钩吻总碱对肺腺癌细胞增殖和凋亡作用的研究

Effects of total alkaloids of Gelsemium elegans Benth. on proliferation and apoptosis of lung adenocarcinoma cells

摘要:

目的 以人肺腺癌细胞（A549、SPCA1）为研究对象，通过在培养液中加入不同浓度的钩吻总碱（TAG）研究其抗肿瘤作用。方法 采用加热回流、萃取等方法提取TAG，利用薄层色谱法鉴定TAG的提取，通过Incucyte S3活细胞动态分析系统拟合不同浓度的TAG作用A549、SPCA1细胞的细胞融合度并观察A549、SPCA1细胞的形态，采用CCK-8法、集落形成实验检测细胞增殖，Hoechst33258染色、Rhodamine123染色检测细胞凋亡，碘化丙啶单染法检测细胞周期，Western blotting检测凋亡指标蛋白Bax、Bcl-2、Caspase-3蛋白表达。结果 经薄层色谱法鉴定成功提取TAG。Incucyte S3活细胞动态分析系统拟合出TAG作用A549、SPCA1细胞的EC50值分别是99.2、82.0 μg/mL，由此确定TAG作用A549、SPCA1细胞的低、中、高浓度依次为50、100、150 μg/mL，且细胞拍照观察到随着药物浓度增加细胞融合度下降、细胞数目减少。CCK-8法和集落形成实验结果表明TAG抑制A549、SPCA1细胞增殖（P <0.05）。Hoechst 33258细胞核染色实验发现给药组细胞出现核碎裂；流式细胞术检测Rhodamine123探针发现给药组出现荧光信号强度增加，检测细胞周期发现TAG使A549、SPCA1细胞周期阻滞在G₂/M期；Western blot结果显示TAG诱导Bcl-2蛋白表达下调，Bax表达上调、Caspase-3蛋白切割活化增加，通过细胞染色实验、流式细胞术及Western blotting表明TAG促进A549、SPCA1细胞凋亡（P <0.05）。结论 TAG抑制肺腺癌细胞增殖，诱导细胞凋亡。

Abstract:

Objective To explore the antitumor effects of total alkaloids from Gelsemium elegans Benth. (TAG) on human lung adenocarcinoma cells (A549, SPCA1) by incorporating various concentrations of TAG into the culture medium.Methods TAG was extracted using heating reflux and extraction methods, and identified through thin-layer chromatography. The Incucyte S3 Live Cell Analysis System was used to fit the concentration-response of TAG on A549 and SPCA1 cells and to observe changes in cell morphology. Cell proliferation was measured using the CCK-8 assay and colony formation tests. Cell apoptosis was assessed using Hoechst 33258 staining and Rhodamine 123 staining, while the cell cycle was examined using propidium iodide staining. Western blotting was employed to detect the expression of apoptosis-related proteins Bax, Bcl-2, and cleaved Caspase-3.Results Successful extraction of TAG was confirmed via thin-layer chromatography. The EC50 values of TAG for A549 and SPCA1 cells were 99.2 and 82.0 μg/mL, respectively, determining the low, medium, and high concentrations of TAG as 50, 100, and 150 μg/mL. Observations showed a decrease in cell confluence and a reduction in cell numbers with increasing concentrations of TAG. The CCK-8 and colony formation assays indicated that TAG inhibited the proliferation of A549 and SPCA1 cells (P <0.05). Hoechst 33258 staining revealed nuclear fragmentation in treated cells, and flow cytometry using Rhodamine123 probe showed increased fluorescence intensity, indicating apoptosis. Cell cycle analysis revealed that TAG induced cell cycle arrest at the G₂/M phase in A549 and SPCA1 cells. Western blot results showed that TAG induced a decrease in Bcl-2 expression, an increase in Bax expression, and activation of Caspase-3 cleavage, suggesting that TAG promotes apoptosis in A549 and SPCA1 cells (P < 0.05).Conclusion TAG inhibits the proliferation of lung adenocarcinoma cells and induces apoptosis.