连翘叶茶对肝癌细胞增殖和迁移功能的影响及其作用机制

doi:10.13560/j.cnki.biotech.bull.1985.2023-0935

摘要/Abstract

摘要：

【目的】探究连翘叶茶粗提物及其主要成分连翘苷和连翘酯苷A对肝癌细胞LM3增殖和迁移功能的影响及其作用机制。【方法】利用水提法提取连翘叶绿茶和红茶，HPLC方法检测连翘绿茶和连翘红茶中有效成分连翘苷、连翘酯苷A的含量。采用CCK8实验，探究两种连翘叶茶粗提物、连翘苷和连翘酯苷A对肝癌细胞LM3增殖的影响；利用两种连翘叶茶粗提物、连翘苷和连翘酯苷A培养肝癌细胞LM3，通过细胞划痕实验，观察24、48、72、96 h细胞迁移情况，检测两种连翘叶茶粗提物、连翘苷和连翘酯苷A对肝癌细胞LM3迁移的影响；使用RT-PCR技术，检测增殖迁移相关基因Ki-67、Erk、PI3K和mTOR的表达，揭示连翘叶茶粗提物及其主要成分连翘苷和连翘酯苷A影响肝癌细胞增殖迁移功能的分子机制。【结果】连翘叶绿茶和红茶粗提物、连翘苷均显著抑制肝癌细胞LM3的增殖和迁移，连翘酯苷A显著抑制肝癌细胞LM3的迁移。其中，连翘绿茶粗提物在低、中和高浓度，培养时间24、48、72 h，均可明显抑制肝癌细胞增殖和迁移。连翘红茶粗提物随着浓度升高对LM3细胞增殖和迁移的抑制效果不断增强，在高浓度72 h抑制效果最佳。RT-PCR结果分析，其主要作用机制可能是通过降低Ki-67基因表达量来实现的。【结论】连翘叶绿茶和连翘叶红茶粗提物及连翘苷和连翘酯苷A均能够通过降低Ki-67表达抑制肝癌细胞LM3的增殖或迁移。

关键词: 连翘叶茶, 连翘苷, 连翘酯苷A, 肝癌细胞, CCK-8, 划痕实验, Ki-67

Abstract:

【Objective】This work aims to investigate the effects and mechanisms of crude extract of F. suspensa leaves tea and its main components, phillyrin and Forsythia glycoside A, on the proliferation and migration function of hepatocellular carcinoma LM3. 【Method】 Water extraction method was used to extract green tea and black tea from Forsythia suspensa leaves tea, HPLC was used to detect the content of effective components such as phillyrin and Forsythia glycoside A in F. suspensa green tea and F. suspensa black tea. CCK8 experiment was used to explore the effects of crude extracts of two types of F. suspensa leaves tea, phillyrin and Forsythia glycoside A on the proliferation of hepatocellular carcinoma LM3. Two crude extracts of Forsythia suspensa leaves tea, phillyrin and Forsythia glycoside A, were used to culture hepatocellular carcinoma LM3. The migration of cells was observed at 24, 48, 72, and 96 h, and the effects of the two crude extracts of F. suspensa leaves tea, phillyrin and Forsythia glycoside A on the migration of hepatocellular carcinoma LM3 were detected through the cell scratch test. To reveal the molecular mechanism of crude extract of F. suspensa leaves tea and its main components phillyrin and Forsythia glycoside A affect the proliferation and migration of hepatocellular carcinoma. The expressions of proliferation and migration-related gene Ki-67, Erk, PI3K and mTOR were detected using RT-PCR technology.【Result】Both the green tea and black tea extracts of F. suspensa leaves tea, as well as phillyrin and Forsythia glycoside A, significantly inhibited the proliferation and migration of hepatocellular carcinoma LM3, while Forsythia glycoside A significantly inhibited the migration of hepatocellular carcinoma line LM3. Among them, the crude extract of F. suspensa green tea significantly inhibited the proliferation and migration of HCC at low, medium, and high concentrations when 24, 48, and 72 h. The inhibitory effect of the crude extract of F. suspensa black tea on the proliferation and migration of LM3 cells continued to increase with increasing concentration, and the best inhibitory effect was achieved at high concentration for 72 h. The analysis of RT-PCR results suggests that its main mechanism of action may be achieved by reducing the expression levels of proliferation and migration related genes such as Ki-67.【Conclusion】In summary, the crude extracts of F. suspensa green tea and F. suspensa black tea, as well as phillyrin and Forsythia glycoside A, can inhibit the proliferation or migration of HCC LM3 by reducing the expression of Ki-67.

Key words: Forsythia suspensa leaves tea, phillyrin, Forsythia glycoside A, hepatocellular carcinoma, CCK-8, scratch experiment, Ki-67

滕文龙, 吴永娜, 王德富, 牛颜冰. 连翘叶茶对肝癌细胞增殖和迁移功能的影响及其作用机制[J]. 生物技术通报, 2024, 40(4): 287-296.

TENG Wen-long, WU Yong-na, WANG De-fu, NIU Yan-bing. Effect of Forsythia suspensa Leaves Tea on HCC Proliferation and Migration Function and Its Mechanism of Action[J]. Biotechnology Bulletin, 2024, 40(4): 287-296.

图/表 8

表1 RT-PCR引物序列

Table 1 Primer sequences for RT-PCR

引物名称 Primer name	序列 Sequence（5'-3'）
GAPDH-F	ATGTTGCAACCGGGAAGGAA
GAPDH-R	AGGAAAAGCATCACCCGGAG
Ki-67-F	CTGACCCTGATGAGAGTGAGGGA
Ki-67-R	GGACAGGTGGAGTGTGCATTA
Erk-F	TCCTTTGAGCCGTTTGGAGG
Erk-R	AGTACATACTGCCGCAGGTC
PI3K-F	GGACCCGATGCGGTTAGAG
PI3K-R	ATCAAGTGGATGCCCCACAG
mTOR-F	GCCGCGCGAATATTAAAGGA
mTOR-R	CTGGTTTCCTCATTCCGGCT

图1 连翘绿茶和连翘红茶中连翘苷含量对比 A：连翘苷标品；B：连翘绿茶样品；C：连翘红茶样品

Fig. 1 Comparison of phillyrin content between F. suspen-sa green tea and F. suspensa black tea A: Phillyrin standard; B: Forsythia suspensa green tea sample; C: F. suspensa black tea sample

图2 连翘绿茶和连翘红茶中连翘酯苷A含量对比 A：连翘酯苷A标品；B：连翘绿茶样品；C：连翘红茶样品

Fig. 2 Comparison of F. glycoside A content between F. suspensa green tea and F. suspensa black tea A: Forsythia glycoside A standard; B: F. suspensa green tea sample; C: F. suspensa black tea sample

图3 连翘叶茶粗提物对肝癌细胞LM3的影响 A：连翘绿茶对肝癌细胞LM3增殖作用的影响；B：连翘红茶对肝癌细胞LM3增殖作用的影响。C：对照组；LQLCT-L：连翘绿茶粗提物低浓度；LQLCT-M：连翘绿茶粗提物中浓度；LQLCT-H：连翘绿茶粗提物高浓度；LQHCT-L：连翘红茶粗提物低浓度；LQHCT-M：连翘红茶粗提物中浓度；LQHCT-H：连翘红茶粗提物高浓度；与对照组比较，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1，下同

Fig. 3 Effect of crude extract of F. suspensa leaves tea on hepatocellular carcinoma line LM3 A: Effect of F. suspensa green tea on the proliferation of HCC LM3. B: Effect of F. suspensa black tea on the proliferation of HCC LM3. C: Control group. LQLCT-L: Low concentration of crude extract from F. suspensa green tea. LQLCT-M: Medium concentration in the crude extract of F. suspensa green tea. LQLCT-H: High concentration of crude extract from F. suspensa green tea. LQHCT-L: Low concentration of crude extract from F. suspensa black tea. LQHCT-M: Medium concentration in the crude extract of F. suspensa black tea. LQHCT-H: High concentration of crude extract from F. suspensa black tea; Comparison with control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. The same below

图4 连翘苷和连翘酯苷A对肝癌细胞LM3的影响 A：连翘苷对肝癌细胞LM3增殖的影响；B：连翘酯苷A对肝癌细胞LM3增殖作用的影响。C：对照组；LQG-L：连翘苷低浓度组；LQG-M：连翘苷中浓度组；LQG-H：连翘苷高浓度组；LQZGA-L：连翘酯苷A低浓度组；LQZGA-M：连翘酯苷A中浓度组；LQZGA-H：连翘酯苷A高浓度组

Fig. 4 Effects of phillyrin and F. glycoside A on hepatocellular carcinoma line LM3 A: Effect of Phillyrin on the proliferation of HCC LM3. B: Effect of Forsythia glycoside A on the proliferation of HCC LM3. C: Control group. LQG-L: Low concentration group of Phillyrin. LQG-M: Medium concentration group of Phillyrin. LQG-H: High concentration group of Phillyrin. LQZGA-L: Low concentration group of Forsythia glycoside A. LQZGA-M: Medium concentration group of Forsythia glycoside A. LQZGA-H: High concentration group of Forsythia glycoside A

图5 连翘绿茶粗提物（A）、连翘红茶粗提物（B）、连翘苷（C）和连翘酯苷A（D）对LM3 细胞迁移能力的影响

Fig. 5 Effect of crude extract of F. suspensa green tea (A) and black tea (B), phillyrin (C), and Forsythia glycoside A (D) on the migration ability of LM3 cells

图6 连翘叶茶粗提物对肝癌细胞LM3增殖和迁移的影响 A：连翘绿茶和连翘红茶对肝癌细胞LM3 Ki-67表达的影响；B：连翘绿茶和连翘红茶对肝癌细胞LM3 Erk表达的影响；C：连翘绿茶和连翘红茶对肝癌细胞LM3 PI3K表达的影响；D：连翘绿茶和连翘红茶对肝癌细胞LM3 mTOR表达的影响。CK：对照组；LQLCT：连翘绿茶粗提物组；LQHCT：连翘红茶粗提物组

Fig. 6 Effects of crude extract of F. suspensa leaves tea on the proliferation and migration of HCC LM3 A: Effects of F. suspensa green tea and F. suspensa black tea on the expression of Ki-67 in HCC LM3. B: Effects of F. suspensa green tea and F. suspensa black tea on the expression of Erk in HCC LM3. C: Effects of F. suspensa green tea and F. suspensa black tea on the expression of PI3K in HCC LM3. D: Effects of F. suspensa green tea and F. suspensa black tea on the expression of mTOR in HCC LM3. CK: Control group. LQLCT: F. suspensa green tea crude extract group; LQHCT: F. suspensa black tea crude extract group

图7 连翘苷和连翘酯苷A对肝癌细胞LM3增殖和迁移的影响 A：连翘苷、连翘酯苷A对肝癌细胞LM3 Ki-67表达的影响；B：连翘苷、连翘酯苷A对肝癌细胞LM3 Erk表达的影响；C：连翘苷、连翘酯苷A对肝癌细胞LM3 PI3K表达的影响；D：连翘苷、连翘酯苷A对肝癌细胞LM3 mTOR表达的影响。CK：对照组；LQG：连翘苷组；LQZGA：连翘酯苷A组

Fig. 7 Effect of phillyrin and Forsythia glycoside A on the proliferation and migration of HCC LM3 A: Effects of phillyrin and Forsythia glycoside A on the expression of Ki-67 in HCC LM3. B: Effects of phillyrin and Forsythia glycoside A on the expression of Erk in HCC LM3. C: Effects of phillyrin and Forsythia glycoside A on the expression of PI3K in HCC LM3. D: Effects of phillyrin and Forsythia glycoside A on the expression of mTOR in HCC LM3. CK: Control group. LQG: Phillyrin group; LQZGA: Forsythia glycoside A group

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