莱菔硫烷对铝及D-半乳糖联合染毒小鼠阿尔茨海默病样损伤的干预作用研究

目的探讨莱菔硫烷(sulforaphane,SFN)对铝及D-半乳糖联合染毒小鼠阿尔茨海默病(Alzheimer’s disease,AD)样损伤的干预作用,为防治AD提供理论依据。方法健康成年昆明种小鼠按体重随机分为3组,每组18只,雌雄各半。模型组和干预组小鼠饮用含铝水(浓度为0.4 g/100 ml),并隔日皮下注射200mg/kg D-半乳糖。干预组小鼠灌胃给予25 mg/kg SFN 1/d,模型组小鼠灌胃等量蒸馏水,同时设立溶媒对照组,连续处理90 d。采用Morris水迷宫测定小鼠的学习记忆能力,免疫组织化学染色法检测脑组织β-淀粉样肽(Aβ)沉积,分光光度法检测大脑皮质丙二醛(MDA)、谷胱甘肽(GSH)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性,Real-time q RT-PCR法检测大脑皮层Cu-Zn SOD、GSH-Px m RNA的表达水平。结果与对照组比,模型组小鼠学习记忆能力下降,海马、大脑皮质Aβ斑块数量增多,差异均有统计学意义(P0.05);与模型组比,干预组小鼠学习记忆能力增强,海马、大脑皮质Aβ斑块减少,差异均有统计学意义(P0.05)。模型组和干预组小鼠大脑皮质SOD活性及SOD m RNA水平高于对照组,差异均有统计学意义(分别为P0.05,P0.01);干预组小鼠大脑皮质SOD活性及SOD m RNA水平与模型组比较,差异无统计学意义(P0.05)。各组小鼠大脑皮质MDA、GSH含量,活性及GSH-Px m RNA水平,差异均无统计学意义(P0.05)。结论 SFN可减少铝及D-半乳糖联合染毒小鼠脑内的Aβ斑块沉积,改善其学习记忆能力;SFN是否通过氧化应激影响AD有待深入研究。     

莱菔硫烷诱导人肝癌HepG2细胞凋亡与Fas/FasL途径的相关性研究

目的 研究莱菔硫烷对人肝癌HepG2细胞Fas、FasL、Bid蛋白表达的影响,探讨其诱导HepG2细胞凋亡过程中Fas/FasL途径的作用。方法 Caspase-8试剂盒检测莱菔硫烷对HepG2细胞Caspase-8活性的影响;流式细胞仪检测莱菔硫烷对HepG2细胞中Fas、FasL、Bid蛋白表达的影响。结果 莱菔硫烷10、20、40 μmol/L作用于HepG2细胞48 h,可显著升高HepG2细胞Caspase-8活性（P＜0.01）;可使HepG2细胞 Fas、FasL蛋白的表达显著升高（P＜0.01）;莱菔硫烷20、40 μmol/L时,可显著提高Bid蛋白的表达（P＜0.01）,上述作用均呈剂量相关性。结论 激活Fas/FasL途径可能是莱菔硫烷诱导人肝癌HepG2细胞凋亡的重要机制之一。     

萝卜硫烷对乳腺癌细胞增殖、细胞周期、细胞凋亡的影响及作用机制研究

目的:研究萝卜硫烷(sulforaphane,SUL)对不同类型乳腺癌细胞增殖和细胞周期细胞凋亡的影响及其作用机制。方法:采用MTT法、流式细胞术和Western印迹法,研究不同浓度受试物对F3Ⅱ、MCF-7、MDA-MB-231和ZR-75-1细胞增殖抑制、细胞周期阻滞、F3Ⅱ细胞凋亡和p34cdc2及Cdc25C表达的影响。结果:(1)SUL对所选的四个细胞系均有很强的增殖抑制作用,其中雌激素正响应型(ERP)细胞对SUL的敏感性明显强于雌激素负响应型(ERN)细胞和扩散性较强的F3Ⅱ细胞;(2)SUL对F3Ⅱ细胞和两种ERP细胞均呈现G2/M期阻滞作用,而对ERN细胞周期则无影响;(3)SUL阻滞F3Ⅱ细胞从G2期向M期转化的作用机制是提高Cdc2的磷酸化水平,降低Cdc25C磷酸化酶的表达,从而抑制细胞周期素B1-Cdc2复合物激酶的去磷酸化作用;(4)在实验条件下,SUL不引起F3Ⅱ细胞凋亡。结论:SUL对四个乳腺癌细胞系细胞增殖抑制活性和细胞周期影响存在明显的差异,不引起F3Ⅱ细胞凋亡。对F3Ⅱ细胞周期G2/M阻滞作用的机制是提高Cdc2的磷酸化水平,降低Cdc25C磷酸化酶的表达。     

A mutated p53 status did not prevent the induction of apoptosis by sulforaphane,a promising anti-cancer drug

Summary We investigated apoptosis induction by sulforaphane on three cell lines characterized by a different p53 status. In particular, we used p53-knock-out fibroblasts from newborn mice transfected with the p53-Ser220 mutation, observed in Li-Fraumeni Syndrome patients, as a model of mutated p53 status. Moreover, immortalized fibroblasts from newborn mice expressing or lacking p53 (p53 +/+ andp53-/-, respectively) have been used to verify whether mutated p53 status could prevent sulforaphane-induced apoptotic events. Sulforaphane was able to induce apoptosis on all three cell lines. Indeed, the caspase-3 assays and poly(ADP-ribose)polymerase (PARP) cleavage data indicated that sulforaphane stimulated caspase-3-like activity and degradation of PARP. However, cells with a wild-type or mutated p53 appeared to be more sensitive to the effects of sulforaphane than cells lacking p53. Taken together, our results suggest that sulforaphane could act by a p53-independent pathway. For this reason, sulforaphane can be viewed as a novel agent useful not only in the treatment of Li-Fraumeni-associated tumors but also drug resistant tumors where p53 dysregulation is a feature.     

Sulforaphane inhibits oral carcinoma cell migration and invasion in vitro

Sulforaphane is a predominant isothiocyanate in Brassica oleracea, a family of cruciferous vegetables, and is known to be inversely related to the risk of various types of human carcinomas. Studies using oral carcinoma cell lines are scarce, however, and the role of sulforaphane on oral carcinoma cell metastasis is yet to be determined. In this study, the growth inhibition of oral carcinoma cell lines by sulforaphane was determined using aqueous soluble tetrazolium salts, and the growth of various oral cancer cell lines was attenuated. The migration and invasion activities of the cells also decreased, as observed in monolayer scratch assays and transwell invasion experiments. The molecular change behind the impairment of the migration and invasion was investigated via secreted metalloprotease level detection using Multiplex protein analysis kits. At the molecular level, the secreted forms of MMP-1 and MMP-2 were down-regulated. The expressions of MMP-1 and MMP-2 did not change when a conventional tumoricidal agent paclitaxel was used. These findings indicate that sulforaphane may have therapeutic potential as an inhibitor of metastasis in oral carcinoma patients.     

不同品种西兰花种子中莱菔硫烷的HPLC分析

目的建立西兰花种子中莱菔硫烷的HPLC含量测定方法。方法色谱柱:Waters公司XBridgeTMC18(150 mm×4.6 mm,5μm);流动相:甲醇-水(体积比30∶70);流速:1.0 mL/min;PDA检测器;检测波长:240 nm;柱温:30℃。结果莱菔硫烷在14.6~292.5μg/mL范围内线性关系良好,r=0.9994,平均回收率为97.32%(RSD=1.2%)。结论本法简便、准确,重复性好,可用作西兰花种子的质量控制,为不同产地不同品种的西兰花种子的收购及栽培等提供有效监控依据。     

Genotoxicity studies of organically grown broccoli (Brassica oleracea var. italica) and its interactions with urethane,methyl methanesulfonate and 4-nitroquinoline-1-oxide genotoxicity in the wing spot test of Drosophila melanogaster

Broccoli (Brassica oleracea var. italica) has been defined as a cancer preventive food. Nevertheless, broccoli contains potentially genotoxic compounds as well. We performed the wing spot test of Drosophila melanogaster in treatments with organically grown broccoli (OGB) and co-treatments with the promutagen urethane (URE), the direct alkylating agent methyl methanesulfonate (MMS) and the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the standard (ST) and high bioactivation (HB) crosses with inducible and high levels of cytochrome P450s (CYPs), respectively. Larvae of both crosses were chronically fed with OGB or fresh market broccoli (FMB) as a non-organically grown control, added with solvents or mutagens solutions. In both crosses, the OGB added with Tween–ethanol yielded the expected reduction in the genotoxicity spontaneous rate. OGB co-treatments did not affect the URE effect, MMS showed synergy and 4-NQO damage was modulated in both crosses. In contrast, FMB controls produced damage increase; co-treatments modulated URE genotoxicity, diminished MMS damage, and did not change the 4-NQO damage. The high dietary consumption of both types of broccoli and its protective effects in D. melanogaster are discussed.