Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells

Objective:To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer ceils.Methods:Human non-small-cell lung carcinoma cell lines H460,AS49 and H157 were cultured in vitro.The cytotoxic activities were determined using MTT assay.The apoptotic cells death was examined by flow cytometry using PI staining and DMA agarose gel electrophoresis.The activities of caspase-3, -8 and -9 were measured via ELISA.Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential.Bcl-2/Bcl-XL/ XIAP/Bid/ DR5 and DR4 proteins were analyzed using western blot.Results:The concentrations required for a 50%decrease in cell growth(IC50) ranged from 1.8 to 3.2 Jt M.Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway,including the depolarization of the mitochondrial membrane,release of cytochrome c from mitochondria,activation of procaspase-9 and -3,and increase of DNA fragments.Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zOEVD-FMK suppressed casticin-induced apoptosis.In addition,casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage.In H157 cell line,casticin increased expression of DRS at protein levels but not affect the expression of DR4.The prelreatmenl with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells.No correlation was found between cell sensitivity to casticin and that to p53 status,suggesting that casticin induce a p53- independent apoptosis.Conclusions:Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DRS in human lung cancer cells.

Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells

AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.

Proliferation inhibition of human cervical cancer HeLa cells by Casticin in vitro

Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa ceils was evaluated by the MTT assay. The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results： Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group （P 〈 0.05）. The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyctin B1 was depressed by Casticin in a concentration dependent manner. Conclusion： Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.

Casticin对宫颈癌细胞凋亡和线粒体凋亡途径的影响

目的探讨紫花牡荆素(Casticin,CAS)是否通过线粒体凋亡途径诱导人宫颈癌HeLa细胞凋亡。方法体外培养人宫颈癌HeLa细胞。ELISA法检测细胞组蛋白/DNA碎片;碘化丙啶(propidium iodide,PI)染色流式细胞术(flow cytometry,FCM)分析细胞凋亡率;比色法测定细胞半胱天冬酶caspase-9,-3活性;罗丹明123探针FCM检测细胞线粒体跨膜电位。结果 CAS以浓度依赖方式增高HeLa细胞组蛋白/DNA碎片水平和凋亡率(P<0.05)。CAS增强HeLa细胞caspase-9,-3活性(P<0.05)和诱导Hela细胞线粒体膜电位下降(P<0.05),呈浓度依赖性。结论 CAS诱导HeLa细胞凋亡涉及线粒体凋亡途径的激活。

The flavonoid casticin enhances TRAIL-induced apoptosis of colon cancer cells through endoplasmic reticulum stress-mediated up-regulation of DR5

Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.

Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1

Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were used as CCSLCs.DNA methyltransferase 1(DNMT1)activity and m RNA levels,self-renewal capability(Nanog and Sox2),and cancer stem cell markers(CD133 and CD44)were detected by a colorimetric DNMT activity/inhibition assay kit,quantitative real-time reverse transcription-polymerase chain reaction,sphere and colony formation assays,and immunoblot,respectively.Knockdown and overexpression of DNMT1 by transfection with sh RNA and c DNA,respectively,were performed to explore the mechanism for action of CAS(0,10,30,and 100 nmol/L).Results:DNMT1 activity was increased in CCSLCs compared with He La and Ca Ski cells(P<0.05).In addition,He La-derived CCSLCs transfected with DNMT1 sh RNA showed reduced sphere and colony formation abilities,and lower CD133,CD44,Nanog and Sox2 protein expressions(P<0.05).Conversely,overexpression of DNMT1 in He La cells exhibited the oppositive effects.Furthermore,CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in He La-derived CCSLCs(P<0.05).Interestingly,DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness.As expected,DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in He La cells.Conclusion:CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation,suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

紫花牡荆素对脂多糖诱导的BEAS-2B细胞损伤和NF-κB-Keap1-Nrf2/ARE通路的影响

目的:探讨紫花牡荆素(CAS)对脂多糖诱导的人正常肺上皮细胞(BEAS-2B)损伤和NF-κB-Keap1-Nrf2/ARE通路的影响。方法:利用不同浓度的CAS预处理BEAS-2B细胞1 h、2 h、4 h,用1μg/ml脂多糖处理BEAS-2B细胞,构建细胞损伤模型,ELISA检测细胞上清炎症因子含量,筛选CAS最适作用浓度和时间;再将细胞分为正常组、模型组、Casticin组、ML385组、Casticin+ML385组、地塞米松组;流式细胞术检测细胞凋亡,ELISA检测细胞上清炎症因子的浓度,Western blot检测细胞NF-κB p65、p-NF-κB p65、Keap1、Nrf2以及细胞核中的Nrf2蛋白水平。结果:CAS最适作用条件为10μmol/L、2 h;与正常组相比,模型组的细胞凋亡率、炎症因子含量、p-NF-κB p65和Keap1蛋白表达水平显著升高(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著降低(P<0.01);与模型组相比,Casticin组和地塞米松组的细胞凋亡率和炎症因子含量显著降低(P<0.01),Casticin组p-NF-κB p65和Keap1蛋白表达水平显著降低(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著升高(P<0.01);与ML385组相比,Casticin+ML385组的细胞凋亡率,炎症因子含量,p-NF-κB p65和Keap1蛋白表达水平显著降低(P<0.01),细胞和细胞核中Nrf2蛋白的表达水平显著升高(P<0.01)。结论:CAS通过调节NF-κB-Keap1-Nrf2/ARE信号通路,抑制脂多糖诱导的细胞炎症。

紫花牡荆素抗肿瘤作用机制的研究进展

多甲氧基黄酮类化合物紫花牡荆素是中国传统中草药蔓荆子的主要活性成分之一,具有抗炎、抗肿瘤等功效。本文归纳总结紫花牡荆素抗肿瘤作用机制的研究进展,以期为紫花牡荆素抗肿瘤的进一步开发利用提供一定参考。

紫花牡荆素调控PI3K/AKT通路对肝癌细胞增殖和凋亡的影响

目的:研究紫花牡荆素调控磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)通路对肝癌细胞增殖和凋亡的影响。方法:肝癌细胞株HepG2、Hep3B经过培养并分为对照组、不同浓度紫花牡荆素(10μmol/L、20μmol/L、40μmol/L、80μmol/L)组、溶剂对照(体积分数0.1%的DMSO)组、溶剂+40μmol/L紫花牡荆素组、AKT激动剂SC-79(5μmol/L)+40μmol/L紫花牡荆素组。分组处理48 h后,检测细胞活力、细胞克隆数、细胞凋亡率及B淋巴细胞瘤基因(Bcl-2)、细胞色素C(CytC)、Bcl-2相关X蛋白(Bax)、裂解型caspase-3(Cleaved caspase-3)、磷酸化PI3K(p-PI3K)、磷酸化AKT(p-AKT)的表达水平。结果:10μmol/L、20μmol/L、40μmol/L紫花牡荆素以浓度依赖的方式降低HepG2、Hep3B的细胞活力;40μmol/L紫花牡荆素组HepG2、Hep3B的细胞克隆数及Bcl-2、CytC、p-PI3K、p-AKT的表达水平均低于对照组,Bax、Cleaved caspase-3的表达水平高于对照组(P<0.05);AKT激动剂SC-79削弱40μmol/L紫花牡荆素对HepG2、Hep3B的调控作用。结论:紫花牡荆素通过抑制PI3K/AKT通路抑制肝癌细胞增殖。

紫花牡荆素通过上调miR-342-3p抑制宫颈癌HeLa细胞糖酵解

目的:探讨紫花牡荆素对宫颈癌HeLa细胞miR-342-3p表达和糖酵解的影响,验证紫花牡荆素是否通过上调miR-342-3p表达抑制宫颈癌HeLa细胞糖酵解。方法:使用不同浓度(0,10,30,100 nmol/L)的紫花牡荆素处理HeLa细胞,采用紫花牡荆素(30 nmol/L)联合miR-342-3p模拟物或抑制物转染HeLa细胞,随后用实时荧光定量PCR分析HeLa细胞miR-342-3p的表达水平,通过测定相对葡萄糖消耗量和相对乳酸产物,分析紫花牡荆素及紫花牡荆素联合miR-342-3p模拟物或抑制物转染对宫颈癌HeLa细胞糖酵解的影响。结果:紫花牡荆素以浓度依赖方式上调HeLa细胞miR-342-3p表达水平,同时降低HeLa细胞的葡萄糖消耗量和抑制乳酸产生。此外,miR-342-3p模拟物协同紫花牡荆素抑制HeLa细胞的葡萄糖消耗和乳酸产生;相反miR-342-3p抑制物减弱紫花牡荆素上调miR-342-3p表达水平的作用和减弱紫花牡荆素抑制HeLa细胞的葡萄糖消耗和乳酸产生作用。结论:紫花牡荆素通过上调miR-342-3p表达水平抑制HeLa细胞糖酵解。

紫花牡荆素通过miR-148a-3p/Wnt1信号通路抑制肝癌MHCC97H细胞的迁移和侵袭

目的用紫花牡荆素(casticin,CAS)处理MHCC97H细胞,观察其迁移和侵袭能力的变化,并初步探讨CAS的分子作用机制。方法CCK-8试剂盒检测不同浓度CAS对MHCC97H细胞活力的影响;分组开展细胞迁移和侵袭实验,评估MHCC97H细胞的迁移和侵袭能力;逆转录荧光定量PCR(RT-qPCR)检测CAS处理后,MHCC97H细胞的miR-148a-3p和Wnt1 mRNA表达变化;迁移侵袭相关蛋白(MMP2、MMP9)和Wnt1蛋白表达量采用Western blot检测;Dual-Luciferase报告基因检测miR-148a-3p与Wnt13′-UTR的结合情况。结果CAS明显抑制MHCC97H细胞的生存活力,其对这种细胞的IC_(50)约为10.0μmol·L^(-1),亚细胞毒浓度的CAS(3.0μmol·L^(-1))可减弱这种细胞的迁移和侵袭能力;miR-148a-3p mimic或CAS均能抑制MHCC97H细胞迁移和侵袭能力,且两者联合处理后的抑制作用强于单独使用;过表达Wnt1能有效减弱CAS的抑制作用;CAS能明显上调MHCC97H细胞miR-148a-3p表达,同时Wnt1、MMP2和MMP9的表达呈现下降;Dual-Luciferase报告基因发现,miR-148a-3p可以直接靶向Wnt13′-UTR。结论CAS可通过miR-148a-3p/Wnt1信号通路减弱肝癌细胞的迁移和侵袭。

紫花牡荆素体内抗炎作用的研究(英文)

目的:探讨中药蔓荆子中主要成分紫花牡荆素的抗炎作用。方法:通过急性抗炎模型——二甲苯致小鼠耳廓肿胀实验、鸡蛋清致大鼠足跖肿胀实验以及腹腔注射0.7%醋酸致小鼠腹腔毛细血管通透性增高的实验,观察紫花牡荆素的抗炎作用。结果:紫花牡荆素对二甲苯所致小鼠耳廊肿胀、鸡蛋清所致大鼠足肿胀以及醋酸所致小鼠毛细血管通透性增加均有明显的抑制作用。结论:紫花牡荆素具有明显的体内抗炎作用,为蔓荆子抗炎作用的有效成分。

紫花牡荆素体外抑制人宫颈癌HeLa细胞增殖的研究

背景与目的:紫花牡荆素(Casticin)是一种从蔓荆子中提取的具有广泛药理活性的多甲基黄酮化合物。有研究表明Casticin能诱导多种肿瘤细胞凋亡,但其对人宫颈癌细胞的作用尚缺乏文献资料。本研究旨在探讨Casticin抑制体外培养人宫颈癌HeLa细胞增殖作用及其机制。方法:MTT法检测Casticin对人宫颈癌HeLa细胞活性的抑制作用;平皿集落形成法检测Casticin对HeLa细胞集落形成率的影响;流式细胞术(FCM)检测Casticin对HeLa细胞周期的影响;采用Western blot分析蛋白表达的变化。结果:Casticin对人宫颈癌HeLa细胞增殖活性有较强的抑制作用,呈剂量和时间依赖性,作用48h的IC50值为2.82μg/mL;与对照组比较,细胞集落形成率明显下降(P<0.05);Casticin作用48h,以浓度依赖方式阻滞HeLa细胞于G2/M期,同时降低CyclinB1蛋白表达,增高P21蛋白表达。结论:Casticin抑制HeLa细胞的增殖作用可能与其降低CyclinB1蛋白表达、活化P21蛋白有关。

高速逆流色谱分离纯化蔓荆子中的活性成分

应用高速逆流色谱法(HSCCC)分离纯化蔓荆子中的活性成分。以石油醚-乙酸乙酯-甲醇-水(体积比为3:6:3.6:3)为两相溶剂体系,在转速为800r/min、流速为1.5mL/min、检测波长为254nm的条件下进行分离,所得馏分经高效液相色谱法(HPLC)检测,并经电喷雾电离(ESI)质谱和核磁共振谱(NMR)鉴定化合物的结构。从250mg蔓荆子粗提物中一次性分离得到4个化合物,分别为23mg对羟基苯甲酸、15mg3,6,7-三甲基槲皮万寿菊素、24mg蔓荆子黄素和5mg蒿黄素,其纯度约为93.1%、97.3%、98.7%和98.5%。该法具有简便、快速、重复性好的优点,为分离蔓荆子中的活性成分提供了新的方法。

紫花牡荆素对肺癌A549细胞增殖及凋亡的影响

目的探讨紫花牡荆素对肺癌A549细胞（简称肺癌细胞）增殖及凋亡的影响,为其用于临床治疗肺癌提供依据。方法取对数生长期的肺癌细胞,分别加入终浓度为0.1、0.5、1、5、10、25、50、100μmol/L紫花牡荆素溶液,分别作用24、48 h,采用SRB法检测增殖抑制率,计算50%抑制浓度（IC50）。取对数生长期的肺癌细胞,分别加入最终浓度为0、5、15μmol/L紫花牡荆素溶液,Hoechst33258染色后共聚焦显微镜下观察细胞凋亡形态;采用流式细胞术检测细胞周期时相分布。结果随紫花牡荆素浓度升高,作用24、48 h的肺癌细胞增殖抑制率均呈上升趋势。经0.5～100μmol/L紫花牡荆素作用48 h的肺癌细胞增殖抑制率均明显高于同一浓度作用24 h的肺癌细胞（P均〈0.05）。紫花牡荆素作用24、48 h时的IC50分别为14.74、8.16μmol/L。随紫花牡荆素浓度升高,作用24 h的肺癌细胞核染色质固缩偏向一边、核裂解和细胞碎片等细胞凋亡形态学改变越来越明显;与同浓度紫花牡荆素作用24 h比较,作用48 h的肺癌细胞凋亡形态学改变更明显。经0、5、15μmol/L紫花牡荆素作用24、48 h的肺癌细胞凋亡率均逐渐升高,处于G1、S期的比例均逐渐降低,处于G2/M期的比例均逐渐升高（P均〈0.05）。与同一浓度紫花牡荆素作用24 h的肺癌细胞比较,经5、15μmol/L紫花牡荆素作用48 h的肺癌细胞凋亡率均升高,处于G1、S期的比例均降低,处于G2/M期的比例均升高（P均〈0.05）。结论紫花牡荆素可将肺癌细胞阻滞于G2/M期,具有增殖抑制作用和凋亡促进作用,并呈时间、浓度依赖性。

紫花牡荆素对佐剂性关节炎小鼠足肿胀度和细胞因子的影响

目的研究紫花牡荆素对佐剂性关节炎小鼠血浆炎症相关细胞因子的作用及其与足跖肿胀度的相关性。方法 36只Bal b/c小鼠,随机分为6组:空白对照组、模型组、阳性药物组、紫花牡荆素12.5,25.0和50.0mg·kg^(-1)剂量组;小鼠右足垫皮内注射弗氏完全佐剂后连续灌胃给药12d,每日1次。第13天测量小鼠后足爪的肿胀度,第14天采血,用流式细胞仪的微球阵列(CBA)法检测佐剂性关节炎小鼠外周血中的致炎性细胞因子(IL-2、IFN-γ和TNF-α)和抗炎性细胞因子(IL-4和IL-5)的水平。结果紫花牡荆素可剂量依赖性地抑制Bal b/c小鼠后足爪的肿胀度;明显降低致炎性Th1细胞因子IL-2、IFN-γ和TNF-α的水平,显著提高抗炎性Th2细胞因子IL-4和IL-5的水平;小鼠后足爪肿胀度与血清细胞因子IL-2、IFN-γ和TNF-α的水平呈正相关,与IL-4和IL-5的水平呈负相关。结论紫花牡荆素改善佐剂性关节炎小鼠足跖肿胀度,可能是通过增加血液IL-4和IL-5含量,降低IL-2、IFN-γ和TNF-α含量,调节细胞因子平衡的结果。

紫花牡荆素体外抑制人卵巢癌HO-8910细胞增殖和诱导凋亡的研究

目的:探讨紫花牡荆素(casticin)体外抑制人卵巢癌HO-8910细胞增殖和诱导凋亡的效应及其机制。方法:MTT法检测casticin对人卵巢癌HO-8910细胞增殖的抑制;Hoechest33258染色观察细胞凋亡形态学改变;流式细胞术(FCM)检测casticin处理HO-8910细胞的凋亡率;Western blotting分析caspase-3、CyclinB1、p21蛋白表达变化。结果:casticin对人卵巢癌HO-8910细胞增殖有较强的抑制作用,呈剂量和时间依赖性。经casticin作用48h后HO-8910细胞表现出典型的凋亡形态特征,并剂量依赖性地增加亚二倍峰,降低CyclinB1蛋白表达,增高caspase-3和p21表达。结论:casticin通过降低CyclinB1表达、活化p21和caspase-3抑制HO-8910细胞增殖并诱导凋亡。

紫花牡荆素抑制FOXM1诱导乳腺癌细胞凋亡的研究

背景与目的:紫花牡荆素分离自传统中草药蔓荆子,具有广泛的抗肿瘤活性。转录因子FOXM1在多种肿瘤中高表达,对肿瘤发生发展具有重要影响。本研究旨在探讨紫花牡荆素诱导乳腺癌MDA-MB-231细胞凋亡及其作用机制是否涉及FOXM1。方法:碘化丙啶(Propidium iodide,PI)流式细胞术(flow cytometry,FCM)检测经不同浓度紫花牡荆素处理的体外培养MDA-MB-231细胞的凋亡率。免疫酶联试验(ELISA)测定细胞组蛋白/DNA碎片水平。DNA琼脂糖凝胶电泳观察细胞凋亡梯形条带图谱。蛋白质印迹法(Western blot)分析细胞FOXM1和Survivin蛋白表达。结果:紫花牡荆素以浓度依赖的方式(0、0.1、0.5和1.0μmol/L)增加MDA-MB-231细胞凋亡率[(4.36±0.12)%、(6.37±0.28)%、(11.30±0.62)%和(34.50±0.85)%]和增高细胞组蛋白/DNA碎片水平。紫花牡荆素处理MDA-MB-231细胞基因DNA琼脂糖电泳图谱呈现典型凋亡"梯型条带",Western blot检测结果显示,紫花牡荆素以浓度依赖方式下调FOXM1和Survivin蛋白表达。结论:紫花牡荆素能有效诱导乳腺癌MDA-MB-231细胞细胞凋亡,其作用机制涉及下调FOXM1和Survivin表达。

正交试验优选蔓荆子提取工艺研究

目的:依据蔓荆子中有效成分的理化性质,优选蔓荆子的提取工艺条件。方法:采用L9(34)正交试验设计,以药材中总黄酮和紫花牡荆素的提取率作为考察指标,采用UV法和HPLC法测定,所得结果进行方差分析,综合两个指标结果确定最佳工艺。结果:蔓荆子药材粗粉,用20倍量的95％乙醇作提取溶剂,回流提取3次,每次1 h为最佳工艺。结论:优选出的工艺稳定,方法可行。

紫花牡荆素诱导人肝癌HepG2细胞凋亡的研究

目的研究紫花牡荆素(Casticin)对肝癌HepG2细胞增殖抑制和凋亡诱导的作用,并探讨其作用机制。方法用终浓度为0、0.5、1.0及2.0μmol/L的Casticin作用于HepG2细胞,于12、24及48h后采用MTT法检测细胞增殖抑制率;Hoechst33342核染色,观察细胞形态学变化;24h后收集各组肝癌HepG2细胞,流式细胞术检测细胞周期及凋亡率;RT-PCR检测survivinmRNA表达。结果 MTT法检测显示,Casticin对肝癌HepG2细胞有增殖抑制作用,并存在浓度和时间依赖关系;Hoechst33342染色后,可见核染色质凝集,凋亡细胞呈致密浓染。与对照组相比,Casticin处理后凋亡细胞比例增加。Casticin作用24h后,细胞被阻滞于G2/M期,随药物质量浓度的增加,细胞凋亡率逐渐增加。RT-PCR结果显示,Casticin下调肝癌HepG2细胞sur-vivinmRNA表达。结论 Casticin在体外对肝癌HepG2细胞有明显的增殖抑制和凋亡诱导作用,初步推断Casticin诱发肝癌细胞凋亡的作用与其对survivin基因表达的抑制有关。