Development of an Antibody against Diosgenin and Spiroaminoketal Alkaloids

An antibody was developed that exhibited affinity for diosgenin as well as the spiroaminoketal alkaloids solasodine and tomatidine. Rabbits were immunized with a diosgenin conjugate prepared by succinylation of diosgenin, followed by purification with a silica solid-phase extraction cartridge and active ester conjugation to Limulus polyphemus hemocyanin (LPH). Rabbit serum was evaluated using an indirect competitive ELISA with diosgenin-BSA as the coating conjugate. A reduction in absorbance of 50% could be obtained using concentrations of ca. 0.8 μM diosgenin, 30 μM solasodine and 100 μM tomatine. The antibody exhibited no affinity for solanidine, a non-spiral glycoalkaloid.     

龙葵生物碱体外抑制肿瘤细胞增殖作用的实验研究

目的:研究龙葵生物碱提取物对人肺癌A549细胞株增殖的抑制作用。方法:采用盐酸-乙醇混合溶剂加热回流法从龙葵中提取分离生物碱。以MTT法考察龙葵生物碱不同浓度对人肺癌A549细胞株增殖的抑制作用,采用倒置显微镜观察药物对肿瘤细胞株形态的影响。结果:龙葵生物碱提取物对人肺癌A549细胞株具有显著的细胞增殖抑制作用,且呈剂量依赖关系,并可使肿瘤细胞形态发生显著变化。结论:龙葵生物碱具有对肺癌细胞的抑制作用。     

龙葵碱对Panc-1细胞增殖和血管形成能力的影响及对AKT-mTOR通路的调节作用

目的 探讨龙葵碱对胰腺癌细胞Panc-1的增殖和血管形成能力的影响及其相关机制。方法 实验分为对照组和药物组,药物组分别采用浓度为3.5、7.0 和10.5 μmol/L的龙葵碱对细胞进行干预,软琼脂克隆试验观察龙葵碱对胰腺癌细胞Panc-1非贴壁依赖性增殖能力的影响;脉管形成实验观察龙葵碱对胰腺癌细胞Panc-1血管形成能力的影响;蛋白质免疫印迹（Western blotting）法检测Panc-1细胞总蛋白中蛋白激酶B（protein kinase B,AKT）、雷帕霉素靶蛋白（target of rapamycin,mTOR）和血管内皮生长因子 （vascular endothelial growth factor,VEGF）的表达变化。结果 龙葵碱可明显抑制Panc-1细胞非贴壁依赖性增殖能力,且呈剂量依赖性[100% vs （42.1±9.6）%,（24.3±8.5）%,（14.4±1.7）%;P ＜0.05];龙葵碱亦可抑制VEGF蛋白表达[100% vs （74.9±5.5）%,（31.9±6.8）%,（16.5±7.5）%,P ＜0.05],并且抑制脉管形成[100% vs （82.3±9.5）%,（76.9±8.9）%,（56.0±12.1）%,P ＜0.05];龙葵碱可下调Panc-1细胞AKT、mTOR磷酸化蛋白表达[100% vs （72.4±0.8）%,（59.4±1.3）%,（40.7±2.9）%;100% vs （96.7±0.4）%,（77.5±3.4）%,（34.1±7.6）%,P ＜0.05]。结论 龙葵碱可能通过抑制AKT-mTOR细胞信号通路而抑制Panc-1细胞的增殖及血管形成能力。     

龙葵碱对胰腺癌细胞裸鼠移植瘤的抑制作用及机制研究

目的 探讨龙葵碱对胰腺癌细胞SW1990裸鼠移植瘤的抑制作用并初步研究其作用机制。方法 建立胰腺癌细胞SW1990的裸鼠荷瘤模型,随机分为对照组和实验组,每组10只。肿瘤接种成功第2周起对照组和实验组分别腹腔注射溶媒（含1‰二甲亚砜）1 mL、龙葵碱10 mg/kg,隔天给药1次,共给药2周。每次给药结束用游标卡尺记录肿瘤大小。给药结束后颈椎脱臼处死裸鼠,测量瘤体重量并计算抑瘤率,留取新鲜肿瘤组织用RT-PCR和免疫组化检测Bcl-2、Bax和Caspase-3基因mRNA和蛋白表达情况。结果 与对照组相比,实验组裸鼠肿瘤生长受到显著抑制（P＜0.05）,实验组的肿瘤组织Bcl-2表达阳性率明显降低（实验组90% vs 对照组46%,P＜0.01）,Bax（实验组38% vs 对照组70%,P＜0.01）和Caspase-3（实验组 22% vs 对照组74%,P＜0.01）的表达阳性率明显升高。结论 龙葵碱在裸鼠体内能显著抑制人胰腺癌细 胞SW1990的增殖,此抑制作用呈明显的时间依赖性。龙葵碱发挥上述作用的机制可能在于诱导胰腺癌细胞凋亡。     

茄碱通过调控Stat3信号通路影响胰腺癌细胞Panc-1中MMP-2和MMP-9表达

目的 探讨茄碱对胰腺癌细胞Panc-1中基质金属蛋白酶2（MMP-2）和基质金属蛋白酶9（MMP-9）表达的影响及Stat3信号通路在此过程中的作用。方法 实验分为对照组和药物组,药物组分别采用浓度为3.5、7.0和10.5 μmol/L的茄碱对Panc-1细胞进行干预,明胶酶谱法观察茄碱对细胞中MMP-2和MMP-9活性的影响;实时荧光定量PCR检测MMP-2和MMP-9基因的表达变化;Western blotting法检测总蛋白中信号转导及转录激活因子3（Stat3）蛋白的磷酸化表达变化。结果 与对照组相比,药物组（7.0、10.5 μmol/L）MMP-2活性明显下降（t值分别为2.4、6.4,P<0.05）;MMP-9活性亦下降（t值分别为3.2、4.8,P<0.05）。实时荧光定量PCR结果显示,药物组（7.0 μmol/L、10.5 μmol/L）MMP-2基因表达下调（t值分别为8.7、13,P<0.05）;MMP-9基因表达下调（t值分别为3.2、16.3,P<0.05）。Western blotting结果显示,随着茄碱浓度的增加,Panc-1细胞中Stat3蛋白磷酸化受到明显抑制（t值分别为10.2、6.4、6.2,P<0.05）,延长药物处理的时间（12 h、18 h、24 h）亦可抑制Stat3蛋白磷酸化（t值分别为11.4、26.1、20.4,P<0.05）。结论 茄碱可抑制胰腺癌细胞Panc-1中MMP-2和MMP-9的表达,发挥其抗肿瘤转移能力。其具体机制可能与调控Sata3信号通路有关。     

龙葵素的生殖毒性研究进展

龙葵素广泛存在于马铃薯、番茄及茄子等茄科植物中。因其具有抗肿瘤和潜在的毒性作用而引起广泛关注。本文主要介绍了龙葵素在生殖毒性方面的研究进展。     

龙葵碱对肝癌Treg细胞介导的肿瘤免疫逃逸的影响

目的 研究龙葵碱对荷肝癌小鼠体内调节性T细胞(Treg细胞)介导肝癌细胞免疫逃逸的影响及其作用机制。方法 建立肝癌H22细胞移植瘤小鼠模型,并设对照组、龙葵碱组、TGF-β1抑制剂组和龙葵碱联合TGF-β1抑制剂组。14 d处理称取各组小鼠移植瘤质量,ELISA法检测各组小鼠外周血中IL-2、IL-10、TGF-β1等免疫性抑制细胞因子的含量变化,流式细胞术检测各组小鼠脾淋巴细胞培养液中CD4+、CD4+CD25+Foxp3+Treg的表达水平,免疫荧光法检测小鼠脾淋巴细胞培养液中Foxp3+免疫荧光强度。结果 14 d后,与对照组相比,龙葵碱组、TGF-β1抑制剂组和联合组的肝癌荷瘤小鼠肿瘤瘤重量均显著低于对照组,差异有统计学意义(P0.05);龙葵碱组、TGF-β1抑制剂组、联合组3组小鼠外周血中免疫性抑制细胞因子IL-2、IL-10、TGF-β1均有明显降低(P0.05);龙葵碱组、TGF-β1抑制剂组、联合组三组CD4+表达水平均高于对照组,CD4+CD25+Treg的比例则明显下降(P0.05);免疫荧光染色结果显示,龙葵碱组、TGF-β1抑制剂组、联合组3组荷瘤小鼠Treg细胞中Foxp3+的表达明显低于对照组,差异有统计学意义(P0.05)。结论 龙葵碱可能通过降低荷瘤小鼠Treg细胞含量来改善免疫逃逸,从而发挥抗肿瘤作用。     

Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein

The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumor activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of anti-tumor and mechanism of solanine. The MTT assay was used to evaluate the IC(50) on the three digestive system tumor cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC(50) for HepG(2), SGC-7901, and LS-174 were 14.47, >50, and >50 microg/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG(2) cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G(2)/M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG(2) cells seems to be mediated by the inhibition in the expression of Bcl-2 protein.     

Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca^2＋]i in the cells

AIM: To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2 ]i in the cells, and to uncover the mechanism by which solanine induces apoptosis. METHODS: HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy (LCSM). HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca2 ]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2 ]i in the cells were observed using LCSM. RESULTS: Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca2 in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca2 in the cells at the same time as it lowered the membrane potential of mitochondria. CONCLUSION: Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2 being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2 in the cell, turning on the mechanism for apoptosis.     

澳洲茄碱对压力超负荷诱导的小鼠心肌纤维化的影响

目的 探讨澳洲茄碱(SS)对压力超负荷诱导的小鼠心肌纤维化(MF)的影响.方法 使用主动脉缩窄术(AB)建立小鼠心肌纤维化模型,采用随机数字表法将40只8～10周雄性C57/B6小鼠随机分为假手术组、假手术+SS组、AB组、AB+SS组,每组10只.AB手术3周后,AB+SS组每天腹腔注射SS(10 mg/kg),AB...