氧化苦参碱对人非小细胞肺癌A549细胞凋亡的影响及其机制

Effect of oxymatrine on apoptosis and regulatory mechanism in human non-small cell lung carcino- ma A549 cells

目的观察氧化苦参碱（OXY）对人非小细胞肺癌A549细胞凋亡的影响并探讨其机制。方法噻唑蓝（M1rr）法测定OXY对A549人肺癌细胞活力的作用。流式细胞术测定A549细胞凋亡率。荧光定量聚合酶链反应（FQ—PCR）和Westernblot方法测定OXY对A549细胞B淋巴细胞／白血病-2（bcl-2）、bcl-2相关X蛋白（bax）mRNA和蛋白表达的影响。Westernblot方法测定OXY对A549细胞的细胞外信号调节蛋白激酶（ERK）、c—Jun氨基端激酶（JNK）、p38丝裂原活化激酶（p38MAPK）、磷酸化ERK（p-ERK）、p-JNK、p-p38MAPK蛋白表达的影响。结果OXY可抑制A549细胞增殖。与对照组[（5．63±0．76）％]比较，OXY组细胞凋亡率[（13．26±2．15）％]上升（P〈0．01）；与对照组（1．02±0．27、0．23±0．04）比较，OXY组baxmRNA（1．61±0．19）和蛋白（0．71±0．10）表达上升（P〈0．05，P〈0．01）；与对照组比较（0．94±0．14、0．64±0．09），OXY组bcl-2mRNA（0．42±0．05）和蛋白（0．21±0．04）表达下降（P〈0．01）。与对照组（0．29±0．03）比较，OXY组p-JNK表达水平（0．66±0．06）明显升高（P〈0．01），其他蛋白表达均无明显变化。与OXY组（0．66±0．07、0．28±0．03、0．65±0．08）比较，同时给予OXY和p-JNK抑制剂SP600125组bax蛋白表达（0．39±0．05）下降，bcl-2表达（0．50±0．08）上升，p-JNK蛋白（0．26±0．04）表达下降（P〈0．01）。结论OXY碱可诱导A549细胞凋亡，可能与升高p-JNK水平有关。

Objective To investigate the effects of oxymatrine （OXY） on apoptosis and the regu- latory mechanism in the human non-small cell lung carcinoma A549 cells. Methods A549 cells were cul- tured. Methyl thiazol tetrazolium （MTF） assay was used to determine cell proliferation. The apoptosis rate was detected by using flow cytometry. Real-time fluorescence quantitative polymerase chain reaction （FQ-PCR） and Western blotting were used to examine OXY mRNA and protein expression. Extracellular signal-regulated kinase （ERK）, C-Jun N-terminal kinase （JNK）, p38 mitogen-activated protein kinase （p38MAPK）, p-ERK, p-JNK and p-p38MAPK protein expression levels were tested by using Western blotting. Results OXY could inhibit the proliferation of A549 cells. As compared with control group [ （5.63 ± 0. 76） % ], the apoptosis rate was increased [ （ 13.26 ± 2. 15） % ] in OXY group （P 〈 0. 01 ）. As compared with control group （ 1.02 ± 0. 27, 0. 23 ± 0. 04 ）, B lymphocytes/leukemia-2 （bcl-2） associ- ated X protein （bax） mRNA （1.61 ± 0. 19） and protein （0. 71 ± 0. 10） expression levels were increased in OXY group （ P 〈 0. 05, P 〈 0. 01 ）. As compared with control group （ 0. 94 ± 0. 14, 0. 64 ± 0. 09 ） , bcl-2 mRNA （0. 42 ± 0. 05 ） and protein （0. 21 ± 0. 04） expression levels were decreased in OXY group （P 〈0. 01 ）. As compared with control group （0. 29 ± 0. 03）, the expression of p-JNK was significantly increased in OXY group （0. 66 ±0. 06） （P 〈0. 01）. bax expression （0. 39 ±0.05） and p-JNK （0. 26 ± 0. 04） were reduced, and bcl-2 expression （0. 50 ± 0. 08 ） was increased in OXY ± SP600125 group as compared with those in OXY group （0. 66 ± 0. 07, 0. 28 ± 0.03 and 0. 65 ± 0. 08 respectively） （ P 〈 0. 01 ）. Conclusion OXY could induce apoptosis of A549 cells by up-regulating p-JNK pathway.