双术汤通过P53/SLC7A11/GPX4通路诱导胃癌细胞铁死亡

doi:10.12122/j.issn.1673-4254.2025.07.02

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (7): 1363-1371.doi: 10.12122/j.issn.1673-4254.2025.07.02

双术汤通过P53/SLC7A11/GPX4通路诱导胃癌细胞铁死亡

陈鑫源¹(), 吴成挺¹, 李瑞迪², 潘雪芹³, 张耀丹³, 陶俊宇¹^,⁴, 林才志¹^,²()

^1.广西中医药大学，广西南宁 530299
^2.广西中医药大学第一附属医院仙葫院区脾胃病科，广西南宁 530200
^3.广西中医药大学赛恩斯新医药学院，广西南宁 530299
^4.广西高发传染病中西医结合转化医学重点实验室，广西南宁 530299

收稿日期:2025-01-25 出版日期:2025-07-20 发布日期:2025-07-17
通讯作者: 林才志 E-mail:839029828@qq.com;lincaizhi710103@163.com
作者简介:陈鑫源，在读博士研究生，E-mail: 839029828@qq.com
基金资助:
国家自然科学基金(82060834);广西自然科学基金(2024GXNSFAA010240)

Shuangshu Decoction inhibits growth of gastric cancer cell xenografts by promoting cell ferroptosis via the P53/SLC7A11/GPX4 axis

Xinyuan CHEN¹(), Chengting WU¹, Ruidi LI², Xueqin PAN³, Yaodan ZHANG³, Junyu TAO¹^,⁴, Caizhi LIN¹^,²()

^1.Guangxi University of Chinese Medicine, Nanning 530299, China
^2.Department of Spleen and Gastroenterology, Xianhu Campus of First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530200, China
^3.Faculty of Chinese Medicine Science, Guangxi University of Chinese Medicine, Nanning 530299, China
^4.Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine, Nanning 530299, China

Received:2025-01-25 Online:2025-07-20 Published:2025-07-17
Contact: Caizhi LIN E-mail:839029828@qq.com;lincaizhi710103@163.com
Supported by:
National Natural Science Foundation of China(82060834)

摘要/Abstract

摘要：

目的探讨双术汤调控胃癌铁死亡的机制。方法基于网络药理学筛选双术汤与胃癌铁死亡的共同靶点，通过生物信息学分析（差异基因、HPA数据库、生存分析）及分子对接验证核心靶点。细胞实验分为对照组（未经任何干预的AGS细胞）、双术汤组（经双术汤含药血清干预的AGS细胞）、双术汤+铁死亡抑制剂组Fer-1（经双术汤含药血清和铁死亡抑制剂Fer-1共同干预的AGS细胞）、铁死亡抑制剂组（仅经Fer-1干预的AGS细胞）4组，检测细胞活力、铁死亡相关指标（活性氧、铁离子、谷胱甘肽）、P53/SLC7A11/GPX4表达水平及线粒体形态的改变。动物实验分为对照组（生理盐水灌胃）、双术汤组（双术汤灌胃）、双术汤+Fer-1组（双术汤灌胃+腹腔注射Fer-1）、Fer-1组（腹腔注射Fer-1），检测裸鼠肿瘤体积与质量、HE染色评估肿瘤病理情况，评估P53/SLC7A11/GPX4表达水平。结果双术汤主要活性成分为槲皮素、汉黄芩素，与核心靶点P53结合紧密。双术汤能够抑制AGS细胞增殖，以高剂量浓度的抑制作用最明显（P<0.05）；与对照组及双术汤+Fer-1组比较，双术汤组ROS和Fe²⁺均升高（P<0.05），P53蛋白及mRNA表达水平升高，SLC7A11、GPX4蛋白及基因水平降低（P<0.05）；与双术汤组相比，Fer-1组ROS和Fe ²⁺ 均降低（P<0.05），P53蛋白及mRNA表达水平降低（P<0.05），SLC7A11、GPX4蛋白、基因水平升高（P<0.05）。对照组线粒体形态正常，双术汤组线粒体皱缩、嵴模糊甚至消失、膜密度升高；双术汤+Fer-1组线粒体损伤程度较前减轻，Fer-1组线粒体形态与对照组相似。双术汤组肿瘤体积和质量低于对照组（P<0.05），肿瘤排列紊乱并出现坏死，而联合Fer-1后坏死减轻。与对照组相比，双术汤组P53蛋白及mRNA表达升高，SLC7A11和GPX4蛋白及基因表达降低（P<0.05）；与双术汤组相比，双术汤+Fer-1组ROS和Fe²⁺水平降低（P<0.05），P53表达下降，SLC7A11和GPX4表达升高（P<0.05）。结论双术汤能够抑制胃癌皮下移植瘤及胃癌细胞生长，其机制与P53/SLC7A11/GPX4轴诱导胃癌铁死亡有关。

关键词: 胃癌, 网络药理学, 生物信息学, 分子对接, 双术汤, 铁死亡, P53/SLC7A11/GPX4通路

Abstract:

Objective To explore the mechanism of Shuangshu Decoction (SSD) for inhibiting growth of gastric cancer xenografts in nude mice. Methods Network pharmacology analysis was conducted to identify the common targets of SSD and gastric cancer cell ferroptosis, and bioinformatics analysis and molecular docking were used to validate the core targets. In the cell experiment, AGS cells were treated with SSD-medicated serum, Fer-1 (a ferroptosis inhibitor), or both, and the changes in cell viability, ferroptosis markers (ROS, Fe²⁺ and GSH), expressions of P53, SLC7A11 and GPX4, and mitochondrial morphology were examined. In a nude mouse model bearing gastric cancer xenografts, the effects of gavage with SSD, intraperitoneal injection of Fer-1, or their combination on tumor volume/weight, histopathology, and expressions of P53, SLC7A11 and GPX4 levels were evaluated. Results The active components in SSD (quercetin and wogonin) showed strong binding affinities to P53. In AGS cells, SSD treatment dose-dependently inhibited cell proliferation, increased ROS and Fe²⁺levels, upregulated P53 expression, and downregulated the expressions of SLC7A11 and GPX4, but these effects were effectively attenuated by Fer-1 treatment. SSD also induced mitochondrial shrinkage and increased the membrane density, which were alleviated by Fer-1. In the tumor-bearing mouse models, gavage with SSD significantly reduced tumor size and weight, caused tumor cell necrosis, upregulated P53 and downregulated SLC7A11 and GPX4 expression in the tumor tissue, and these effects were obviously mitigated by Fer-1 treatment. Conclusion SSD inhibits gastric cancer growth in nude mice by inducing cell ferroptosis via the P53/SLC7A11/GPX4 axis.

Key words: gastric cancer, network pharmacology, bioinformatics, molecular docking, Shuangshu Decoction, ferroptosis, P53/SLC7A11/GPX4 pathway

陈鑫源, 吴成挺, 李瑞迪, 潘雪芹, 张耀丹, 陶俊宇, 林才志. 双术汤通过P53/SLC7A11/GPX4通路诱导胃癌细胞铁死亡[J]. 南方医科大学学报, 2025, 45(7): 1363-1371.

Xinyuan CHEN, Chengting WU, Ruidi LI, Xueqin PAN, Yaodan ZHANG, Junyu TAO, Caizhi LIN. Shuangshu Decoction inhibits growth of gastric cancer cell xenografts by promoting cell ferroptosis via the P53/SLC7A11/GPX4 axis[J]. Journal of Southern Medical University, 2025, 45(7): 1363-1371.

图/表 6

表1 引物序列和产物长度

Tab.1 Primers sequences and the product length

Primer	The sequence (5'→3')
P53-F	TCAGATAGCGATGGTCTGGC
P53-R	CTCATAGGGACCCACCACAC
SLC7A11-F	GGCAGTTGCTGGGCTGATTT
SLC7A11-R	GGGCAACCATGAAGAGGCAT
GPX4-F	GCGGGCTACAACGTCAAATTC
GPX4-R	TCCACTTGATGGCATTTCCCAG
β-actin-F	CCTGGCACCCAGCACAAT
β-actin-R	GGGCCGGACTCGTCATAC

表2 双术汤排名前10的活性成分

Tab.2 Top 10 active ingredients of Shuangshu Decoction

Molecule name	Degree	Molecular weight	Atom-based log P	Oral bioavailability (%)	Drug-likeness	Main source
Quercetin	199	302	1.5	46.43	0.28	Yanhusuo, Chuanlianzi
Wogonin	146	284.28	2.59	30.68	0.23	Cangshu
Naringenin	127	314.31	2.57	41.17	0.30	Chenpi
Cavidine	125	353.45	3.72	35.64	0.8	Banxia, Yanhusuo
Hyndarin	107	355.47	3.6	73.93	0.64	Yanhusuo
Mandenol	108	308.56	6.99	41.99	0.19	Yanhusuo, Chuanlianzi
Cryptopin	109	369.45	3.15	78.74	0.72	Yanhusuo
(r)-canadine	106	339.42	3.40	55.37	0.77	Yanhusuo
Bicuculline	105	367.38	2.83	69.67	0.88	Yanhusuo
Berberine	105	336.39	3.39	36.86	0.77	Yanhusuo

图1 胃癌相关基因筛选及双术汤作用机制的网络药理学分析

Fig.1 Network pharmacology analysis of gastric cancer-related genes and the mechanism of Shuangshu Decoction. A: Differential gene screening for gastric cancer. B: Wayne diagram of Shuangshu Decoction, gastric cancer (GC) and ferroptopsis. C: PPI diagram of the core targets. D: Differential analysis of P53. E: Representative P53 protein expression pattern in human gastric cancer and normal tissues (Original magnification: ×400). F: Survival analysis of gastric cancer patients with different P53 expression levels. G: Molecular docking analysis of the interaction between the compounds and the core proteins. ****P<0.0001.

图2 双术汤含药血清促进AGS细胞铁死亡的作用及机制

Fig.2 Shuangshu Decoction-medicated serum promotes ferroptosis of AGS cells. A: Determination of the optimal concentration and time of the medicated serum for cell treatment. B: Effect of Shuangshu Decoction on ferroptosis of AGS cells. C: Effect of Shuangshu Decoction-medicated serum on AGS cell morphology (×100). D: Observation of ROS production in the cells under fluorescence microscope (×200). E: Influence of serum of Shuangshu Decoction on ROS production analyzed by flow cytometry and fluorescence intensity. F: Expression of GSH in AGS cells in each group. G: Expression of Fe2+ in AGS cells in each group. *P<0.05 vs control group; #P<0.05 vs SST group; ▲P<0.05 vs SST+Fer-1 group.

图3 双术汤通过P53/SLC7A11/GPX4通路调控AGS细胞铁死亡的分子机制

Fig.3 Shuangshu Decoction promotes ferroptosis in AGS cells via the P53/SLC7A11/GPX4 pathway. A: Protein expression levels of P53, SLC7A11 and GPX4 in the treated cells. B: P53, SLC7A11 and GPX4 mRNA expressions in the treated cells. C: Mitochondrial matrix density in each group. D: Mitochondrial damage in each group (TEM, ×20000). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs SST group; ▲P<0.05 vs SST+Fer-1 group; M: Mitochondria.

图4 双术汤抑制裸鼠移植瘤生长的作用及对P53/SLC7A11/GPX4通路的影响

Fig.4 Inhibitory effect of Shuangshu Decoction on xenograft tumor growth in nude mice and its regulation of the P53/SLC7A11/GPX4 pathway. A: Tumor volume in each group. B: Tumor weight in each group. C: Tumor growth curve in each group. D: Pathological examination of the tumors in each group (HE staining, ×200). E: Protein expression levels of P53, SLC7A11, and GPX4 in the tumor tissues from each group detected by Western blotting. F: SLC7A11, GPX4, and P53 mRNA expression levels in the tumor tissues. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.05 vs SST group; ▲P<0.05 vs SST+Fer-1 group.

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双术汤通过P53/SLC7A11/GPX4通路诱导胃癌细胞铁死亡

Shuangshu Decoction inhibits growth of gastric cancer cell xenografts by promoting cell ferroptosis via the P53/SLC7A11/GPX4 axis

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