没食子酸对人肝癌HepG2细胞增殖、迁移、侵袭和凋亡的影响及其机制

王志茹; 赵文静; 陈曦

doi:10.12449/JCH250315

没食子酸对人肝癌HepG2细胞增殖、迁移、侵袭和凋亡的影响及其机制

DOI: 10.12449/JCH250315

吉林省肝胆病医院中心实验室，长春 130062

基金项目:

吉林省卫生与健康技术创新项目 (2020J082)

伦理学声明：本研究方案于2023年9月11日经由吉林省肝胆病医院伦理委员会审批，批号：2023-科研伦理审查-004。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：王志茹负责课题设计，资料分析，撰写论文；陈曦参与收集数据，修改论文；赵文静负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
赵文静， xingyuewj@163.com （ORCID： 0000-0002-0841-9362）

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出版历程
- 收稿日期: 2024-07-09
- 录用日期: 2024-09-19
- 出版日期: 2025-03-25

Effect and mechanism of gallic acid on the proliferation， migration， invasion， and apoptosis of human hepatocellular carcinoma HepG2 cells

Central Laboratory，Hepatobiliary Hospital of Jilin，Changchun 130062，China

Research funding:

Health Science and Health Technology Innovation Project of Jilin Province (2020J082)

More Information

Corresponding author: ZHAO Wenjing， xingyuewj@163.com （ORCID： 0000-0002-0841-9362）

摘要

摘要: 目的观察没食子酸（GA）对人肝癌HepG2细胞增殖、迁移、侵袭和凋亡的影响，并探讨其作用机制。方法用不同浓度GA（0、5、10、20、30、40、50 μg/mL）处理肝癌HepG2细胞24 h和48 h后，CCK-8法检测细胞活性并计算IC₅₀值；实验分为对照组（HepG2细胞）、5 μg/mL GA组、10 μg/mL GA组、20 μg/mL GA组，平板克隆形成实验检测GA对细胞增殖能力的影响，细胞划痕和Transwell小室侵袭实验检测GA对细胞迁移和侵袭能力的影响，流式细胞仪检测GA对细胞凋亡的影响；Western Blot检测基质金属蛋白酶2（MMP-2）、MMP-9和凋亡相关蛋白表达情况。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 GA作用HepG2细胞24 h和48 h的IC₅₀值为（38.02±2.58）μg/mL和（18.36±1.54）μg/mL。对照组、5 μg/mL GA组、10 μg/mL GA组、20 μg/mL GA组的细胞克隆形成数分别为（239.00±29.45）个、（210.00±19.00）个、（144.33±16.03）个、（57.00±9.55）个，与对照组比较，各实验组细胞克隆形成能力均明显下降（P值均<0.05）。处理24 h后，各组细胞的迁移率分别为42.62%±7.82%、35.34%±6.42%、21.85%±4.42%、12.57%±3.54%，穿膜细胞数目分别为（230.30±15.30）个、（182.12±12.60）个、（137.20±7.50）个、（124.40±6.80）个，与对照组比较，各实验组细胞的相对迁移率和穿膜细胞数均明显下降（P值均<0.05）。处理48 h后，各组细胞凋亡率分别为0.67%±0.08%、13.27%±1.07%、20.94%±2.45%、40.74%±2.63%，与对照组比较，各实验组细胞凋亡率均明显升高（P值均<0.05）。与对照组比较，各实验组细胞MMP-2、MMP-9表达水平均明显降低（P值均<0.05），Bcl-2关联X蛋白（Bax）、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）蛋白表达水平均明显升高（P值均<0.05）。结论 GA可抑制HepG2细胞增殖、迁移和侵袭，促进其凋亡，作用机制可能与调控Bax/Bcl-2以及迁移相关蛋白MMP-2、MMP-9有关。
- 没食子酸 /
- 肝肿瘤，实验性 /
- 细胞增殖 /
- 细胞运动 /
- 细胞凋亡
Abstract: Objective To investigate the effect of gallic acid （GA） on the proliferation， migration， invasion， and apoptosis of human hepatocellular carcinoma HepG2 cells and its mechanism. Methods HepG2 cells were treated with different concentrations of GA （0， 5， 10， 20， 30， 40， and 50 μg/mL） for 24 and 48 hours， and CCK8 assay was used to measure cell viability and calculate IC₅₀. The experiment was divided into control group （HepG2 cells）， 5 μg/mL GA group， 10 μg/mL GA group， and 20 μg/mL GA group. Plate colony formation assay was used to evaluate the effect of GA on cell proliferation； wound healing assay and Transwell chamber assay were used to observe the effect of GA on cell migration and invasion； flow cytometry was used to observe the effect of GA on cell apoptosis； Western blot was used to measure the expression of matrix metallopeptidase-2 （MMP-2）， matrix metallopeptidase-9 （MMP-9）， and apoptosis-related proteins. A one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results The mean IC₅₀ value of GA on HepG2 cells was 38.02±2.58 μg/mL at 24 hours and 18.36±1.54 μg/mL at 48 hours. The number of cell colonies was 239.00±29.45 in the control group， 210.00±19.00 in the 5 μg/mL GA group， 144.33±16.03 in the 10 μg/mL GA group， and 57.00±9.55 in the 20 μg/mL GA group， suggesting that compared with the control group， each GA group had a significant reduction in cell colony formation ability （all P<0.05）. After 24 hours of treatment， the cell migration rate was 42.62%± 7.82% in the control group， 35.34%±6.42% in the 5 μg/mL GA group， 21.85%±4.42% in the 10 μg/mL GA group， and 12.57%± 3.54% in the 20 μg/mL GA group， respectively， in these four groups， and the number of transmembrane cells in these four groups was 230.30±15.30， 182.12±12.60， 137.20±7.50， and 124.40±6.80， respectively， suggesting that compared with the control group， each GA group had significant reductions in migration rate and the number of transmembrane cells （all P<0.05）. After 48 hours of treatment， the cell apoptotic rate was 0.67%±0.08% in the control group， 13.27%±1.07% in the 5 μg/mL GA group， 20.94%± 2.45% in the 10 μg/mL GA group， and 40.74%±2.63% in the 20 μg/mL GA group， and compared with the control group， each GA group had a significant increase in cell apoptosis rate （all P<0.05）. Compared with the control group， each GA group had significant reductions in the protein expression levels of MMP-2 and MMP-9 （all P<0.05） and significant increases in the protein expression levels of Bax and cleaved caspase-3 （all P<0.05）. Conclusion GA can inhibit the proliferation， migration， and invasion of HepG2 cells and promote the apoptosis of HepG2 cells， possibly by regulating MMP-2， MMP-9， and the apoptosis-related proteins Bax/Bcl-2.
- Gallic Acid /
- Liver Neoplasms， Experimental /
- Cell Proliferation /
- Cell Movement /
- Apoptosis

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图 1 GA对HepG2细胞活性的影响

Figure 1. Effect of GA on activity of HepG-2 cells

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图 2 GA对HepG2细胞增殖能力的影响

Figure 2. Effect of GA on proliferation of HepG2 cells

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图 3 GA对对HepG2细胞迁移能力的影响

Figure 3. Effect of GA on migration activity of HepG2 cells

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图 4 GA对HepG2细胞侵袭能力的影响（结晶紫，✕100）

Figure 4. Effect of GA on invasion activity of HepG2 cells （crystal violet， ✕100）

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图 5 GA对HepG2细胞凋亡的影响

Figure 5. Effect of GA on apoptosis of HepG2 cells

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注：与对照组比较，*P<0.05。

图 6 GA对HepG2细胞MMP-2、MMP-9和凋亡相关蛋白表达的影响

Figure 6. Effect of GA on MMP-2、MMP-9 and apoptosis-related proteins of HepG-2 cells

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表 1 各组HepG2细胞迁移率和穿膜细胞数

Table 1. Migration rate and number invasion of HepG2 cells in each group

组别	迁移率（η/%）	穿膜细胞数（个）
对照组	42.62±7.82	230.30±15.30
5 μg/mL GA组	35.34±6.42^1）	182.12±12.60^1）
10 μg/mL GA组	21.85±4.42^1）	137.20±7.50^1）
20 μg/mL GA组	12.57±3.54^1）	124.40±6.80^1）
F值	40.030	82.926
P值	<0.001	<0.001
注：与对照组比较，1）P<0.05。

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