Effect of Forsythia suspensa Leaves Tea on HCC Proliferation and Migration Function and Its Mechanism of Action

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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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