铁皮石斛共性黄酮类成分新西兰牡荆苷Ⅱ的体外抗氧化与诱导HepG2细胞凋亡的作用
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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.
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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[15] Nagaprashantha L D,Vatsyayan R,Singhal J,et al.Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer[J]. Biochem Pharmacol,2011,82(9):1100-1109.
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[20]李娜,高俊岩,刘敏.细胞凋亡和肿瘤的关系研究进展[J].当代医学,2009,15(16):13-14.
[21] Platanias L C. Map kinase signaling pathways and hematologic malignancies[J]. Blood,2003,101(12):4667-4679.
[22]陈建勇,王聪,王娟,等. MAPK信号通路研究进展[J].中国医药科学,2011,1(8):32-34.
[23] Bachegowda L,Gligich O,Mantzaris I,et al. Signal transduction inhibitors in treatment of myelodysplastic syndromes[J]. J Hematol Oncol,2013,6:50.
[24] Eckhart L,Ballaun C,Hermann M,et al. Identification of novel mammalian Caspases reveals an important role of gene loss in shaping the human Caspase repertoire[J].Mol Biol Evol,2008,25(5):831-841.
[2]包雪声,顺庆生,陈立钻,等.中国药用石斛[M].上海:上海医科大学出版社,2001:56.
[3]周桂芬,吕圭源.铁皮石斛不同部位黄酮碳苷类成分及清除DPPH自由基能力比较研究[J].中国中药杂志,2012,37(11):1536-1540.
[4]黄月纯,谢镇山,任晋,等. 3种种源铁皮石斛叶黄酮类成分HPLC特征图谱比较[J].中国实验方剂学杂志,2015,21(24):37-40.
[5]魏述永.葛根素心血管保护作用及其机制研究进展[J].中国中药杂志,2015,40(12):2278-2284.
[6]朱开梅,陈丹,李美波,等.构树叶总黄酮调控Bcl-2与Bax蛋白表达及Caspase-3活性诱导Hep G-2细胞凋亡的研究[J].中国实验方剂学杂志,2014,20(19):128-133.
[7]张骏艳,张磊,李俊,等.野菊花总黄酮对佐剂性关节炎大鼠氧自由基代谢的影响[J].中国中药杂志,2010,35(3):344-347.
[8] KU S K. Vicenin-2 and scolymoside inhibit high-glucoseinduced vascular inflammation in vitro and in vivo[J].Can J Physiol Pharm,2016,94(3):287-295.
[9] Hassan N,Ali A,Withycombe C,et al. TET-2 upregulation is associated with the anti-inflammatory action of vicenin-2[J]. Cytokine,2018,108:37-42.
[10] Lee I C. Anti-inflammatory effects of vicenin-2 and scolymoside on polyphosphate-mediated vascular inflammatory responses[J]. Inflamm Res,2016,65(3):203-212.
[11] Lee W,Ku S K,Bae J S. Ameliorative effect of vicenin-2 and scolymoside on TGFBI p-induced septic responses[J]. Inflammation,2015,38(6):2166-2177.
[12] Lee W. Antithrombotic and antiplatelet activities of vicenin-2[J]. Blood Coagul Fibrin,2015,26(6):628-634.
[13] Islam M N,Ishita I J,Jung H A,et al. Vicenin 2 isolated from Artemisia capillaris exhibited potent anti-glycation properties[J]. Food Chem Toxicol,2014,69:55-62.
[14] Verspohl E J,Fujii H,Homma K,et al. Testing of Perilla frutescens extract and Vicenin 2 for their antispasmodic effect[J]. Phytomedicine,2013,20(5):427-431.
[15] Nagaprashantha L D,Vatsyayan R,Singhal J,et al.Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer[J]. Biochem Pharmacol,2011,82(9):1100-1109.
[16]刘延祥,张学武.中药诱导肿瘤细胞凋亡的研究进展[J].时珍国医国药,2007,18(7):1563-1565.
[17]王华清.茶叶籽中黄酮类化合物的分离鉴定及其抗氧化活性研究[D].南京:南京师范大学,2012.
[18] LIU Y,CHANG C C,Marsh G M,et al. Population attributable risk of aflatoxin-related liver cancer:systematic review and Meta-analysis[J]. Eur J Cancer,2012,48(14):2125-2136.
[19]邓鹏裔.芝麻素对人肝癌细胞系Hep G2的抑制作用及机理研究[D].武汉:华中科技大学,2013.
[20]李娜,高俊岩,刘敏.细胞凋亡和肿瘤的关系研究进展[J].当代医学,2009,15(16):13-14.
[21] Platanias L C. Map kinase signaling pathways and hematologic malignancies[J]. Blood,2003,101(12):4667-4679.
[22]陈建勇,王聪,王娟,等. MAPK信号通路研究进展[J].中国医药科学,2011,1(8):32-34.
[23] Bachegowda L,Gligich O,Mantzaris I,et al. Signal transduction inhibitors in treatment of myelodysplastic syndromes[J]. J Hematol Oncol,2013,6:50.
[24] Eckhart L,Ballaun C,Hermann M,et al. Identification of novel mammalian Caspases reveals an important role of gene loss in shaping the human Caspase repertoire[J].Mol Biol Evol,2008,25(5):831-841.