脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响

doi:10.3760/cma.j.cn371439-20230523-00099

摘要/Abstract

摘要：

目的研究异戊烯基类黄酮化合物脱水淫羊藿素（AHI）对人肝癌细胞株MHCC-97H的增殖、迁移和凋亡的影响。方法体外培养人肝癌细胞株MHCC-97H及人正常肝细胞株L02，分别用0、20、40、80、120、160、200 μg/ml浓度的AHI处理MHCC-97H细胞，用0、25、50、100、150、200、400、500 μg/ml浓度的AHI处理L02细胞。采用CCK-8和克隆形成实验检测细胞增殖情况；划痕实验检测细胞迁移情况；Hoechst33342实验和流式细胞术检测细胞凋亡情况；蛋白质印迹法检测细胞凋亡相关蛋白表达情况。结果经0、20、40、80、120、160、200 μg/ml AHI处理24 h后，MHCC-97H细胞的存活率分别为（100.00±0.00）%、（97.41±2.10）%、（96.58±3.23）%、（87.72±4.85）%、（78.33±3.76）%、（56.97±2.61）%、（15.25±2.51）%，差异有统计学意义（F=429.20，P<0.001）；80、120、160、200 μg/ml AHI与0 μg/ml AHI相比，差异均有统计学意义（均P<0.001）。经0、25、50、100、150、200、400、500 μg/ml AHI处理24 h后，L02细胞存活率分别为（100.00±0.00）%、（96.82±3.79）%、（95.36±3.43）%、（90.79±5.75）%、（77.67±5.66）%、（63.98±5.22）%、（34.22±4.01）%、（33.84±4.41）%，差异有统计学意义（F=233.20，P<0.001）；100、150、200、400、500 μg/ml AHI与0 μg/ml AHI相比，差异均有统计学意义（均P<0.05）。AHI处理L02细胞24 h半抑制浓度（IC₅₀）为（300.20±17.10） μg/ml，明显大于MHCC-97H细胞IC₅₀（158.60±5.50） μg/ml，差异有统计学意义（t=13.65，P<0.001）。经0、120、160、200 μg/ml AHI处理24 h后，MHCC-97H细胞克隆数分别为1 993.00±46.29、1 355.00±54.84、998.33±21.03、218.33±35.95，差异有统计学意义（F=954.80，P<0.001）；120、160、200 μg/ml AHI与0 μg/ml AHI相比，差异均有统计学意义（均P<0.001）。经0、120、160、200 μg/ml AHI处理24 h后，MHCC-97H细胞愈合率分别为（51.68±1.93）%、（16.04±0.73）%、（8.88±0.31）%、（-6.94±0.46）%，差异有统计学意义（F=1 616.00，P<0.001）；120、160、200 μg/ml AHI与0 μg/ml AHI相比，差异均有统计学意义（均P<0.001）。Hoechst33342实验显示，经0 μg/ml AHI处理的MHCC-97H细胞于倒置显微镜下显示出均匀的暗蓝色，并呈现出完整的细胞核状态；相较于0 μg/ml，120、160、200 μg/ml AHI处理的细胞出现皱缩、破碎的现象，细胞核形态异常，部分细胞核被染为亮蓝色，且该情况随着浓度的增加愈为明显。经0、120、160、200 μg/ml AHI处理24 h后，MHCC-97H细胞凋亡率分别为（10.51±0.56）%、（42.23±0.87）%、（61.92±0.52）%、（72.05±0.74）%，差异有统计学意义（F=4 677.00，P<0.001）；120、160、200 μg/ml AHI与0 μg/ml AHI相比，差异均有统计学意义（均P<0.001）。经0、120、160、200 μg/ml AHI处理的MHCC-97H细胞Bax、Cleaved Caspase-3/Caspase-3、Cleaved Caspase-9/Caspase-9、Bcl-2蛋白相对表达量差异均有统计学意义（F=30.43，P<0.001；F=212.80，P<0.001；F=475.30，P<0.001；F=10.75，P=0.004）；160、200 μg/ml AHI Bax蛋白表达与0 μg/ml AHI相比，均显著升高（均P<0.001）；120、160、200 μg/ml AHI Cleaved Caspase-3/Caspase-3、Cleaved Caspase-9/Caspase-9蛋白表达与0 μg/ml AHI相比，均显著升高（均P<0.001）；120、160、200 μg/ml AHI Bcl-2蛋白表达与0 μg/ml AHI相比，均显著降低（均P<0.05）。结论 AHI可抑制人肝癌细胞株MHCC-97H的增殖、迁移，并促进其凋亡。

关键词: 肝肿瘤, 细胞增殖, 细胞运动, 细胞凋亡, 脱水淫羊藿素

Abstract:

Objective To investigate the effects of Anhydroicaritin （AHI）， an isopentenylated flavo-noid compound， on proliferation， migration and apoptosis of human hepatocarcinoma cell line MHCC-97H. Methods Human hepatocarcinoma cell line MHCC-97H and human normal liver cell line L02 were cultured in vitro. MHCC-97H cells were treated with 0， 20， 40， 80， 120， 160， 200 μg/ml of AHI respectively and L02 cells were treated with 0， 25， 50， 100， 150， 200， 400， 500 μg/ml of AHI respectively. CCK-8 and clone formation assay were used to detect cell proliferation. Scratch test was used to explore cell migration ability. Hoechst33342 assay and flow cytometer were used to detect cell apoptosis. The expressions of apoptosis-related proteins were detected by Western blotting. Results The cell viabilities of MHCC-97H cells treated with 0， 20， 40， 80， 120， 160， 200 μg/ml of AHI for 24 h were （100.00±0.00）%，（97.41±2.10）%，（96.58±3.23）%，（87.72±4.85）%，（78.33±3.76）%，（56.97±2.61）% and （15.25±2.51）% respectively， and there was a statistically significant difference （F=429.20， P<0.001）. There were statistically significant differences between 0 μg/ml and 80， 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. The cell viabilities of L02 cells treated with 0， 25， 50， 100， 150， 200， 400， 500 μg/ml of AHI for 24 h were （100.00±0.00）%，（96.82±3.79）%，（95.36±3.43）%，（90.79±5.75）%，（77.67±5.66）%，（63.98±5.22）%，（34.22±4.01）% and （33.84±4.41）% respectively， and there was a statistically significant difference （F=233.20， P<0.001）. There were statistically significant differences between 0 μg/ml and 100， 150， 200， 400， 500 μg/ml of AHI treatment （all P<0.05）. The 24 h half maximal inhibitory concentration （IC₅₀） value of AHI treated L02 cells was （300.20±17.10） μg/ml， which was significantly higher than that of MHCC-97H cells [（158.60±5.50） μg/ml]， and there was a statistically significant difference （t=13.65， P<0.001）. The cell clone numbers of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml of AHI for 24 h were 1 993.00±46.29， 1 355.00±54.84， 998.33±21.03 and 218.33±35.95 respectively， and there was a statistically significant difference （F=954.80， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. The healing rates of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml of AHI for 24 h were （51.68±1.93）%，（16.04±0.73）%，（8.88±0.31）% and （-6.94±0.46）% respectively， and there was a statistically significant difference （F=1 616.00， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. Hoechst33342 experiment showed that MHCC-97H cells treated with 0 μg/ml AHI showed uniform dark blue with a complete nuclear state under inverted microscope. Compared with 0 μg/ml AHI treated cells， cells in the 120， 160， 200 μg/ml AHI treatment groups wrinkled and broken， and nuclei were also morphologically abnormal， with some nuclei stained bright blue， and the situation became more obvious with increasing dose. The apoptosis rates of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml AHI for 24 h were （10.51±0.56）%，（42.23±0.87）%，（61.92±0.52）% and （72.05±0.74）% respectively， and there was a statistically significant difference （F=4 677.00， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. There were statistically significant differences among the different expression levels of Bax， Cleaved Caspase-3/Caspase-3， Cleaved Caspase-9/Caspase-9， and Bcl-2 proteins in MHCC-97H cells of 0， 120， 160， and 200 μg/ml of AHI treatment （F=30.43， P<0.001； F=212.80， P<0.001； F=475.30， P<0.001； F=10.75， P=0.004）. The Bax protein expression of 160 and 200 μg/ml was significantly increased than that of 0 μg/ml AHI （both P<0.001）. The Cleaved Caspase-3/Caspase-3， Cleaved Caspase-9/Caspase-9 protein expressions of 120， 160 and 200 μg/ml were significantly increased than those of 0 μg/ml AHI （all P<0.001）. The Bcl-2 protein expression of 120， 160， 200 μg/ml was significantly decreased compared with that of 0 μg/ml AHI （all P<0.05）. Conclusion AHI can inhibit the proliferation and migration of hepatocellular carcinoma cell line MHCC-97H， and promote its apoptosis.

Key words: Liver neoplasms, Cell proliferation, Cell movement, Apoptosis, Anhydroicaritin

向玉玲, 谭佳杰, 熊远果, 赵丽蓉, 黎晨, 张洪. 脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(9): 513-519.

Xiang Yuling, Tan Jiajie, Xiong Yuanguo, Zhao Lirong, Li Chen, Zhang Hong. Effects of Anhydroicaritin on the proliferation， migration and apoptosis of hepatocellular carcinoma cells[J]. Journal of International Oncology, 2023, 50(9): 513-519.

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浓度（μg/ml）	24 h	48 h	72 h
0	100.00±0.00	100.00±0.00	100.00±0.00
20	97.41±2.10	96.37±2.97	94.60±2.45^c
40	96.58±3.23	92.91±5.22^b	82.92±3.69^a
80	87.72±4.85^a	80.79±4.60^a	55.42±5.20^a
120	78.33±3.76^a	57.48±3.12^a	38.72±2.82^a
160	56.97±2.61^a	26.91±3.78^a	23.81±3.01^a
200	15.25±2.51^a	9.85±1.25^a	1.63±0.62^a
F值	429.20	656.80	921.80
P值	<0.001	<0.001	<0.001

浓度（μg/ml）	Bax	Cleaved Caspase-3/Caspase-3	Cleaved Caspase-9/Caspase-9	Bcl-2
0	0.45±0.05	0.10±0.02	0.08±0.01	0.77±0.03
120	0.51±0.02	0.57±0.05^a	0.26±0.01^a	0.71±0.02^b
160	0.68±0.03^a	0.68±0.02^a	0.61±0.03^a	0.70±0.01^c
200	0.77±0.07^a	0.89±0.06^a	0.91±0.05^a	0.68±0.01^d
F值	30.43	212.80	475.30	10.75
P值	<0.001	<0.001	<0.001	0.004