effect and mechanism of Biejiajian Pill on subcutaneous xenograft tumor model of hepatocellular carcinoma Huh7 cells

1.
Graduate School of Guangxi University of Chinese Medicine，Nanning 530022，China
2.
Department of Spleen and Stomach Hepatology，Xianhu Branch of The First Affiliated Hospital of Guangxi University of Chinese Medicine，Nanning 530022，China
3.
Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine，Nanning 530022，China
4.
Department of Chinese Internal Medicine，The First Affiliated Hospital of Guangxi University of Chinese Medicine，Nanning 530022，China
5.
Department of Hepatology，The First Affiliated Hospital of Guangxi University of Chinese Medicine，Nanning 530022，China

Research funding:

National Natural Science Foundation of China (8246150205);

Guangxi Natural Science Foundation (2022GXNSFAA035460);

Guangxi Natural Science Foundation (2024GXNSFDA010005)

More Information

Corresponding author: HUANG Jingjing， 55869563@qq.com （ORCID： 0000-0002-4932-0838）

摘要

摘要: 目的评估鳖甲煎丸对肝癌生长的抑制作用，并阐明其通过线粒体能量代谢介导AMP活化的蛋白质激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）通路调控的潜在机制。方法采用人肝癌Huh-7细胞建立裸鼠皮下移植瘤模型，将18只荷瘤裸鼠随机分为模型组、鳖甲煎丸组（2.2 g/kg）和二甲双胍组（250 mg/kg），灌胃给药14 d。实验期间监测肿瘤体积与质量；采用HE染色观察组织病理学变化；检测瘤组织活性氧（ROS）与三磷酸腺苷（ATP）水平；采用免疫组化法、蛋白质免疫印迹法检测AMPK/mTOR通路相关蛋白表达水平。满足正态分布的计量资料采用单因素方差分析进行多组间比较，进一步两两比较采用Tukey’s法检验；不满足正态分布的计量资料采用Kruskal-Wallis H检验进行多组间比较，进一步两两比较采用Dunn’s法。结果与模型组比较，鳖甲煎丸组相对肿瘤体积与质量均显著降低（P值均<0.01），抑瘤率达45.73%。病理学显示，与模型组比较，鳖甲煎丸组肿瘤细胞数量减少，并出现广泛坏死；机制研究表明，与模型组比较，鳖甲煎丸组ROS水平显著升高（P<0.001），ATP水平显著降低（P<0.001）；同时，p-AMPK/AMPK比值（0.81±0.20 vs0.13±0.04）与p-ULK1/ULK1比值（0.69±0.17 vs0.18±0.13）均显著升高（P值均<0.01），而p-mTOR/mTOR比值（1.34±0.16 vs 3.20±0.62）显著降低（P<0.01）。结论鳖甲煎丸可能通过诱导线粒体能量代谢障碍，即升高ROS水平并降低ATP水平，进而激活AMPK/mTOR信号通路，抑制肝癌生长。
- 鳖甲煎丸 /
- 肝肿瘤 /
- AMP活化的蛋白质激酶 /
- 哺乳动物雷帕霉素靶蛋白
Abstract: Objective To investigate the inhibitory effect of Biejiajian Pills （BJJW） on the growth of liver cancer， as well as its potential mechanism in mediating the AMP-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） pathway through mitochondrial energy metabolism. Methods Human hepatoma Huh7 cells were used to establish a nude mouse model of subcutaneous xenograft tumor. A total of 18 tumor-bearing nude mice were randomly divided into model group， BJJW group （2.2 g/kg）， and metformin group （250 mg/kg）， and the corresponding drug was given by gavage for 14 consecutive days. Tumor volume and weight were monitored during the experiment； HE staining was used to observe histopathological changes； the levels of reactive oxygen species （ROS） and adenosine triphosphate （ATP） in tumor tissue were measured； immunohistochemistry and Western blotting were used to measure the expression levels of proteins associated with the AMPK/mTOR pathway. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups， and the Tukey’s test was used for further comparison between two groups； the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups， and the Dunn’s test was used for further comparison between two groups. Results Compared with the model group， the BJJW group had a tumor inhibition rate of 45.73%， with significant reductions in both tumor volume and weight （P<0.01）. Pathological examination showed that compared with the model group， the BJJW group had a significant reduction in the number of tumor cells and the presence of extensive necrosis. Mechanistic studies showed that compared with the model group， the BJJW group had a significant increase in ROS level （P<0.001） and a significant reduction in ATP level （P<0.001）， as well as significant increases in p-AMPK/AMPK ratio （0.81±0.20 vs 0.13±0.04， P<0.01） and p-ULK1/ULK1 ratio （0.69±0.17 vs 0.18±0.13， P<0.01） and a significant reduction in p-mTOR/mTOR ratio （1.34±0.16 vs 3.20±0.62， P<0.01）. Conclusion BJJW may inhibit the growth of liver cancer by inducing mitochondrial energy metabolism dysfunction， increasing the level of ROS， reducing the level of ATP， and activating the AMPK/mTOR signaling pathway.
- Biejiajian Pills /
- Liver Neoplasms /
- AMP-activated Protein Kinase /
- Mammalian Target of Rapamycin

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图 1 各药物组对Huh7裸鼠移植瘤的抑制作用

Figure 1. Inhibitory effect of various drugs groups on subctaneous xenografts in nude mice

下载: 全尺寸图片幻灯片

注：黑色箭头，坏死；蓝色箭头，出血；黄色箭头，核分裂现象；红色箭头，淋巴细胞浸润；绿色箭头，嗜酸性物质渗出；棕色箭头，肿瘤细胞水样变性。

图 2 鳖甲煎丸对Huh7裸鼠移植瘤组织形态学的影响（HE染色）

Figure 2. Effect of Biejiajian Pills on morphological changes of Huh7 nude mice transplanted tumors（H&E staining）

下载: 全尺寸图片幻灯片

3 鳖甲煎丸对Huh7 裸鼠移植瘤组织ROS 水平的影响（DHE 荧光法染色，×200）

3. Effect of Biejiajian Pills on ROS content inHuh7 nude mice transplanted tumors（DHE fluorescence staining，×200）

下载: 全尺寸图片幻灯片

图 4 鳖甲煎丸对Huh7裸鼠移植瘤组织ATP水平的影响

Figure 4. Effect of Biejiajian Pills on ATP content in Huh7 nude mice transplanted tumors

下载: 全尺寸图片幻灯片

图 5 鳖甲煎丸对Huh7裸鼠移植瘤组织p-AMPK、p-mTOR蛋白表达水平的影响

Figure 5. Effect of Biejiajian Pills on p-AMPK， p-mTOR protein in Huh7 nude mice transplanted tumors

下载: 全尺寸图片幻灯片

图 6 各组Huh7裸鼠移植瘤组织AMPK、p-AMPK、mTOR、p-mTOR、ULK1、p-ULK1蛋白印迹

Figure 6. Western Blott bands AMPK， p-AMPK， mTOR， p-mTOR， ULK1 and p-ULK1 of graft tumours in Huh7 nude mice transplanted tumors

下载: 全尺寸图片幻灯片

表 1 鳖甲煎丸对Huh7裸鼠移植瘤体积变化的影响

Table 1. Effect of Biejiajian Pills on hepatocellular subcutaneous tumor mice

组别	动物数（只）	第1天	第3天	第5天	第7天	第9天	第12天	第14天
MOD组	6	106.17±4.19	126.87±8.55	183.98±4.98	291.84±22.53	459.73±83.46	632.40±101.37	829.73±115.17
BJJW组	6	106.61±5.05	117.67±7.66	125.26±12.83^1）	174.88±28.46^1）	251.81±85.24^1）2）	303.09±112.98^1）2）	408.44±141.95^1）2）
MET组	6	104.47±3.64	124.73±8.78	144.88±18.62^1）2）	147.09±27.96^1）2）	180.80±48.03^1）	222.08±63.49^1）	272.74±81.03^1）
F值		0.409	1.996	30.004	50.589	22.870	31.401	37.979
P值		0.672	0.170	<0.001	<0.001	<0.001	<0.001	<0.001
注：与MOD组比较，1）P<0.01；与MET组比较，2）P<0.05。

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表 2 鳖甲煎丸对Huh7裸鼠移植瘤生长的影响

Table 2. Effect of Biejiajian Pills on the growth of transplanted tumours

组别	动物数（只）	RTV	移植瘤质量（g）	肝脏指数（%）	脾脏指数（%）
MOD组	6	7.81±1.02	0.61±0.09	40.46±5.42	3.67±0.43
BJJW组	6	3.82±1.35^1）	0.33±0.113^1）	42.77±3.64	3.54±0.27
MET组	6	2.60±0.74^1）	0.23±0.08^1）	41.78±2.63	4.15±0.76
F值		39.249	25.342	0.486	2.244
P值		<0.001	<0.001	0.624	0.140
注：与MOD组比较，1）P<0.01。

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表 3 鳖甲煎丸对Huh7裸鼠移植瘤组织p-AMPK、p-mTOR蛋白表达水平的影响

Table 3. Effect of Biejiajian Pills on p-AMPK、p-mTOR protein in Huh7 nude mice transplanted tumors

组别	动物数（只）	p-AMPK	p-mTOR
MOD组	6	2.59（1.57～3.48）	7.74（4.13～13.16）
BJJW组	6	7.72（5.54～9.47）^1）2）	1.05（0.60～1.67）^1）2）
MET组	6	17.31（15.69～21.47）^1）	0.22（0.14～0.51）^1）
χ²值		40.48	41.62
P值		<0.001	<0.001
注：与MOD组比较，1）P<0.01；与MET组比较，2）P<0.05。

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表 4 鳖甲煎丸对Huh7裸鼠移植瘤组织AMPK/mTOR信号通路相关蛋白表达水平的影响

Table 4. Effect of Biejiajian Pills on expression levels of AMPK/mTOR signaling pathway-related proteins in Huh7 nude mice transplanted tumors

组别	动物数（只）	AMPK	p-AMPK	p-AMPK/ AMPK	mTOR	p-mTOR	p-mTOR/ mTOR	ULK1	p-ULK1	p-ULK1/ ULK1
MOD组	6	0.41±0.03	0.05±0.02	0.13±0.04	0.17±0.02	0.55±0.09	3.20±0.62	0.40±0.10	0.06±0.04	0.18±0.13
BJJW组	6	0.44±0.07	0.36±0.10^1）2）	0.81±0.20^1）2）	0.18±0.04	0.24±0.06^1）2）	1.34±0.16^1）2）	0.42±0.10	0.28±0.05^1）2）	0.69±0.17^1）2）
MET组	6	0.35±0.07	0.74±0.13^1）	2.10±0.09^1）	0.20±0.04	0.07±0.05^1）	0.33±0.19^1）	0.40±0.04	0.71±0.10^1）	1.79±0.33^1）
F值		4.00	79.41	374.79	0.65	75.88	84.72	0.06	137.75	78.28
P值		0.040	<0.001	<0.001	0.538	<0.001	<0.001	0.943	<0.001	<0.001
注：与MOD组比较，1）P<0.01；与MET组比较，2）P<0.05。

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