胡黄连苷Ⅱ对H2O2损伤L-02细胞的保护作用

目的:探讨胡黄连苷Ⅱ(picroside Ⅱ)对氧化应激损伤L-02细胞的保护作用.方法:H2O2损伤的L-02细胞作为氧化应激损伤模型,MTT法检测细胞增殖状况,膜联蛋白(Annexin-Ⅴ)和碘化丙啶(PI)染色流式细胞术(flow cytometry,FCM)检测细胞凋亡,罗丹明123染色FCM检测细胞线粒体膜电位(mitochondrial potential membrane,△Ψm),双氢罗丹明123染色FCM检测细胞内活性氧(reactive oxygen species,ROS)的含量.结果:0.6 mmol/L H2O2可诱导L-02细胞凋亡.细胞内ROS浓度增加,细胞线粒体跨膜电位明显下降.预先经过0.05,0.5,5 mmol/L浓度的胡黄连苷Ⅱ处理后,H2O2诱导的L-02细胞凋亡明显减少(30.8%±9.09%,10.2%±9.82%,8.2%±7.10%vs 42.8%±8.28%,均P＜0.01),同时明显减弱H2O2对细胞内ROS浓度和线粒体跨膜电位的影响.结论:胡黄连苷Ⅱ对氧化应激损伤L-02细胞具有保护作用,其机制可能与降低细胞内ROS含量,进而抑制△Ψm的降低有关.     

白藜芦醇对小细胞肺癌C-myc基因表达的调控研究

目的研究白藜芦醇对小细胞肺癌C-myc基因表达的影响,探讨其在小细胞肺癌中的可能抑癌机制。 方法以人小细胞肺癌H446细胞系为研究对象,采取Western blot法和Real-Time PCR法检测不同浓度白藜芦醇及白藜芦醇作用不同时间后C-myc的表达变化,分别以NAC和顺铂为干预进行验证。 结果Western blot和Real-time PCR检测结果显示白藜芦醇可调控人小细胞肺癌H446细胞C-myc的表达,并随着作用时间的延长和作用浓度的增加,C-myc的表达进一步下降,具有时间依赖和浓度依赖的特点,差异有统计学意义(P<0.05)。给予NAC干预后,可抑制白藜芦醇对C-myc蛋白表达的调控影响,呈现拮抗作用,差异均有统计学意义(P<0.05)。顺铂可抑制C-myc的表达,与白藜芦醇联用对C-myc调控具有协同作用,Western blot检测结果显示在胞浆蛋白中差异有统计学意义(P<0.05),但在胞核蛋白中,与白藜芦醇组相比,白藜芦醇与顺铂联用,差异无统计学意义(P>0.05)。Real-time PCR检测结果显示顺铂可使C-myc表达水平下调,与白藜芦醇联用具有协同作用,差异有统计学意义(P<0.05)。 结论白藜芦醇对小细胞肺癌H446细胞的C-myc表达具有明显的影响,间接说明白藜芦醇对小细胞肺癌中的抑癌作用可能与myc基因有关。     

肺康饮口服液对Ncl-H446细胞生长及凋亡的影响

目的通过观察肺康饮口服液对人小细胞肺癌Ncl-H446生长及凋亡的影响,探讨其体外的抗肿瘤效应。方法体外培养Ncl-H446细胞,以不同浓度的肺康饮口服液作用为实验组,并设对照组,CCK8法测细胞增殖抑制效应、透射电镜观察药物对细胞形态学的影响,流式细胞术检测Ncl-H446细胞凋亡。结果不同浓度肺康饮口服液作用Ncl-H446细胞24 h增殖抑制明显(P<0.05),其抑制效应具有剂量依赖的特点;电镜及流式显示Ncl-H446细胞凋亡。结论肺康饮口服液可抑制Ncl-H446细胞的生长且增殖抑制效应具有剂量依赖性,并可诱导肿瘤细胞凋亡。     

隐丹参酮对U937细胞的生长抑制作用及机制

目的探讨隐丹参酮对U937细胞的生长抑制作用及其作用机制。方法以不同浓度的隐丹参酮作用于体外培养的U937细胞,MTT法检测细胞生长抑制率,收集药物作用48 h后的细胞行AnnexinⅤ染色,并通过流式细胞术(FCM)检测细胞凋亡率和细胞周期,JC-1法检测细胞线粒体膜电位的变化,RT-PCR法检测细胞凋亡前后Bcl-2表达水平的变化。结果隐丹参酮可显著抑制细胞生长及诱导细胞发生凋亡,显示出明显的量—效与时—效关系。FCM检测结果表明,不同浓度的药物作用于48 h后,随药物浓度增加细胞凋亡率逐渐升高,亚G1期细胞逐渐增多,线粒体膜电位逐渐下降。RT-PCR结果表明,Bcl-2表达水平显著下降。结论隐丹参酮能抑制U937细胞的生长及诱导细胞发生凋亡,降低线粒体膜电位及Bcl-2水平可能是其重要作用机制之一。     

中药清毒栓对宫颈癌Hela细胞的抑制作用

目的探讨中药清毒栓诱导宫颈癌Hela细胞凋亡及增殖抑制作用。方法采用胆囊收缩素-8肽(CCK-8)法检测清毒栓对Hela细胞增殖的作用,线粒体膜电位法检测Hela细胞凋亡的变化。结果高剂量清毒栓在24 h时对Hela细胞增殖具有明显的抑制及诱导细胞凋亡的作用,分别与空白对照组及清毒栓中、低剂量组比较差异有统计学意义(P<0.05),清毒栓中剂量组与低剂量组比较无显著性差异(P>0.05)。结论中药清毒栓对宫颈癌Hela细胞抑制作用可能通过抑制肿瘤血管内皮细胞的增殖及降低线粒体膜电位诱导肿瘤细胞凋亡有关。     

美洲大蠊提取物诱导人肝癌细胞SMMC-7721凋亡及作用机制

目的 探讨美洲大蠊提取物对人肝癌细胞SMMC-7721增殖、凋亡的影响并探讨其相关机制.方法 取对数生长期状态良好的SMMC-7721细胞分为三组,观察组常规培养24h后加入美洲大蠊提取物,使终浓度分别为300、100、33.3 μg/mL,顺铂组加入顺铂20 μg/mL作用细胞48 h,对照组不做任何处理.采用MTT比色法检测细胞增殖抑制率;采用流式细胞仪分析细胞凋亡率;JC-1染色法检测细胞线粒体膜电位变化;分光光度法检测Caspase-3活性变化.结果 观察组和顺铂组细胞抑制率明显高于对照组,IC50为100 μg/mL,并呈现时间一剂量依赖关系(P＜0.05);观察组和顺铂组细胞凋亡率明显高于对照组,呈现剂量依赖性(P＜0.05).凋亡过程中线粒体膜电位下降,Caspase-3活性升高(P＜0.05).结论 美洲大蠊提取物具有抑制人肝癌细胞SMMC-7721增殖并诱导其凋亡作用,线粒体途径可能是其诱导肝癌细胞凋亡的主要机制之一.     

旋毛虫肌幼虫虫体蛋白对小细胞肺癌H446细胞凋亡的影响

目的 探讨旋毛虫肌幼虫虫体蛋白对小细胞肺癌H446细胞凋亡的影响及其作用的可能机制。方法 将H446细胞与0.2 mg/mL、0.4 mg/mL、0.6 mg/mL、0.8 mg/mL、1.0 mg/mL、1.2 mg/mL虫体蛋白分别培养并设为实验组,不处理的H446细胞设为对照组,采用噻唑兰(MTT)比色法检测旋毛虫肌幼虫虫体蛋白对H446细胞增殖活性的抑制作用;采用流式细胞术(FCM)检测旋毛虫肌幼虫虫体蛋白诱导H446细胞株凋亡的情况;采用Real-timePCR及Western blot法检测细胞色素C(cytochrome C,Cyt-C)和凋亡蛋白酶活化因子(apoptotic protease activating factor 1,Apaf-1) mRNA及蛋白的表达。结果 MTT比色法结果表明虫体蛋白能够抑制H446细胞增殖活性;流式细胞术结果表明虫体蛋白作用于H446细胞24 h后,有明显的促凋亡作用;Real-timePCR及Western blot法结果显示,与阴性对照组相比,促凋亡基因Cyt-C和Apaf-1表达均上调。结论 旋毛虫肌幼虫虫体蛋白能够抑制H446细胞增殖活性,并诱导细胞凋亡,其作用可能与Cyt-C和Apaf-1表达上调有关。     

葛根粗提物、葛根素对肺癌H446细胞增殖的抑制作用及其机制

目的 观察葛根粗提物(CP)和葛根素纯品(SP)对小细胞肺癌H446细胞增殖的影响,并探讨其机制.方法 用含不同浓度的SP和CP培养H446细胞,用MTT法检测细胞生长抑制率.用流式细胞术检测细胞周期和凋亡率、细胞免疫化学技术检测Bel-2和Bax蛋白.另设空白对照组和溶剂对照组.结果 CP作用72 h的半数抑制浓度(ICSO)为435μg/ml,SP为1 403μg/ml.此浓度作用72 h,细胞凋亡率分别为14.71%和2.61%,两组相比P<0.05;G0/G1期细胞构成比分别为79.20%和72.20%,G2/M期细胞构成比分别为8.94%和10.50%,与对照组相比P均<0.05;CP作用于H446细胞后,细胞内Bel-2蛋白表达量降低,Bax的蛋白表达量增加,与对照组相比P均<0.05.结论 SP和CP对小细胞肺癌H446细胞具有增殖抑制作用,呈浓度相关性;SP和CP可诱导细胞凋亡且CP强于SP,其机制可能与阻滞细胞周期于G0/G1期、上调Bax表达、下调Bel-2表达有关.     

拓扑替康诱导肺癌细胞HTB-56凋亡早期线粒体膜电位的变化

目的探讨拓扑替康诱导肺癌细胞HTB-56凋亡早期线粒体膜电位的改变。方法选择人肺癌细胞HTB-56细胞株,JC-1荧光染色标记线粒体,用早期流式细胞仪检测拓扑替康作用于HTB．56细胞不同时间细胞线粒体膜电位。采用琼脂糖凝胶电泳检测拓扑替康作用不同时间引起HTB-56细胞凋亡情况。结果经拓扑替康处理后48h、72hHTB-56细胞出现了明显的凋亡带,对照组未见明显亮带影。4h后细胞线粒体膜电位下降,8h后细胞膜电位明显下降,且下降程度随时间延长而增大。且药物组绿／红荧光比值与对照组比较均有显著性差异（P〈0．05）。结论拓扑替康诱导肺癌细胞HTB-56可使之凋亡,其机制可能与线粒体膜电位在凋亡极早期发生改变有关。     

黄芩苷诱导人肝癌HepG-2细胞凋亡作用观察

目的研究黄芩苷对人肝癌HepG-2细胞凋亡的影响,并探讨其作用机制。方法以不同浓度的黄芩苷与人肝癌HepG-2细胞共同培养,采用MTT法测定细胞增殖抑制率;采用Hoechst33258/PI染色法在荧光显微镜下观察凋亡细胞形态学变化;采用TUNEL法检测细胞凋亡率;采用Western blot法检测细胞凋亡相关蛋白Caspase-9、Caspase-3和Bcl-2表达的变化。结果在25~100μg/ml的药物浓度作用下,细胞增殖被抑制。药物浓度在50μg/ml下作用48h,细胞抑制率达50.63%,药物浓度在75μg/ml下作用48h细胞,抑制率达77.62%。抑制率呈浓度和时间依赖性;药物浓度在50μg/ml时作用48h,可见HepG-2细胞皱缩、细胞核碎裂成碎片,呈现典型的凋亡改变;随着黄芩苷作用浓度的增加,细胞凋亡率增高(P<0.05);随药物浓度的增加,Caspase-9和Caspase-3蛋白表达量呈增加趋势,而Bcl-2表达减少。结论黄芩苷能通过诱导细胞凋亡进而抑制人肝癌细胞增殖,其诱导凋亡机制可能与线粒体通路有关。     

白藜芦醇抑制缺氧复氧诱导心肌微血管内皮细胞凋亡的研究

目的探讨白藜芦醇对缺氧复氧诱导心肌微血管内皮细胞(CMEC)凋亡的抑制作用及其可能的分子机制。方法体外培养大鼠CMEC,建立缺氧复氧损伤模型,随机分为对照1组、缺氧复氧1组、缺氧复氧+白藜芦醇组(白藜芦醇1组),白藜芦醇分别选择5、10、20、30及40μmol/L浓度,MTT法检测CMEC增殖能力。20μmol/L白藜芦醇为最适浓度,使用磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002,后续实验又分为对照2组、缺氧复氧2组、缺氧复氧+白藜芦醇2组(白藜芦醇2组)、缺氧复氧+白藜芦醇+LY294002组(LY294002组)。PI-AnnexinⅤ检测CMEC的凋亡;Western blot法检测细胞蛋白激酶B(Akt)、磷酸化Akt、缺氧诱导因子1α(HIF-1α)蛋白表达或磷酸化水平。结果与缺氧复氧1组比较,不同浓度白藜芦醇1组均可增加CMEC增殖能力,呈剂量依赖性,以白藜芦醇1组20μmol/L保护作用最显著(P<0.05)。与白藜芦醇2组比较,LY294002组CMEC凋亡率明显升高,磷酸化Akt和Akt蛋白、HIF-1α蛋白表达明显下降(P<0.05)。结论白藜芦醇可显著抑制缺氧复氧诱导的CMEC凋亡,其机制可能与PI3K/Akt介导的HIF-1α上调相关。     

Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner

Resveratrol, a phytoalexin, reduced the viability of MH7A cells, a human rheumatoid arthritis synovial cell line. In the apoptosis assay, resveratrol increased TUNEL-positive cells and stimulated H2A.X phosphorylation. Resveratrol disrupted mitochondrial membrane potentials in MH7A cells and stimulated cytochrome c release from the mitochondria to the cytosol. Resveratrol activated caspase-3 and caspase-9 but not caspase-8 in MH7A cells. Resveratrol upregulated the expression of the NAD-dependent deacetylase sirtuin 1 mRNA and downregulated the expression of the Bcl-X_L mRNA, and resveratrol-induced MH7A cell death, mitochondrial damage, and caspase-3/-9 activation were prevented by sirtinol, an inhibitor of sirtuin 1. The results of the present study show that resveratrol induces MH7A cell apoptosis by activating caspase-9 and the effector caspase-3 along mitochondrial disruption as a result of reduced Bcl-X_L expression, allowing cytochrome c release from the mitochondria into the cytosol, in a sirtuin 1-dependent manner. This suggests that resveratrol could suppress hyperplasia of synovial cells, a critical factor of rheumatoid arthritis.     

Antitumor and immunomodulatory activity of resveratrol onexperimentally implanted tumor of H22 in Balb／c mice

AIM: To study the antitumor and immunomodulatory activity of resveratrol on experimentally implanted tumor of H22 in Balb/c miceMETHODS: The cytotoxicity of peritoneal macrophages (M φ ) against H22 cells was measured by the radioactivity of [3H]TdR assay, mice with H22 tumor were injected with different concentrations of resveratrol, and the inhibitory rates were calculated and IgG contents were determined by single immunodiffusion method. the plaque forming cell (PFC) was measured by improved Cunningham method, the levels of serum tumor necrosis factor-α (TNF-α) were measured by cytotoxic assay against L929 cells.implanted tumor of H22 in mice. The inhibitory rates were 31.5 %, 45.6 % and 48.7 %, respectively (P＜0.05), which could raise the level of serum IgG and PFC response to production of serum TNF-α in mice H22 tumor. However,the effect of resveratrol was insignificant (P ＞0.05).CONCLUSION: Resveratrol could inhibit the growth of H22tumor in Balb/c mice. The antitumor effect of resveratrol might be related to directly inhibiting the growth of H22cells and indirectly inhibiting its potential effect on nonspecific host immunomodulatory activity.     

The antioxidant resveratrol protects against chondrocyte apoptosis via effects on mitochondrial polarization and ATP production

OBJECTIVE: To determine the effects of the antioxidant resveratrol on the functions of human chondrocytes in osteoarthritis (OA). METHODS: Chondrocytes and cartilage explants were isolated from OA patients undergoing knee replacement surgery. Effects of resveratrol in the presence or absence of interleukin-1beta (IL-1beta) stimulation were assessed by measurement of prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) synthesis, cyclooxygenase (COX) activity, matrix metalloproteinase (MMP) expression, and proteoglycan production. To explore the mechanisms of action of resveratrol, its effects on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, cytochrome c release, and annexin V staining. RESULTS: Resveratrol inhibited both spontaneous and IL-1beta-induced PGE(2) production by >20% (P < 0.05) and by 80% (P < 0.001), respectively; similarly, LTB(4) production was reduced by >50% (P < 0.05). The production of PGE(2) was inhibited via a 70-90% suppression of COX-2 expression and enzyme activity (P < 0.05). Resveratrol also promoted anabolic effects in OA explant cultures, by elevating proteoglycan synthesis and decreasing production of MMPs 1, 3, and 13. Pretreatment of OA chondrocytes with resveratrol blocked mitochondrial membrane depolarization, loss of mitochondrial biomass, and IL-1beta-induced ATP depletion. Similarly, IL-1beta-mediated induction of the apoptotic markers cytochrome c and annexin V was also inhibited by resveratrol. Exogenous addition of PGE(2) abolished the protective effects of resveratrol on mitochondrial membrane integrity, ATP levels, expression of apoptotic markers, and DNA fragmentation. CONCLUSION: Resveratrol protects against IL-1beta-induced catabolic effects and prevents chondrocyte apoptosis via its inhibition of mitochondrial membrane depolarization and ATP depletion. These beneficial effects of resveratrol are due, in part, to its capacity to inhibit COX-2-derived PGE(2) synthesis. Resveratrol may therefore protect against oxidant injury and apoptosis, which are main features of progressive OA.     

白藜芦醇对白血病K562细胞增殖、凋亡的影响及机制探讨

目的 探讨白藜芦醇对白血病K562细胞生长的影响及相关作用机制.方法 用不同浓度白藜芦醇作用于K562细胞,CCK-8法观察白藜芦醇对K562细胞生长增殖的影响,AnnexinV-FITC/PI双染法观察白藜芦醇对K562细胞凋亡的影响,PI染色法观察白藜芦醇对K562细胞周期的影响,Western blot法检测K562细胞中的Bcl-2、Bcl-xl、Bax、Cyclin D1蛋白.结果 白藜芦醇作用后的K562细胞的增殖被抑制并且凋亡增加,呈时间-剂量依赖性;同时,凋亡相关分子Bcl-2、Bcl-xl表达下调,Bax表达上调;白藜芦醇作用K562细胞后,G1期细胞增多,S期细胞减少,细胞周期调节蛋白Cyclin D1表达下降.结论 白藜芦醇可抑制K562细胞增殖并诱导其凋亡,其机制可能与下调细胞内Bcl-2、Bcl-xl、Cyclin D1表达并上调Bax的表达有关.     

Anti-hepatoma activity of resveratrol in vitro

AIM: To study the anti-tumor effect of resveratrol alone and the synergistic effects of resveratrol with 5-FU on the growth of H22 cells line in vitro. METHODS: The number of cells was measured by MTT method the morphological changes of H22 cells were investigated under microscopy and electron microscopy examination. RESULTS: Resveratrol inhibited the growth of hepatoma cells line H22 in a dose- and time-dependent manner, IC50 of the resveratrol on H22 cells was 6.57mg x L(-1),The synergistic anti-tumor effects of resveratrol with 5-FU increased to a greater extent than for H22 cells treated with 5-FU alone (70.2% vs 28.4%) P<0.05 .Under microscope and electron microscope, characteristics of apoptosis such as typical apoptotic bodies were commonly found in tumor cells in the drug-treated groups. CONCLUSION: Resveratrol can suppresses the growth of H22 cells in vitro,its anti-tumor activity may occur through the induction of apoptosis.     

Melatonin potentiates chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor cells

Abstract: Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant action. Thus, melatonin has proven useful in the treatment of tumors in association with chemotherapeutic drugs. This study was performed to evaluate the effect of melatonin on the cytotoxicity and apoptosis induced by three different chemotherapeutic agents, namely 5‐fluorouracil (5‐FU), cisplatin, and doxorubicin in the rat pancreatic tumor cell line AR42J. We found that both melatonin and the three chemotherapeutic drugs induce a time‐dependent decrease in AR42J cell viability, reaching the highest cytotoxic effect after 48 hr of incubation. Furthermore, melatonin significantly augmented the cytotoxicity of the chemotherapeutic agents. Consistently, cotreatment of AR42J cells with each of the chemotherapeutic agents in the presence of melatonin increased the population of apoptotic cells, elevated mitochondrial membrane depolarization, and augmented intracellular reactive oxygen species (ROS) production compared to treatment with each chemotherapeutic agent alone. These results provide evidence that in vitro melatonin enhances chemotherapy‐induced cytotoxicity and apoptosis in rat pancreatic tumor AR42J cells and, therefore, melatonin may be potentially applied to pancreatic tumor treatment as a powerful synergistic agent in combination with chemotherapeutic drugs.     

人小细胞肺癌细胞H446及其MDR细胞H446/DDP受顺铂冲击后乳酸代谢与活性氧产生情况

目的通过检测人小细胞肺癌(SCLC)细胞H446及其获得性多药耐药(MDR)细胞H446/DDP受顺铂(DDP)冲击后乳酸代谢与活性氧(ROS)水平的变化,了解SCLC获得性MDR细胞的分子生物学变化,为逆转耐药提供理论和实验依据。方法体外常规培养SCLC细胞H446及H446/DDP细胞,经相同浓度(0.5μg/ml)、不同时间点的顺铂刺激后,分别应用干化学方法和激光共聚焦显微镜法检测乳酸浓度和ROS水平。结果顺铂刺激24h内,H446细胞和H446/DDP细胞乳酸浓度没有差异,但随着时间的延长,H446细胞乳酸产量显著性高于H446/DDP细胞。此外,顺铂刺激40min内,H446/DDP细胞ROS产量呈衰减趋势,而H446细胞ROS产量随着时间的延长而增加,15min时达到峰值,随后逐渐降低,并伴随细胞皱缩这一现象。结论较强的抗氧化损伤能力可能是SCLC获得性MDR抵御顺铂损伤的重要因素。