摘要
目的:对不同道地种源铁皮石斛的共性黄酮类成分新西兰牡荆苷Ⅱ(芹菜素-6,8-二-C-葡萄糖苷,viceninⅡ)进行体外抗氧化及诱导肝癌Hep G2细胞凋亡的活性研究。方法:采用2,2-联苯基~(-1)-苦基肼法(DPPH),2-联氨-双-3-乙基苯并噻唑啉-6-磺酸法(ABTS)和铜离子还原法对viceninⅡ(0. 005~1 g·L~(-1))进行体外的抗氧化活性研究;采用噻唑蓝(MTT)比色法检测viceninⅡ(12. 5~100μmol·L~(-1))对人6种不同的肿瘤细胞体外的增殖抑制作用;后续凋亡实验viceninⅡ浓度为75μmol·L~(-1),采用Hoechst33258染色,荧光显微镜进行Hep G2细胞形态学观察;应用流式细胞术,磷脂结合蛋白V/碘化丙啶(Annexin V-FITC/PI)检测Hep G2细胞凋亡率;采用实时荧光定量逆转录聚合酶链反应(Real-time PCR)检测丝裂原活化蛋白激酶(MAPK)通路及相关凋亡基因的mRNA表达。结果:ViceninⅡ质量浓度为1 g·L~(-1)时,对DPPH的清除率和Cu2+还原率为48. 82%和22. 01%(P <0. 01);当viceninⅡ质量浓度为0. 5 g·L~(-1)时,对ABTS清除率为86. 88%(P <0. 01); viceninⅡ显著抑制Hep G2细胞增殖并诱导凋亡,75μmol·L~(-1)的viceninⅡ作用48 h后,细胞存活率为45. 69%(P <0. 01),细胞凋亡率为14. 57%(P <0. 01),升高B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax)/Bcl-2,半胱氨酸蛋白酶(Caspase)-8,p38,细胞外信号调节激(ERK),c-Jun氨基末端酶(JNK),核转录因子-κB(NF-κB) mRNA表达(P <0. 01)。结论:铁皮石斛不同道地种源的共性黄酮类成分新西兰牡荆苷Ⅱ在体外具有一定的抗氧化作用,能有效抑制Hep G2细胞增殖,并可能通过调控MAPK通路及Bax/Bcl-2途径诱导Hep G2细胞凋亡。
Objective: To study the antioxidation activities in vitro of a comment flavonoid component named vicenin Ⅱ(Apigenin 6,8-di-C-glucoside) in Dendrobii Officinalis Caulis from different origin places and investigate its effects on apoptosis of HepG2 cells. Method: The antioxidation activities in vitro of vicenin Ⅱ(0. 005~(-1) g·L~(-1)) were evaluated by 2,2-diphenyl~(-1)-picrylhydrazyl(DPPH),salicylic acid and 2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) and copper ion reduction assays. Methye thiazolye telrazlium(MTT) assay was used to test the inhibitory effect of vicenin Ⅱ(12. 5 ~ 100 μmol·L~(-1)) on proliferation of 6 tumour cells in vitro. In subsequent apoptosis experiment,the concentration of vicenin Ⅱ was 75 μmol·L~(-1). The morphological changes of HepG2 cells were evaluated by Hoechst 33258 under fluorescence microscope; and the cell apoptosis rate was detected by flow cytometry with AnnexinV/PI apoptosis assay kit. The mRNA expressions of mitogen activated protein kinase(MAPK) pathway related apoptotic genes were detected by Real-time PCR assay.Result: The 1 g·L~(-1) vicenin Ⅱ showed 48. 82% and 22. 01% for DPPH scavenging rate and Cu2 +reduction rate respectively(P < 0. 01). 0. 5 g·L~(-1) vicenin Ⅱ showed 86. 88% for ABTS scavenging rate(P < 0. 01). viceninⅡ could significantly inhibit the proliferation and increase the apoptosis rate on HepG2 cells; after treatment for48 h with 75 μmol·L~(-1) Vicenin Ⅱ,the cells survival rate was 45. 69%(P < 0. 01) and apoptotic rate was14. 57%(P < 0. 01). Meanwhile,the mRNA expression levels of B-cell lymphoma-2(Bcl-2) related X protein(Bax)/Bcl-2,Caspase-8,p38,extracellular signal-regulated kinases(ERK),c-Jun N-terminal kinase(JNK),and nuclear transcription factor(NF)-κB were increased(P < 0. 01). Conclusion: The general flavone glycosides component vicenin Ⅱ of Dendrobii Officinalis Caulis from different origins has a certain antioxidation effect and significant inhibitory effect on proliferation,and could induce apoptosis on HepG2 cells probably by regulating the expression of related genes in MAPK pathway and Bax/Bcl-2.
引文
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