目的 研究小檗胺体外对人肺腺癌A549细胞凋亡及TNF-α-JNK信号通路的影响。方法 MTT比色法检测小檗胺对A549细胞增殖的影响；显微镜观察A549细胞形态变化；Annexin V/PI双标法检测细胞的凋亡率；Western blotting法检测细胞凋亡标记蛋白Bcl-x、Caspase-3和PARP表达的变化，c-jun氨基末端激酶（JNK）的蛋白表达及其磷酸化表达水平；荧光定量PCR和ELISA检测肿瘤坏死因子-α（TNF-α）表达的变化；通过JNK通路抑制剂进一步验证TNF-α-JNK信号通路在小檗胺药效中的作用。结果 小檗胺能够显著抑制A549细胞增殖，并呈浓度相关性，小檗胺给药后24 h的IC₅₀值为9.01 μmol/L；经小檗胺处理后，A549细胞可见典型的凋亡形态学改变，Annexin V/PI双标法检测结果也显示小檗胺可诱导A549细胞凋亡，小檗胺10 μmol/L组细胞早期凋亡率为13.8%，为对照组的6.6倍；小檗胺可显著降低抗凋亡蛋白Bcl-x的表达，明显增强促凋亡蛋白Caspase-3和PARP的活性，显著提高TNF-α基因和蛋白表达及JNK蛋白的磷酸化表达水平，激活TNF-α-JNK通路。结论 小檗胺能抑制A549细胞的增殖，诱导其凋亡，其作用机制可能与TNF-α-JNK信号通路的激活有关。

Objective To investigate the effect of berbamine (BBM) on the induction of apoptosis in human lung cancer cell A549 cells and the activity of TNF-α-JNK signaling pathway. Methods After the A549 cells being treated with BBM at different concentration, the inhibitory effect of BBM on proliferation was detected by MTT assay. Cell morphological changes were detected by light microscope. Alteration of apoptosis rate of A549 cells was determined by Annexin V/PI double staining. Western blotting was used to detect the activity of apoptosis-related proteins, including Bcl-x, Caspase-3, and PAPR. The expressions of c-jun N-terminal kinase (JNK) and p-JNK were determined by Western blotting. Changes of tumor necrosis factor-alpha (TNF-α) were detected by fluorescent quantitative PCR and ELISA, respectively. Finally, the impacts of BBM on the proliferation and apoptosis of A549 cells were detected in the absence or presence of a JNK inhibitor. Results BBM significantly inhibited the growth of A549 cells in a dose-dependent manner. The IC₅₀ of 24 h was 9.01 μmol/L. Cells treated with BBM showed the typically morphological characteristics of apoptotic cells. Annexin V/PI double staining test indicated that BBM could induce the apoptosis of A549 cells in a dose-dependent manner. The early apoptotic population of cells treated with 10 μmol/L BBM was 13.8%, which was 5.6 times higher than that of the control. BBM decreased the expression of anti-apoptotic protein Bcl-x and increased the activity of proapoptotic proteins of caspase-3 and PARP. The mRNA and the protein expression levels of TNF-α were significantly increased by BBM treatment. The p-JNK expression was also dramatically up-regulated after BBM treatment. The effects of BBM on the proliferation and apoptosis in A549 cells were significantly reduced when JNK pathway was blocked. Conclusion BBM could inhibit the growth and induce the apoptosis of A549 cells, and its mechanism may be related to the activated TNF-α-JNK signaling pathway.

国家自然科学基金资助项目（81202820）；浙江省自然科学基金资助项目（LQ12H28005）；浙江省中医药管理局项目（2012ZQ006）；浙江省教育厅科研项目（Y201223804）；浙江省2012年第一批省级重点科技创新团队项目（2010R50044-12）；浙江中医药大学人才专项项目（2012ZR05）