姜黄素对顺铂所致大鼠肾毒性的防护作用

目的研究姜黄素(CMN)对顺铂(CDDP)所致肾损害的防护作用,并探讨其可能机制。方法将42只大鼠按体重随机分6组,分别为对照组、CMN组、CDDP组、CMN(204、0和80 mg/kg) CDDP组。CMN连续给予大鼠灌胃3 d,第2天灌胃后1 h腹腔注射CDDP(5.5 mg/kg)。CDDP处理后,分别在第1、3和5天采血,测定血清尿素氮(BUN)和肌酐(CRE)。第5天采血后处死动物,测定肾脏系数、肾皮质匀浆丙二醛(MDA)、谷胱甘肽(GSH)含量和谷胱甘肽过氧化物酶(GSH-Px)活力以及肾组织铂(Pt)含量等。同时利用体外实验观察对抗增殖作用的影响。结果CMN预处理可减轻CDDP引起的肾脏系数升高及BUN、CRE水平升高;能抑制CDDP引起的MDA形成增高;并能提升CDDP引起的GSH含量和GSH-Px活力下降。CMN低剂量的上述作用明显(P<0.05或P<0.01)。CMN防护组与CDDP组的人卵巢癌细胞系和膀胱癌细胞系的半数抑制浓度差异无显著性。结论CMN经口给予能防护CDDP所致的肾损害,其机制可能与其抗氧化作用和清除自由基活性有密切关系。较高剂量CMN未见防护CDDP所致肾毒性的作用,其原因可能与其助氧化作用有关。CMN对CDDP抗肿瘤细胞增殖作用无明显影响。     

小鼠腹水型肝癌淋巴道高转移细胞系HCa-P/L6细胞模型的建立及其药物敏感性的评价

[目的]建立小鼠腹水型肝癌淋巴道高转移细胞系HCa-P/L6细胞模型并应用姜黄素对该模型的药物敏感性进行评价。[方法]将小鼠腹水型肝癌淋巴道低转移细胞株（HCa-P）经615小鼠腹中线皮下接种的方法获得淋巴结转移瘤1代细胞HCa-P/L1,再经小鼠淋巴系统筛选5次,筛选高转移细胞系HCa-P/L6。用15～240μmol·L^-1姜黄素（Curcumin,Cur）分别处理人肝癌细胞系（SMC7721和HepG2）和小鼠腹水型肝癌细胞（HCa-P/L6和HCa-P）48h,以MTT法考察4种细胞的药物敏感性。以非细胞毒性最大剂量Cur作用于HCa-P/L6和HCa-P,以生长曲线和群体倍增时间评估药物对细胞生长的影响,以流式细胞仪检 测药物对细胞周期分布的影响,考察两株小鼠腹水型肝癌细胞与姜黄素作用敏感性的差异。[结果]从HCa-P中筛选出具有多部位转移特性的淋巴道高转移细胞系HCa-P/L6,转移率为100%。Cur明显抑制小鼠腹水型肝癌和人肝癌细胞的增殖,并呈 剂量依赖性（方差分析P〈0.05）。在30～120μmol·L^-1,Cur对小鼠肝癌的抑制作用强于对人肝癌的抑制作用;在60～240μmol·L^-1范围内,Cur对HCa-P/L6的抑制作用强于对HCa-P。在非细胞毒性最大剂量15μmol·L^-1 Cur作用下,HCa-P/L6和HCa-P的生长受到抑制（t检验P〈0.05）,并且呈时间依赖性（方差分析P〈0.01）,HCa-P/L6和HCa-P的群体倍增时间延长（卡方检验P〈0.01）,Cur对HCa-P/L6的抑制作用大于对HCa-P;细胞周期被重新分布,HCa-P/L6被阻滞在S期,HCa-P被阻滞于S期和G2/M期。[结论]从HCa-P中筛选出了淋巴道高转移细胞系HCa-P/L6。小鼠腹水型肝癌比人肝癌对姜黄素作用敏感,而且高转移细胞株系HCa-P/L6较低转移细胞株HCa-P对Cur更为敏感。HCa-P/L6为肿瘤淋巴道转移机制和中药活性成分筛选的研究提供了一个新的敏感模型。     

姜黄素对2型糖尿病大鼠血糖的影响

目的:探讨姜黄素对糖尿病大鼠血糖的干预效应. 方法: 采用高脂高糖饲料诱导4周加链脲佐菌素注射建立糖尿病大鼠模型,姜黄素200mg/kg 进行干预给药6周,实验结束后,分别检测大鼠的血糖和尿量.结果:姜黄素对正常大鼠的血糖没有影响,但是能降低糖尿病大鼠的血糖;姜黄素治疗组实验前后比较血糖有明显下降(P<0.05), 且实验后姜黄素血糖与阳性对照组比较,差异有非常显著性意义(P<0.01).同样,姜黄素治疗组大鼠尿量与阳性对照组比较差异有显著性意义(P<0.01).结论:姜黄素能有效改善II型糖尿病的血糖水平和减少尿量,同时也为糖尿病的临床治疗提供了新的途径与思路.     

姜黄素抑制人肝癌细胞BEL-7402中VEGF和HIF-1α表达通过PI3K／AKT／FRAP信号传导通路

目的探讨MAPK和PI3K信号传导通路在姜黄素调节VEGF和HIF-1α表达中的作用。方法分别加入LY29400225μmol／L、50μmol／L，U012610μmol／L、20μmol／L，rapamycin 5ng／ml、10ng／ml处理人肝癌细胞BEL-7402，30min后加入姜黄素10μmol／L，对照组单独加入0、10μmol／L姜黄素，缺氧环境中培养6h后，行RT—PCR和Western Blot检测VEGF蛋白、mRNA和HIF-1α蛋白表达；分别加入不同浓度的的姜黄素以及LY294002 25 μmol／L处理人肝癌细胞BEL-7402，缺氧环境中培养6h后，行Western Blot检测磷酸化AKT和总AKT蛋白表达。结果姜黄素10μmol／L＋LY29400225μmol／L组、姜黄素10μmol／L＋LY294002 50 μmol／L组、姜黄素10μmol／L＋rapamycin 5 ng／ml组、姜黄素10μmol／L＋rapamycin 10 ng／na组HIF-1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比降低，差异有统计学意义（P〈0．01）；而姜黄素10μmol／L＋U012610μmol／L组、姜黄素10μmol／L＋U0126 20 μmol／L组HIF—1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比差异无统计学意义（P〉0.05）；不同浓度的姜黄素、LY294002 25 μmol／L处理的人肝癌细胞BEL-7402缺氧6h后磷酸化AKT蛋白表达逐渐降低，LY294002 25 μmol／L可以基本阻断磷酸化AKT蛋白的表达，而对总AKT蛋白表达无明显变化。结论姜黄素对人肝癌细胞BEL-7402中HIF-1α蛋白和VEGF的表达通过P13K／AKT／FRAP信号传导通路。     

Growth-inhibitory effects of curcumin on Raji cells and its mechanisms

Objective： To study the growth-inhibitory effects of curcumin on B-NHL cell line Raji cells in vitro and its molecular mechanisms. Methods： The growth inhibition rates of Raji cells, after being treated with 6.25μmol/L - 50μmol/L curcumin for 12 h - 48 h, were examined by MTT assay. The apoptosis rate was detected by flow cytometry （FCM）, the protein expression levels of bcl-2 and p53 in Raji cells were examined by SP immunohistochemistry. The expression of p53 in Raji cell were checked by RT-PCR. Results： After being treated by various concentrations of curcumin, the growth of Raji cells was inhibited significantly. The rates of apoptosis were 11.8% -79.7% （P〈0.01）, the down regulation of p53 expression was observed within 24 h after the treatment of curcumin by RT-PCR. The expression of bcl-2 and p53 was decreased, which depended on the action time. Conclusion： Curcumin could significantly inhibit the growth of Raji cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms.     

Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder

P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons

AIM: To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. METHODS: Primary cultured rat cortical neurons wereperformed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cellapoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Aψm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer.‘ Bcl-2family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected byWestern blot. RESULTS: Exposure of tBHP 100 μmol/L to neurons for 60 rain resulted in △ψm loss and cyto-chrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis.After removal of tBHP and then further treatment with curcumin (2.5-20 μmol/L) for 18 h, curcumin abrogated △ψm loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family.Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly.Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. CONCLUSION: Curcumin mayattenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mito-chondria from oxidative damage.     

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells

Purpose One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3.Materials and methods Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting.Results Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells.Conclusions Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.     

姜黄素与顺铂联合对人肺腺癌A549细胞增殖和凋亡作用

目的:研究姜黄素、顺铂和两者联合应用对人肺癌A549细胞增殖、凋亡和细胞周期的影响。方法:采用MTT法观察姜黄素和顺铂对人肺癌A549细胞增殖的影响;采用流式细胞仪检测姜黄素、顺铂和两者联合应用对人肺癌A549凋亡和细胞周期的影响。结果:①姜黄素及顺铂能抑制人肺腺癌A549细胞的增殖,并呈浓度及时问依赖性;姜黄素10,15,20μmol/L分别与顺铂1,2 mg/L联合24,48,72 h的抑制率显著高于单用顺铂、姜黄素组(均为P<0.05),两者呈现出相加的抗瘤效果。②姜黄素和顺铂均可在一定程度上诱导细胞凋亡,并且凋亡率与浓度呈正相关。两种药物联用后,诱导细胞凋亡作用显著增强。③姜黄素将细胞周期阻滞在G2/M期(P<0.01),顺铂将细胞周期阻滞在S期(P<0.01)。结论:姜黄素、顺铂均可抑制人肺癌A549细胞的增殖、诱导细胞的凋亡,在一定浓度范围内,呈量-效关系,而且两者联合应用具有相加或协同作用。     

姜黄素对内毒素休克兔急性肾损伤的影响

目的探讨姜黄素对内毒素休克兔急性肾损伤(acute kidney injury,AKI)的影响及其可能机制。方法健康雄性新西兰大白兔40只,2月龄,随机分为四组:对照组(C组)、姜黄素对照组(Cur组,姜黄素50 mg/kg)、内毒素休克组(L组,脂多糖5 mg/kg)和姜黄素预处理组(Cur L组,姜黄素50 mg/kg,30 min后予脂多糖5 mg/kg),每组10只。给予脂多糖后6 h测定血尿素氮(BUN)和肌酐(Cr)浓度并处死兔。观察肾组织病理变化并进行肾损伤评分。采用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性,硫代巴比妥酸法测定丙二醛(MDA)浓度,RT-PCR法检测肾组织Nrf2和HO-1 m RNA表达量,Western blot法检测肾组织中Nrf2总蛋白、Nrf2核蛋白及HO-1蛋白含量。结果 C组和Cur组肾单位结构未见明显异常。L组肾小球皱缩,肾小管上皮细胞肿胀、空泡形成或脱落,肾间质水肿,大量炎性细胞浸润。Cur L组病理改变较L组明显减轻。与C组比较,L组及Cur L组肾损伤评分、BUN、Cr及MDA浓度明显升高,SOD活性明显降低,肾组织Nrf2和HO-1m RNA表达量、Nrf2总蛋白、Nrf2核蛋白及HO-1蛋白含量明显升高(P0.05)。与L组比较,Cur L组肾损伤评分、BUN、Cr及MDA浓度明显降低,SOD活性明显升高,肾组织Nrf2和HO-1 m RNA表达量、Nrf2总蛋白、Nrf2核蛋白及HO-1蛋白含量明显升高(P0.05)。结论姜黄素可以减轻内毒素休克诱发兔的AKI,其机制可能与激活Nrf2/HO-1信号通路有关。