正交试验优选血三七总黄酮的提取工艺研究

目的优选血三七Polygonum amplexicaule var.sinense中总黄酮的提取工艺。方法以乙醇体积分数、提取时间、乙醇倍数、提取次数为因素,以总黄酮提取率和浸膏得率为指标,采用正交试验优选提取工艺。结果最佳条件为用10倍70%乙醇,提取3次,每次1.5 h。结论优选的工艺稳定、合理,可用于血三七中总黄酮的提取。     

杠板归总黄酮抗大鼠肝纤维化作用的机制研究

目的在明确杠板归总黄酮(TFP)抗二甲基亚硝胺(DMN)诱导的大鼠肝纤维化(HF)药效基础上,探索TFP抗HF的作用机制。方法将90只SD大鼠随机均分为正常组、模型组、秋水仙碱(0.1 mg·kg~(-1))组和TFP(200、100、50mg·kg~(-1))组。除正常组外,其余各组大鼠均腹腔注射体积分数为0.5%的DMN溶液(2 m L·kg~(-1)),隔天1次,连续8周。从造模d 1起,各给药组分别给予相应剂量的药物进行干预,正常组和模型组给予等体积的溶媒,每天1次。8周后实验结束,取血和肝组织。取相同位置的肝组织,苏木精-伊红(HE)染色观察肝组织病变程度;比色法检测血清ALT、AST、SOD和MDA含量或活性;酶联免疫吸附实验(ELISA)检测血清HA、LN、PCⅢ和Ⅳ-C含量;ELISA法检测肝组织TNF-α、IL~(-1)β和IL-6的含量;蛋白免疫印迹法(Western blot)检测肝组织α-SMA、TGF-β1、JAK2、STAT3、pJAK2和p-STAT3蛋白表达。结果与模型组比较,TFP(200、100、50 mg·kg~(-1))能明显改善肝组织病变,降低ALT、AST、HA、LN、PCⅢ和Ⅳ-C表达,降低MDA水平,增加SOD活性,同时减少TNF-α、IL-1β和IL-6释放,抑制α-SMA、TGF-β1、p-JAK2和p-STAT3表达。结论 TFP能够抗DMN诱导的大鼠HF,其机制可能与抗氧化作用,抑制TGF-β1表达,调控JAK2/STAT3信号通路,并抑制炎症反应有关。     

海蓬子皂苷甲调控SHP/STAT信号通路抗肝癌的研究

目的研究海蓬子皂苷甲(bigelovii A,BA)诱导细胞凋亡的作用及其机制。方法使用MTT实验检测BA对肿瘤细胞增殖的抑制作用;流式细胞仪分析BA对细胞凋亡的影响;Western blot分析p-STAT3、STAT3、p-STAT5、STAT5、SHP-1、SHP-2的表达。结果20和40μmol·L^-1 BA可诱导HepG2细胞发生凋亡,具有剂量依赖性。10、20和40μmol·L^-1 BA可抑制IL6诱导的STAT3和STAT5磷酸化,但酪氨酸磷酸酶抑制剂过钒酸钠可逆转该抑制作用,且20μmol·L^-1 BA能激活蛋白酪氨酸磷酸酶SHP-1和SHP-2。结论BA可能通过SHP-1/SHP-2和STAT3/STAT5通路诱导HepG2细胞凋亡。     

杠板归总黄酮抗肝纤维化大鼠的作用及其机制研究

目的研究杠板归总黄酮对二甲基亚硝胺(DMN)诱导的肝纤维化大鼠的拮抗作用。方法将120只SD大鼠随机均分成正常组、模型组、秋水仙碱组和杠板归总黄酮高、中、低剂量(0.15、0.1、0.5 g·kg-1)组。除正常组外,各组大鼠均ip0.5%DMN溶液2.0 m L·kg-1,隔天注射1次,造模成功后,各给药组均ig给药,qd,连续8周,正常组与模型组ig等量生理盐水。于8周末取血和肝脏,酶联免疫吸附法检测血清透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PCⅢ)和转化生长因子β1(TGF-β1);HE染色观察肝纤维化(HF)形成;Western blot法检测肝组织中TGF-β1的表达。结果与模型组比较,杠板归总黄酮各剂量均可有效降低HF大鼠血清中HA、LN、PCⅢ和TGF-B1的水平;杠板归总黄酮可明显降低HF大鼠肝脏中TGF-β1的表达。结论杠板归总黄酮对DMN诱导的大鼠HF具有良好的拮抗作用,可能通过抗肝损伤和降低TGF-β1的表达来实现。     

氟尿嘧啶通过激活促凋亡信号通路介导人肝癌细胞凋亡的治疗作用

目的研究氟尿嘧啶(5-Fu)通过激活促凋亡信号通路介导人肝癌细胞(HepG-2)凋亡的治疗作用。方法体外培养HepG-2细胞,分别在0(空白对照组)、40、80、160μM浓度5-Fu干预72 h,采用四甲基偶氮唑盐(MTT)法测定5-Fu对HepG-2的抑制作用,DAPI染色检查细胞形态学的改变,Western blot法测定内源性Fas蛋白以及cleaved-caspase-8表达水平。结果与空白对照组比较,5-Fu干预组明显抑制HepG-2的细胞增殖(P<0.01),诱导细胞凋亡。此外,5-Fu可有效上调HepG-2内源性Fas蛋白和cleaved-caspase-8表达水平(P<0.01)。结论氟尿嘧啶介导的抗肝癌作用是通过抑制HepG-2细胞增殖和激活内源性Fas信号通路而诱导癌细胞凋亡。     

肝细胞肝癌中DNMT3 b、STAT3的表达及其相关性

目的 探讨DNA甲基转移酶3b(DNMT3b)、信号转导子与转录激活子3(STAT3)在肝细胞肝癌(HCC)中的表达及其意义.方法 用免疫组化SP法检测48例HCC组织,25例癌旁组织和15例正常肝组织中DNMT3b蛋白和STAT3蛋白的表达,并分析其相关性.结果 DNMT3t在HCC组织中的阳性表达率为81.3%,明显高于正常肝组织(6.7%)以及癌旁组织(44.0%)(P<0.05).STAT3在HCC组织中的阳性表达率为72.9%,显著高于癌旁组织(48.0%)和正常肝组织(20.0%)(P<0.05).在HCC中,DNMT3b和STAT3的表达呈正相关(P<0.05).结论 DNMT3b及STAT3的高表达与HCC的发生发展有关,DNMT3b可能作为转录调控因子与STAT3起协同作用.     

GPC3特异的siRNA筛选和对肝癌细胞增殖能力的影响

目的筛选特异靶向GPC3的siRNA和探讨GPC3在肝癌中的作用机制。方法设计靶向GPC3的siRNA,转染huh7细胞后荧光定量PCR检测其对GPC3表达的抑制效果;选用抑制效果明显的siRNA下调GPC3表达后,WesternBlot检测GPC3的蛋白表达水平,并用Edu检测方法对Huh7细胞的增殖能力进行检测。结果成功筛选出一条抑制效率达到74.87%的特异靶向GPC3的siRNA,其可以显著抑制GPC3的转录翻译;同时,Edu检测发现当GPC3表达下调后,Huh7细胞的增殖能力下降。结论 GPC3的表达有利于HCC的增殖,而有效靶向GPC3的siRNA可为更进一步的研究提供有利手段。     

白藜芦醇调节STAT3与miR-21表达抗肝癌作用的初步研究

目的初步探讨白藜芦醇(Resveratrol,Res)的抗肝癌作用及其分子机制。方法建立移植性肝癌小鼠模型,口服给予Res进行干预,分析肿瘤生长状况。然后采用Real-time PCR、RT-PCR等方法检测转录因子Foxp3及微小核糖核酸-21(miR-21)的基因表达,Western blot分析信号转导子与转录激活子3(STAT3)的活性表达,流式细胞术观察CD4+CD25+T细胞数量的变化。结果与荷瘤对照组相比,Res给药组CD4+CD25+T细胞数量和Foxp3表达均明显降低,同时miR-21表达减少,该效应或许与其下调STAT3磷酸化活性水平密切相关。结论 Res可能通过调控STAT3信号分子及miR-21表达,抑制调节性T细胞产生,改变肿瘤微环境,从而发挥抗肝癌作用。     

藤茶总黄酮体外抗肿瘤实验研究

目的观察藤茶总黄酮在体外对肿瘤的影响作用。方法应用四甲基偶氮唑蓝法研究藤茶总黄酮对肿瘤细胞体外增殖的影响。结果藤茶总黄酮对人乳腺癌细胞MCF-7、人前列腺癌细胞PC3和人前列腺癌细胞LNcap的抑制作用都比较强,且剂量依赖性比较明显,随着药物浓度的增大抑制率也随之增大。72 h半数抑制浓度IC50分别为19.15、163.98、27.54μg/mL。结论藤茶总黄酮在体外对人乳腺癌和人前列腺癌有明显的抑制作用。     

Chemical constituents from the rhizome of Polygonum paleaceum and their antifungal activity

A new compounds neopaleaceolactoside (1), along with nine known compounds phyllocoumarin (2), quercetin (3), quercitrin (4), quercetin-3-methyl ether (5), vincetoxicoside B (6), isoquercitrin (7), kaempferol (8), (-)-epicatechin (9), and chlorogenic acid (10), was isolated from Polygonum paleaceum Wall. Their chemical structures were established based on one-dimensional and two-dimensional nuclear magnetic resonance techniques, mass spectrometry and by comparison with spectroscopic data reported. Some selected compounds were screened for their antifungal activity. Quercetin (3), vincetoxicoside B (6), kaempferol (8), and (-)-epicatechin (9) showed synergistic antifungal activities with the FICI values <0.5. A preliminary structure-activity relationship could be observed that free 3-OH in the structure of flavonoids was important for synergistic antifungal activity.     

古抑菌素对人肝癌细胞作用的研究

目的:通过组蛋白去乙酰化酶(histone deacetylase,HDACS)抑制剂古抑菌素A(Trichostatin A,TSA)对人肝癌细胞HepG2(以下简称HepG2)和正常肝细胞LO2(以下简称LO2)增殖与凋亡作用的比较,探讨TSA对肝癌的作用机制.方法:应用光学显微镜、透射电镜、四氮唑蓝(MTT)法、免疫细胞化学法观察经不同浓度TSA处理后的HepG2和LO2的增殖、凋亡与凋亡相关蛋白的改变.结果:HepG2细胞经TSA处理后,电镜下超微结构发生了凋亡早期改变;小剂量TSA对HepG2细胞具有较强的抑制作用,对LO2细胞影响不明显,当TSA浓度达到1000 nmol/L以上时,对LO2表现出明显的细胞毒性作用;TSA可诱导HeptG2细胞凋亡,并增加凋亡相关蛋白Bax的表达.结论:TSA可明显抑制肝癌细胞HepG2的增殖,其机制可能与上调Bax的表达,诱导细胞凋亡有关.     

IM-94对肝癌细胞的杀伤作用的实验研究

目的 探讨IM-94对人肝癌细胞的杀伤作用。方法 采用MTT法分析IM-94对人肝癌细胞772l生长的影响,用流式细胞术检测细胞有丝分裂周期及细胞凋亡,用H．E．染色分析细胞的核分裂相。结果 IM-94对肝癌细胞有明显的抑制作用,从流式细胞术分析细胞阻滞在G2 M期,并诱发细胞凋亡,在24h后,出现凋亡小峰。结论 IM-94能阻滞细胞分裂,诱导肝癌细胞凋亡。     

苦荞麦总黄酮对软脂酸损伤脐静脉内皮细胞的保护作用

目的研究苦荞麦总黄酮对软脂酸损伤脐静脉内皮细胞的保护作用。方法体外培养人脐静脉内皮细胞EA.hy926,将细胞培养液分为正常对照组、模型组、苦荞麦总黄酮低浓度组、苦荞麦总黄酮中浓度组、苦荞麦总黄酮高浓度组和二甲双胍组。用软脂酸建立胰岛素抵抗状态下的血管内皮细胞模型,MTT法检测细胞增殖率,LDH试剂盒检测细胞LDH的漏出量,流式细胞技术检测细胞的凋亡率。结果与正常组比较,模型组细胞增殖率明显降低(P<0.01),LDH漏出量显著增高(P<0.01),凋亡率明显增高(P<0.01)。同模型组相比,苦荞麦总黄酮低、中、高浓度组,以及二甲双胍组细胞增殖率明显增高(P<0.01),凋亡率显著降低(P<0.01),且苦荞麦总黄酮各浓度组表现出明显的剂量依赖关系;苦荞麦中、高浓度组,以及二甲双胍组LDH漏出量明显减少(P<0.01)。结论苦荞麦总黄酮对软脂酸损伤的脐静脉内皮细胞具有明显的保护作用。     

Inhibitory activity of flavonoids from Ephedrae Herba on hypoxia signaling in PANC-1 cells and the evaluation of their mechanisms

Journal of Natural Medicines - Pancreatic cancer is a lethal disease with a very poor prognosis. Recent reports indicate that hypoxia signaling mediated by hypoxia-inducible factor (HIF)...     

酶解-吸附澄清法提取水辣蓼总黄酮的工艺优化

目的 优化酶解-吸附澄清法提取水辣蓼总黄酮的工艺.方法 采用响应面法,构建多元二次回归数学模型,筛选酶法提取和絮凝澄清的最佳工艺条件.结果 最佳酶法提取工艺为料液比1∶50,pH6.06,复合酶用量0.24％,酶解温度50℃,酶解时间1.62 h;最佳澄清工艺为酶解液的浓缩比1∶5,药液pH5.24,膨润土用量0.45 mL·g-1,43.43℃下絮凝40 min.结论 酶法提取条件温和,膨润土絮凝能有效降低酶解液中总固形物的含量,优于传统醇沉法.     

Cytotoxic effects and pro-apoptotic mechanism of TBIDOM, a novel dehydroabietylamine derivative, on human hepatocellular carcinoma SMMC-7721 cells

We have investigated the antiproliferative effects of TBIDOM (N-(4-(2,2,2-trifluoroethyl) benzylidene) (7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl) meth-anamine) and have explored its possible mechanisms on human hepatocellular carcinoma SMMC-7721 cells. The proliferative status of cells treated with TBIDOM was measured by the colorimetric MTT assay. Cellular apoptosis was analysed using Hoechst 33342 staining and flow cytometry. Reduction of mitochondrial membrane potential (Delta psi(m)) was also detected by flow cytometry. Western blotting assay was used to evaluate the release of cytochrome c and expression of p53, Bcl-2 and Bax proteins. It was shown that TBIDOM displayed a significant inhibitory effect on growth of SMMC-7721 cells in a dose- and time-dependent manner. Hoechst 33342 staining and flow cytometry analysis showed an increase of apoptosis rate and decrease of mitochondrial membrane potential after SMMC-7721 cells were exposed to TBIDOM for 24 h. Pretreatment of SMMC-7721 cells with TBIDOM significantly induced a decrease of Bcl-2 protein expression and an increase of caspase-3 activity and Bax protein expression. The results indicated that TBIDOM could effectively inhibit proliferation by induction of apoptosis and could be a promising candidate in the development of a novel class of antitumour agent.     

A tumour‐promoting role of Th9 cells in hepatocellular carcinoma through CCL20 and STAT3 pathways

Hepatocellular carcinoma (HCC ) is a common and aggressive human malignancy. An imperative demand is present for a better understanding of the functions and regulations of the immune system and how they affect HCC pathogenesis. Recently, a group of interleukin 9 (IL ‐9)‐producing CD 4⁺ T cells, termed Th9 cells, has been described in mice and humans with both tumour‐inhibiting as well as tumour‐promoting effects. The specific roles of Th9 cells in human HCC are not entirely understood. Here, we examined the frequencies and functions of IL ‐9‐producing Th9 cells in HCC patients. We found that the frequencies of circulating IL ‐9‐producing Th9 cells were significantly higher in HCC patients compared to in healthy individuals. In HCC patients, the frequencies of IL ‐9‐producing Th9 cells were significantly higher in peritumour and tumour tissues than in unaffected liver tissues. Interestingly, HCC patients with higher tumour‐infiltrating Th9 frequency had significantly shorter disease‐free survival period after resection. Previously, high expression of CCL 20 was associated with poor prognosis in HCC . CCL 20 also induced epithelial‐mesenchymal transition‐like changes in HCC cells. We found that incubation of primary HCC cells with autologous Th9 significantly elevated the CCL 20 production from tumour cells, which could be partially inhibited by suppressing STAT 3. Together, this study suggested a tumour‐promoting role of Th9 cells in HCC .