合江佛手挥发油调控Bcl-2/Bax蛋白表达诱导人恶性胶质瘤U87细胞凋亡

目的：研究合江佛手挥发油诱导人恶性胶质瘤U87细胞凋亡及对凋亡相关因子Bcl-2和Bax蛋白表达的影响。方法：采用不同浓度的合江佛手挥发油作用于人乳腺癌细胞MCF-7、人恶性胶质瘤细胞U87、人肝癌细胞HepG2、人肺癌细胞A549，以CCK-8法（Cell Counting Kit-8 assay）筛选体外抗肿瘤活性最强的细胞；Hoechst33342/PI、透射电子显微镜观察及Annexin V-FITC/PI双染法相结合检测U87细胞的凋亡；Western Blot检测U87细胞凋亡相关因子Bcl-2、Bax蛋白表达的影响。结果：合江佛手挥发油对MCF-7、U87、HepG2、A549细胞的IC50分别为（173.99±18.68）、（82.19±13.07）、（192.87±19.66）和（215.83±23.27）μg·mL^-1，其中对U87细胞抑制活性最强。合江佛手挥发油作用于U87细胞48 h后，Hoechst33342/PI、透射电子显微镜观察均可见核固缩、碎裂和凋亡小体等凋亡形态学改变。Annexin V-FITC/PI双染法结果显示，合江佛手挥发油和阳性对照维甲酸组U87细胞凋亡率明显增高，与阴性对照比较，P<0.05或P<0.01，药物作用呈现出较好的剂量依赖关系。Western Blot法结果表明，Bax蛋白表达增多，Bcl-2蛋白表达减少，Bcl-2/Bax比值降低。结论：合江佛手挥发油通过下调Bcl-2/Bax蛋白表达的比值，明显诱导人恶性胶质瘤细胞U87细胞凋亡。

Volatile oil of Hejiang Bergamot induces apoptosis through regulating protein expression of Bcl-2/Bax in human malignant glioma cell line U87 cells

OBJECTIVE To explore the effect of volatile oil from Hejiang Bergamot (VO-HB) on apoptosis of human glioma cell line U87 and the expression of apoptosis related factors Bcl-2 and Bax.METHODS Different concentrations of VO-HB were utilized for MCF-7, U87, HepG2 and A549 cells. And the cells with the strongest anti-tumor activity were screened by CCK-8 assay. Hoechst33342/PI, transmission electron microscopy (TEM) and annexin V-FITC/PI double staining were employed for detecting apoptosis in U87 cells; Western Blot was used for detecting the expression of related factors Bcl-2 and Bax.RESULTS The IC₅₀ values of VO-HB for MCF-7, U87, HepG2, A549 were(173.99±18.68), (82.19±13.07), (192.87±19.66) and (215.83±23.27) μg·mL^-1 respectively. Thus the activity to U87 cells was the strongest in vitro. After 48-hour treatment of VO-HB, apoptotic morphological changes such as nuclear pyknosis, fragmentation and apoptotic bodies were observed by hoechst33342/PI and TEM. The results of annexin V-FITC/PI double staining showed that the apoptotic rate of U87 cells in VO-HB and positive control retinoic acid group significantly increased as compared with control (P<0.05 or P<0.01), showing an excellent dose-dependent relationship. Western blot showed that Bax protein expression increased, bcl-2 protein expression decreased and Bcl-2/Bax ratio declined.CONCLUSION VO-HB may significantly induce the apoptosis of human malignant glioma cell line U87 by down-regulating the protein expression ratio of Bcl-2/Bax.