垂盆草诱导 HepG2 细胞凋亡及其作用机制研究。
Promotion of HepG2 cell apoptosis by Sedum emarginatum Migo and the mechanism of action.
机构信息
Guangxi University of Chinese Medicine, Nanning, 530001, China.
Guangxi Superior Chinese Patent Medicine and National Medicine Development Engineering Technology Research Center, Nanning, 530001, China.
出版信息
BMC Complement Med Ther. 2022 Jan 31;22(1):31. doi: 10.1186/s12906-022-03503-6.
BACKGROUND
Sedum emarginatum Migo(S. emarginatum) has anti-tumor and anti-oxidant effects. This study aimed to screen the extractions of S. emarginatum against liver cancer in vitro and explore its anti-liver cancer mechanism.
METHODS
The CCK-8(Cell Counting Kit-8) method was used to detect the inhibitory effect of different extracts of S. emarginatum on the proliferation of liver cancer HepG2 cells. The morphological changes of the cells after administration were observed with microscopy, cell apoptosis was detected by flow cytometry, and the expression of Bax, Bcl-2 and Caspase-3 mRNA in the cells were detected by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) to explore the mechanism of action.
RESULTS
CCK-8 method test results showed that among the different extracts of S. emarginatum, the ethyl acetate extract(1000 μg/ml, 2000 μg/ml, 2500 μg/ml, 3000 μg/ml) and n-butanol extract(1000 μg/ml, 2000 μg/ml, 2500 μg/ml, 3000 μg/ml) have the strongest inhibitory effect on the proliferation of HepG2 cells. In these 4 concentrations, the inhibitory effect increased as the concentration increased. The IC of the ethyl acetate extract on HepG2 cells was less than that of the n-butanol extract, so the ethyl acetate extract has a better proliferation inhibitory effect on HepG2 cells than the n-butanol extract, followed by the 70% ethanol extract(3000 μg/ml) and the water extract(3000 μg/ml), petroleum ether extract was the weakest. The results of microscopy showed that ethyl acetate extract caused hepatocarcinoma HepG2 cell morphology changed, cell density decreased, and suspension cells increased. Moreover, the results of flow cytometry showed that the ethyl acetate extract of S. emarginatum could induce HepG2 cell apoptosis at the concentrations of 2500μg/ml and 3000μg/ml. RT-PCR results showed that the expression of Bax mRNA was up-regulate by the middle(2500 μg/ml) and high(3000 μg/ml) dose groups of ethyl acetate extract. The expression of Caspase-3 mRNA was up-regulated by the low(2000 μg/ml), medium(2500 μg/ml) and high(3000 μg/ml) dose groups of ethyl acetate extract. The expression of Bcl-2 mRNA was down-regulated by the high(3000 μg/ml) dose group of ethyl acetate extract.
CONCLUSION
The ethyl acetate extract of S. emarginatum has the best effect on human liver cancer HepG2 cells. Its anti-hepatocellular mechanism may be related to affect the expression of apoptosis genes (Bax, Bcl-2 and Caspase-3mRNA) and promote the apoptosis of liver cancer cells. It provided a reference for the research and development of drugs for the treatment of liver cancer.
背景
景天三七（Sedum emarginatum Migo，S. emarginatum）具有抗肿瘤和抗氧化作用。本研究旨在体外筛选景天三七提取物对肝癌的抑制作用，并探讨其抗肝癌机制。
方法
采用 CCK-8（细胞计数试剂盒-8）法检测景天三七不同提取物对肝癌 HepG2 细胞增殖的抑制作用。显微镜观察给药后细胞形态的变化，流式细胞术检测细胞凋亡，RT-PCR（逆转录-聚合酶链反应）检测细胞中 Bax、Bcl-2 和 Caspase-3mRNA 的表达，探讨作用机制。
结果
CCK-8 法检测结果显示，在景天三七的不同提取物中，乙酸乙酯提取物（1000μg/ml、2000μg/ml、2500μg/ml、3000μg/ml）和正丁醇提取物（1000μg/ml、2000μg/ml、2500μg/ml、3000μg/ml）对 HepG2 细胞的增殖抑制作用最强。在这 4 种浓度下，抑制作用随浓度增加而增加。乙酸乙酯提取物对 HepG2 细胞的 IC 小于正丁醇提取物，因此乙酸乙酯提取物对 HepG2 细胞的增殖抑制作用优于正丁醇提取物，其次是 70%乙醇提取物（3000μg/ml）和水提取物（3000μg/ml），石油醚提取物作用最弱。显微镜结果显示，乙酸乙酯提取物导致肝癌 HepG2 细胞形态改变，细胞密度降低，悬浮细胞增多。此外，流式细胞术结果表明，在 2500μg/ml 和 3000μg/ml 浓度下，景天三七乙酸乙酯提取物可诱导 HepG2 细胞凋亡。RT-PCR 结果显示，中剂量（2500μg/ml）和高剂量（3000μg/ml）组乙酸乙酯提取物可上调 BaxmRNA 的表达。低剂量（2000μg/ml）、中剂量（2500μg/ml）和高剂量（3000μg/ml）组乙酸乙酯提取物可上调 Caspase-3mRNA 的表达。高剂量（3000μg/ml）组乙酸乙酯提取物可下调 Bcl-2mRNA 的表达。
结论
景天三七乙酸乙酯提取物对人肝癌 HepG2 细胞的抑制作用最好。其抗肝癌机制可能与影响凋亡基因（Bax、Bcl-2 和 Caspase-3mRNA）的表达和促进肝癌细胞凋亡有关。为肝癌治疗药物的研究与开发提供了参考。
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