大豆苷元及其苯磺酸酯衍生物对肺癌细胞增殖和凋亡的影响

为研究大豆苷元及其3种苯磺酸酯衍生物对非小细胞肺癌细胞的增殖和凋亡的影响,利用CCK-8检测大豆苷元及衍生物处理A549、H1299和HELF细胞后细胞活性的时间-效应关系和剂量-效应关系,并通过Tunel流式细胞术检测衍生物B处理的细胞凋亡情况,RT-qPCR分析TP53和Caspase9 mRNA表达。结果表明:在1.0×10~(-5)～1.0×10~3μmol·L~(-1)浓度范围时,加入大豆苷元的浓度每增加10倍,肺正常HELF细胞的活性增加16.11%[95%CI 13.74,18.49](P0.001),肺癌A549与H1299细胞活性则分别降低3.26%(P0.001)、3.33%(P0.001);加入衍生物B的浓度每增加10倍,HELF细胞的活性增加9.92%(P0.001),A549和H1299细胞活性降低5.23%(P0.001)、4.32%(P0.001)。10μmol·L~(-1)大豆苷元浓度下,作用时间每延长1 h,HELF细胞活性增加1.87%(P0.001),A549和H1299细胞活性分别降低1.30%(P0.001)、1.75%(P0.001),苯磺酸酯衍生物B对HELF细胞活性增加1.72%(P0.001),A549和H1299细胞降低1.46%(P0.001)、2.07%(P=0.001)。10μmol·L~(-1)的衍生物B处理6 h后,HELF细胞相对数量增加了42.66%(P=0.03),A549和H1299细胞相对数量减少20.67%(P=0.035)、18.33%(P=0.043)。衍生物B促进A549和H1299细胞的凋亡的作用约为对照组的1.15倍(P0.001)、1.24倍(P=0.001),抑制HELF细胞的凋亡作用为对照组的0.84倍(P=0.005),10μmol·L~(-1)衍生物B处理时肺癌和肺正常细胞的TP53和Caspase9的mRNA表达比对照组高。研究表明:大豆苷元及其3种苯磺酸酯衍生物能够抑制肺癌细胞的增殖活性的同时,增加肺正常细胞增殖活性,大豆苷元苯磺酸酯衍生物B能促进肺癌细胞凋亡并抑制肺正常细胞凋亡,其机制可能与通过P53通路诱导癌细胞凋亡有关。     

EGCG抑制Nicotine诱导肺腺癌H1299细胞JAK2/STAT3 mRNA的表达研究

为探索EGCG对Nicotine诱导肺癌细胞增殖的抑制作用,本研究通过MTT实验筛选出EGCG、Nicotine对肺腺癌细胞H1299的最佳作用浓度,利用实时荧光定量PCR技术检测EGCG、Nicotine对H1299细胞中Bax、Bcl-2、Jak2和Stat3基因mRNA相对表达量的变化。实验结果表明,EGCG对H1299细胞的半抑制浓度IC₅₀值约为32βμmol·L^-1（24βh）、15βμmol·L^-1（48βh）;1βμmol·L^-1的Nicotine对H1299细胞促增殖作用明显;以15βμmol·L^-1的EGCG预处理H1299细胞24βh可显著下调1βμmol·L^-1 Nicotine的促增殖作用（P<0.05）。1βμmol·L^-1的Nicotine处理H1299细胞可明显降低JAK2/STAT3信号通路中Bax基因mRNA的表达,增加Bcl-2、Jak2、Stat3基因的mRNA表达;15βμmol·L^-1 EGCG预处理H1299细胞可反向调控Nicotine诱导所致JAK2/STAT3信号通路中Jak2、Stat3和Bax、Bcl-2基因表达量的变化,结果具有显著性差异（P<0.05）。由此可知,EGCG对Nicotine诱导的肺腺癌H1299细胞增殖及JAK2/STAT3信号通路中促增殖基因mRNA的表达起抑制作用。     

低pH和铝胁迫对胡椒根细胞存活和有机酸分泌的影响

以胡椒插条苗为研究对象,通过在低pH和正常pH营养液中添加不同浓度铝,研究胡椒根系细胞存活和有机酸分泌对低pH和铝胁迫的响应。结果表明:pH5.0(低pH)条件下,不添加铝时,胡椒根细胞即表现出坏死症状,当添加80μmol/L铝时,胡椒根细胞开始凋亡,200μmol/L铝添加导致胡椒根细胞全部死亡;pH6.0(正常pH)条件下,当添加80μmol/L铝时,胡椒根细胞出现坏死症状,200μmol/L铝添加导致根尖细胞大量凋亡。由此可见,低pH和铝的双重胁迫加速胡椒根细胞凋亡。低浓度铝添加致使胡椒根系分泌有机酸速率增加,柠檬酸为胡椒适应低pH和铝胁迫分泌的主要有机酸;在低pH和正常pH条件下,40μmol/L铝添加均可诱导胡椒根系柠檬酸分泌速度的增加,苹果酸仅在正常pH条件下低浓度铝胁迫时分泌速率增加。因此,低pH条件下,铝胁迫引起胡椒根系分泌有机酸种类减少,分泌速率降低。研究还发现Al3+是对胡椒毒害最严重的铝形态,是pH4.5土壤中的主要铝形态,目前海南省胡椒主要植区有50%的土壤pH低于4.5,所以对于胡椒园土壤的铝毒害应引起足够重视。     

镧对镉胁迫下大豆幼苗 光合作用和活性氧代谢的影响

在盆栽条件下,研究了不同浓度的La对30μmol/L和50μmol/L Cd胁迫下的大豆幼苗生理效应。结果 表明,适量的La能在一定程度上缓解Cd胁迫所导致的叶绿素含量、叶绿体可溶性蛋白质含量、光合速率、叶绿体 Mg2 + - ATPase活性的降低;能有效增强或保护叶片抗氧化能力,降低超氧阴离子自由基(O2 · )产生速率、组织自动 氧化速率和H2O2 含量,防止膜透性的增加;适量的La能保持大豆叶片中超氧化物歧化酶( SOD)和抗坏血酸过氧 化物酶(AsA - POD)活性的相对稳定,维持活性氧的代谢平衡。La对30μmol /L Cd胁迫的缓解能力较强,而对 50μmol/L Cd胁迫的缓解能力较弱。La的最佳作用浓度为0. 30μmol/L,高于0. 50μmol/L的La则加剧Cd的毒害。     

TFDG对IL-1β体外诱导大鼠软骨细胞炎性损伤的保护作用研究

体外分离培养SD雄性大鼠膝关节软骨细胞,经甲苯胺蓝及Ⅱ型胶原(Col Ⅱ)免疫荧光染色鉴定后,加入白介素-1β(IL-1β)诱导建立骨关节炎(OA)细胞模型,探讨茶黄素双没食子酸酯(TFDG)对OA软骨细胞的保护作用。细胞形态观察发现TFDG能明显改善OA软骨细胞形态。实时荧光定量PCR(Real-time PCR)结果显示,TFDG不仅可以上调软骨细胞分子标志物Col Ⅱ mRNA的表达,还可以下调炎症因子IL-1β、IL-6mRNA的表达。酶联免疫吸附实验(ELISA)检测结果进一步表明TFDG可明显降低炎症因子的分泌。免疫印迹(Western blot)检测结果证明,TFDG预干扰可降低炎症诱导酶环氧化酶COX-2蛋白表达量。这些结果说明TFDG通过减弱炎症反应,从而对IL-1β体外诱导大鼠软骨细胞炎性损伤起到保护作用。     

儿茶素对ECV304细胞增殖和迁移的影响

利用体外细胞培养技术探讨了四种儿茶素对ECV304细胞增殖和迁移的影响。结果发现,EGCG对ECV304细胞存活率的影响最显著,在EGCG浓度为50~150μmol/L时,细胞存活率达到极显著差异,半数抑制浓度IC50为75~150μmol/L;EGCG对该细胞迁移作用影响明显,且呈剂量依赖性,IC50为150μmol/L,EGCG可能通过抑制内皮细胞的迁移抑制新生血管的生成;ECG与ECV304作用36h和48h,IC50分别为390μmol/L和370μmol/L;C和EC与ECV304作用时,细胞存活率在80%以上。四种儿茶素在浓度低于75μmol/L时,对正常ECV304细胞抑制影响不大。     

鹿茸多肽对TBHP诱导的H9c2心肌细胞凋亡的影响

目的探讨鹿茸多肽(velvet antler peptide,VAP)预处理对叔丁基过氧化氢(TBHP)诱导H9c2大鼠心肌细胞损伤及凋亡的影响。方法采用含10%胎牛血清(FBS)培养液(DMEM)传代培养细胞7d后,将H9c2心肌细胞分为空白对照组(Blank control group)、空白血清组(Blank serum group)、TBHP组(TBHP group)、100 mg·kg^(-1)VAP低剂量含药血清组(TBHP+VAPLgroup)、400 mg·kg^(-1) VAP高剂量含药血清组(TBHP+VAPHgroup)。正常对照组不做任何处理,其余给药组给予VAP处理24h后,给予200μmol·L^(-1)TBHP处理。MTT法检测各组细胞存活率;Hochst染色法检测各组细胞凋亡情况;Western blotting法检测心肌细胞Bax、Bcl-2、Caspase-3蛋白表达水平。结果MTT检测结果表明,与TBHP组比较,VAP高和低剂量组的H9c2细胞活性升高(P<0.01)。Hoechst染色法结果显示,与空白对照组比较,TBHP组的活细胞减少;与TBHP组比较,VAP高和低剂量组的活细胞增多。Western blotting法检测结果表明,与空白对照组比较,TBHP组上调心肌细胞Bax、Caspase-3蛋白表达水平,降低Bcl-2蛋白表达水平(P<0.05);与TBHP组比较,VAP高和低剂量组降低Bax、Caspase-3蛋白表达水平,升高Bcl-2蛋白表达水平(P<0.05)。结论VAP含药血清预处理可保护TPHP诱导的H9c2细胞心肌损伤。通过降低心肌细胞中Caspase-3蛋白的表达或抑制其活性,增加Bcl-2蛋白的表达,降低Bax蛋白的表达,上调Bcl-2/Bax,从而抑制心肌细胞凋亡。     

大豆异黄酮对过氧化氢致L02细胞凋亡的抑制作用

为研究大豆异黄酮对过氧化氢所致L02细胞凋亡的抑制作用及其可能机制,以过氧化氢诱导L02细胞制备L02肝细胞凋亡模型。用Hoechst 33342荧光染色技术观察L02细胞核形态变化,用原位末端标记(TUNEL)技术观察L02细胞凋亡情况,采用免疫印迹法检测L02细胞中Caspase-3活性片段(Cleaved caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、聚腺苷二磷酸核糖聚合酶活性片段(Cleaved PARP)表达,以及c-Jun氨基末端激酶(JNK)和核转录因子-κB(NF-κB)活化情况。结果显示:过氧化氢处理L02细胞能使细胞核Hoechst染色和TUNEL染色增强,提示L02细胞凋亡增多。同时,过氧化氢上调L02细胞Cleaved caspase-3、Cleaved PARP表达(P0.05)、Bax/Bcl-2比值(P0.05)和NF-κB p65核转位水平(P0.05)。与损伤组比较,大豆异黄酮组L02细胞凋亡显著减少,Cleaved caspase-3和Cleaved PARP表达量、Bax/Bcl-2比值和NF-κB p65核转位水平均降低(P0.05)。结果表明大豆异黄酮能抑制过氧化氢所致L02细胞凋亡,其作用可能与NF-κB通路有关。     

茶黄素-3,3'-O-双没食子酸酯对糖尿病大鼠血管内皮损伤及炎症反应的保护作用

探讨茶黄素-3,3'-O-双没食子酸酯（TFDG）对糖尿病大鼠血管内皮损伤及炎症反应的保护作用及相关机制。以高脂饲料喂养加链脲佐菌素注射制造糖尿病大鼠,将其分为模型组（CON）、5 mg·kg^-1和10 mg·kg^-1 TFDG干预组（TFDG5和TFDG10）,另取正常大鼠为对照组（NC）,分组处理8周,监测体重和空腹血糖变化,观察大鼠腹主动脉病理形态学变化,ELISA检测血浆IL-6、IL-1β和TNF-α水平,Western blot检测蛋白表达水平。结果表明,与CON组相比,TFDG干预对体重和血糖无显著影响,病理切片观察显示TFDG干预组大鼠腹主动脉组织损伤较CON组有所改善,同时TFDG干预组大鼠血浆炎症因子水平显著降低,血浆NO水平显著升高,腹主动脉组织MDA水平降低。进一步研究显示,TFDG下调了糖尿病大鼠腹主动脉组织NLRP3、caspase-1和IL-1β的表达,抑制NLRP3炎症通路激活。TFDG能够有效保护糖尿病大鼠血管内皮损伤并抑制炎症反应,其机制可能是通过下调NLRP3炎症通路实现的。     

茶叶提取物对抗肺癌细胞(A549)体外试验研究

观察表没食子儿茶素没食子酸酯(EGCG)、茶黄素-3、3'-双没食子酸酯(TFDG)、茶黄素(TF)、茶黄素复合物(TFs)、葡萄籽提取物(GrapeSeedExtract,GSE)、松树皮提取物(PineBarkExtract,PBE)、咖啡因(Caf)、槲寄生(VCE)和茶氨酸(The)的体外抗癌活性,通过人肺癌细胞(A549)进行体外试验,结果表明除咖啡因、槲寄生、茶氨酸的作用较小以外其他几种均有很强的促进人肺癌细胞(A549)凋亡的作用,并且EGCG的作用最强,顺序为EGCG、TFs>GSE>PBE、TFDG>TF。用作图法求得EGCG、GSE、PBE、TFDG和TF的IC50值分别为1.01μM、21.91μM、31.01μM、32.87μM和279.67μM。     

Anti-proliferative Effect of <Emphasis Type="Italic">Melissa officinalis</Emphasis> on Human Colon Cancer Cell Line

Melissa officinalis L. (Lamiaceae) is consumed as a traditional herbal tea in the Mediterranean region. The cytotoxic effect of the 50% ethanolic and aqueous extract, determined by the MTT and NR assays, was evaluated in vitro on Human Colon Cancer Cell Line (HCT-116), using Triton 10% as positive control. The 50% ethanolic extract showed significant differences after 72 h of treatment, reducing cell proliferation to values close to 40%, even the lowest dose tested (5 μg/ml). In the MTT assay, the same extract caused the lowest cell viability with 13% at a concentration of 1,000 μg/ml after 72 h of treatment, being a value lower than Triton 10%. The antioxidant activity was also confirmed evaluating the capacity of the extracts to scavenge ABTS and DPPH radicals, and IC₅₀ values were highly correlated with the total phenolic and flavonoid content. Bioassay guided fractionation led to the isolation of an anti-proliferative compound, rosmarinic acid. Its structural elucidation was performed by HPLC/DAD/ESI/MS analysis. High dose of rosmarinic acid (1,000 μg/ml) was clearly cytotoxic against HCT-116 cells, with a significant decrease in cell number since the earliest time point (24 h).     

Three New Resveratrol Derivatives from the Mangrove Endophytic Fungus Alternaria sp.

Three new resveratrol derivatives, namely, resveratrodehydes A–C (1–3), were isolated from the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS, 1D and 2D NMR spectroscopic data. All compounds showed broad-spectrum inhibitory activities against three human cancer cell lines including human breast MDA-MB-435, human liver HepG2, and human colon HCT-116 by MTT assay (IC₅₀ < 50 μM). Among them, compounds 1 and 2 both exhibited marked cytotoxic activities against MDA-MB-435 and HCT-116 cell lines (IC₅₀ < 10 μM). Additionally, compounds 1 and 3 showed moderate antioxidant activity by DPPH radical scavenging assay.     

Anti-Proliferative Properties,Biocompatibility, and Chemical Composition of Different Extracts of Plantago major Medicinal Plant

Background:To study the anticancer activity of Plantago major, we assessed the effect of ethanolic, methanolic and acetonic extracts of this plant on HCT-116, SW-480, and HEK-293 cell lines as control. Methods:The cytotoxic activity, biocompatibility, and toxicity were evaluated by MTT assay, hemolysis, and Artemia salina-LD₅₀ (on mice) tests, respectively. The analysis of the extracts was performed by GC-MS analysis. Results:The results showed that all the extracts had the most antiproliferative properties on the HCT-116 cell line. The P. major root extract was more effective than the aerial parts, and IC₅₀ values for ethanolic, methanolic and acetonic root extracts were 405.59, 470.16, and 82.26 µg/mL, respectively on HCT-116 cell line at 72 h. Hemolysis degree of the ethanolic extract of aerial and root parts were approximately 1% at 400 μg/mL.. Using the ethanolic extracts, the Artemia survived every concentration, and no toxicity was observed. One week after the oral administration of different parts of P. major extracts, none of the mice died, even those were administered 2000 mg/kg. The results of GC/MS analysis showed that P. major extracts contain potential anticancer compounds, such as stearic acid (8.61%) in aerial parts of methanolic extract and 1,2- Benzenedicarboxylic acid, mono(2-ethylhexyl)ester (88.07% and 40.63%) in aerial and root parts of acetonic extract of P. major. Conclusions:Our findings suggest that the P. major is a source of potential compounds with antiproliferative properties. Key Words: Gas chromatography-mass spectrometry, HCT-116 cells, Hemolysis, Lethal dose 50     

New Dimeric Members of the Phomoxanthone Family: Phomolactonexanthones A,B and Deacetylphomoxanthone C Isolated from the Fungus Phomopsis sp.

Three new phomoxanthone compounds, phomolactonexanthones A (1), B (2) and deacetylphomoxanthone C (3), along with five known phomoxanthones, including dicerandrol A (4), dicerandrol B (5), dicerandrol (6), deacetylphomoxanthone B (7) and penexanthone A (8), were isolated in the metabolites of the fungus Phomopsis sp. HNY29-2B, which was isolated from the mangrove plants. The structures of compounds 1–3 were established on the basis of spectroscopic analysis. All compounds were evaluated against four human cancer cell lines including human breast MDA-MB-435, human colon HCT-116, human lung Calu-3 and human liver Huh7 by MTT assay. The compounds 4, 5, 7 and 8 showed cyctotoxic activities against tested cancer cell lines (IC₅₀ < 10 μM).     

4-Methylenesterols from a Sponge Theonella swinhoei

Three new 4-methylenesterols, theonellasterol K (1), acetyltheonellasterol (2) and acetyldehydroconicasterol (3), along with two known sterols, theonellasterol (4) and theonellasterone (5), were isolated from the sponge Theonella swinhoei. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. Compound 1 exhibited significant cytotoxic activity against HCT-116, K562 and Molt 4 cancer cell lines.     

根癌农杆菌农杆菌介导抗菌肽基因转化大豆的研究

为利用抗菌肽基因培育转基因大豆抗病材料,采用根癌农杆菌介导法将抗菌肽基因和绿色荧光蛋白基融合因转入大豆胚尖外植体。结果表明,60mg/L的卡那霉素可以对转化植株进行有效的筛选;在共培养时添加200µmol/L乙酰丁香酮有利于抗菌肽基因的转化;在生根培养基中不添加卡那霉素更有利于转化苗的成活。目的基因特异的PCR检测和绿色荧光蛋白表达分析,发现抗菌肽基因已转入大豆,并得到表达。     

Cytotoxic sesterterpenoids from a sponge Hippospongia sp

One new pentacyclic sesterterpene, hippospongide A (1), and one new scalarane sesterterpenoid, hippospongide B (2), along with six previously reported known scalarane-type sesterterpenes (3-8), were isolated from a sponge Hippospongia sp. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. These metabolites are the first pentacyclic sesterterpene and scalarane-type sesterterpenes to be reported from this genus. Compounds 3-5 exhibited significant cytotoxicity against DLD-1, HCT-116, T-47D and K562 cancer cell lines.