连翘脂素调节cAMP/EPAC1/RAP1信号通路对肺癌细胞恶性进展的影响

doi:10.11958/20250063

摘要/Abstract

摘要：

目的探究连翘脂素调节环磷酸腺苷/环磷腺苷激活的交换蛋白1/Ras相关蛋白1（cAMP/EPAC1/RAP1）信号通路对肺癌细胞恶性进展的影响。方法体外培养肺癌细胞A549，并分为对照组，连翘脂素低、中、高剂量组（25、50、100 mg/L），连翘脂素高剂量+百日咳毒素（PTX）组（100 mg/L连翘脂素+5 μmol/L PTX），连翘脂素高剂量+EPAC1拮抗剂（ESI-09）组（100 mg/L连翘脂素+1.5 μmol/L ESI-09）。CCK-8实验检测细胞增殖；划痕实验检测细胞迁移；Transwell检测细胞侵袭；流式细胞术检测细胞凋亡；酶联免疫吸附试验检测细胞上清液cAMP水平；Western blot分析cAMP/EPAC1/RAP1信号通路及凋亡蛋白B淋巴细胞瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）表达。结果相较于对照组，连翘脂素低、中、高剂量组OD450值、划痕愈合率、细胞侵袭数目、cAMP水平、EPAC1、RAP1和Bcl-2表达下降，细胞凋亡率和Bax表达升高，且呈剂量依赖性（P<0.05）；与连翘脂素高剂量组比较，连翘脂素高剂量+PTX组A549细胞中光密度（OD）450值、划痕愈合率、侵袭细胞数目、cAMP水平、EPAC1、RAP1和Bcl-2蛋白表达增加，细胞凋亡率和Bax表达下降（P<0.05）；连翘脂素高剂量+ESI-09组各指标水平均与之相反。结论连翘脂素通过下调cAMP/EPAC1/RAP1信号通路阻碍A549细胞增殖、迁移和侵袭，促进细胞凋亡。

关键词: 肺肿瘤, 连翘脂素, 细胞增殖, 细胞凋亡, cAMP/EPAC1/RAP1信号通路

Abstract:

Objective To investigate the effect of forsythigenin on the malignant progression of lung cancer cells by regulating the cyclic adenosine monophosphate/exchange protein directly activated by cAMP1/Ras-associated protein 1 (cAMP/EPAC1/RAP1) signaling pathway. Methods Lung cancer cell line A549 was cultured in vitro and grouped into the control group, the low dose forsythigenin group (25 mg/L), the medium dose forsythigenin group (50 mg/L), the high dose forsythigenin group (100 mg/L), the high dose forsythigenin+specific increase in intracellular cAMP content (pertussis toxin PTX) group (100 mg/L forsythigenin+5 μmol/L PTX) and high dose forsythigenin+EPAC1 antagonist (ESI-09) group (100 mg/L forsythigenin+1.5 μmol/L ESI-09). CCK-8 experiment was applied to detect cell proliferation. Scratch test was applied to detect cell migration. Flow cytometry was applied to detect cell apoptosis. Transwell was applied to detect cell invasion. ELISA method was applied to detect cAMP level in cell supernatant. Western blot assay was applied to detect expression levels of cAMP/EPAC1/RAP1 signaling pathway proteins and apoptotic proteins [B lymphoblastoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax)]. Results Compared with the control group, the OD450 value of A549 cells, number of cell invasions, scratch healing rate, level of cAMP, expression levels of Bcl-2, EPAC1 and RAP1 proteins were significantly reduced in the low dose, medium dose and high dose forsythigenin groups, and the expression of Bax protein and the rate of cell apoptosis were significantly increased in a dose-dependent manner (P<0.05). Compared with the high-dose forsythigenin group, the OD450 value of A549 cells, scratch healing rate, number of cell invasions, level of cAMP, expression levels of Bcl-2, EPAC1 and RAP1 proteins were obviously increased in the high-dose forsythigenin+PTX group, the expression of Bax protein and the apoptosis rate were obviously reduced (P<0.05). Levels of all indexes in the high dose forsythigenin+ESI-09 group were opposite. Conclusion Forsythigenin inhibits proliferation, migration, and invasion of A549 cells and promotes apoptosis by down-regulating the cAMP/EPAC1/RAP1 signaling pathway.

Key words: lung neoplasms, forsythiaside, cell proliferation, apoptosis, cAMP/EPAC1/RAP1 signaling pathway

中图分类号:

R734.2

图/表 6

图1 各组细胞不同时间点的OD450值比较 a与对照组比较，b与连翘脂素低剂量组比较，c与连翘脂素中剂量组比较，d与连翘脂素高剂量组比较，P<0.05。

Fig.1 Comparison of OD450 values in different time points of cells between different groups

图2 各组A549细胞迁移情况 A：对照组；B：连翘脂素低剂量组；C：连翘脂素中剂量组；D：连翘脂素高剂量组；E：连翘脂素高剂量+PTX组；F：连翘脂素高剂量+ESI-09组；图3-5同。

Fig.2 A549 cell migration in each group

图3 各组A549细胞侵袭情况（结晶紫染色，×200）

Fig.3 A549 cell invasion in each group (Crystal violet staining, ×200)

图4 各组A549细胞凋亡情况

Fig.4 Apoptosis of A549 cells in each group

图5 各组A549细胞cAMP/EPAC1/RAP1通路及凋亡蛋白表达情况

Fig.5 The expression of cAMP/EPAC1/RAP1 pathway and apoptotic protein in A549 cells in each group

表1 Comparison of cAMP/EPAC1/RAP1 pathway and apoptotic protein expression between different groups of A549 cells

Tab.1 各组A549细胞cAMP/EPAC1/RAP1通路及凋亡蛋白表达比较（n=5，$\bar{x} \pm s$）

组别	EPAC1	RAP1	Bax	Bcl-2
对照组	1.13±0.09	1.15±0.10	0.74±0.08	1.11±0.11
连翘脂素低剂量组	0.95±0.09^a	0.93±0.08^a	0.99±0.09^a	0.92±0.08^a
连翘脂素中剂量组	0.72±0.08^ab	0.69±0.07^ab	1.26±0.10^ab	0.78±0.07^ab
连翘脂素高剂量组	0.42±0.05^abc	0.35±0.04^abc	1.57±0.18^abc	0.37±0.04^abc
连翘脂素高剂量+ PTX组	0.69±0.07^d	0.72±0.06^d	1.08±0.12^d	0.66±0.03^d
连翘脂素高剂量+ ESI-09组	0.28±0.03^d	0.21±0.02^d	1.98±0.09^d	0.23±0.05^d
F	96.544^＊＊	137.119^＊＊	74.781^＊＊	116.511^＊＊

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