Glycoprotein isolated from Solanum nigrum L. modulates the apoptotic-related signals in 12-O-tetradecanoylphorbol 13-acetate-stimulated MCF-7 cells

Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We isolated only the SNL glycoprotein from SNL and found that it was cytotoxic at low concentration. With respect to cytotoxicity, we investigated whether purified SNL glycoprotein is able to regulate protein kinase C (PKC) alpha activation and nuclear factor (NF)- kappaB activities in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced tumor promotion, and whether it has an apoptosis-inducing effect in MCF-7 cells using western blot analysis. In addition, to elucidate the relationship between PKCalpha and NF-kappaB, inhibitory studies were performed with staurosporine (an inhibitor of phospholipid/calcium-dependent protein kinase) and pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB activation). To verify induction of apoptosis by the SNL glycoprotein, we performed DNA fragmentation and nuclear staining assays using ethidium bromide and bisbenzamide H33342. The results in this study indicated that SNL glycoprotein induces apoptosis through modulation of PKCalpha and NF-kappaB activity in MCF-7 cells. In fact, SNL glycoprotein interfered with PKCalpha membrane translocation and inhibited NF-kappaB (p50) protein activity in MCF-7 cells stimulated with TPA (61.68 ng/mL, 100 nM) dose-dependently. Regarding the apoptotic-inducing effect, nucleosomal DNA fragmentation and nuclear staining by SNL glycoprotein in MCF-7 cells were shown. Collectively, the data demonstrate that SNL glycoprotein is a potential natural anticancer agent because of its ability to induce apoptosis in MCF-7 cells.     

神经酰胺诱导人结肠癌HT-29细胞凋亡的研究

目的探讨外源性神经酰胺诱导人结肠癌HT-29细胞的凋亡作用及对Bcl-2家族蛋白基因表达的影响。方法不同浓度C2-神经酰胺处理HT-29细胞,采用Hoechest荧光染色和琼脂糖凝胶电泳方法检测细胞凋亡,RT-PCR方法检测Bcl-2、Bcl-xl、Bax、Bad和Bid基因表达水平。结果C2-神经酰胺诱导HT-29细胞发生核染色质断裂、出现凋亡小体等典型凋亡改变,50μmol/L神经酰胺作用12和24小时凋亡细胞率分别达到64·7%和81·3%。同时神经酰胺使Bax、Bad和Bid基因表达水平增高,使Bcl-2和Bcl-xl基因表达水平减弱,Bcl-2与Bax比值下降,呈现剂量-效应关系。结论外源性神经酰胺能通过上调或下调Bcl-2家族基因表达水平诱导人结肠癌HT-29细胞发生凋亡。     

金雀异黄酮对结肠癌细胞HT-29增殖及凋亡的影响

目的观察植物雌激素金雀异黄酮(GS)对人结肠癌细胞HT-29增殖及凋亡的影响,并初步探讨其分子生物学作用机制。方法采用MTT比色法和流式细胞术检测GS对HT-29细胞增殖活力和细胞周期分布的影响;CellDeathELISA和流式细胞术从不同方面检测GS对HT-29细胞凋亡的影响;RT-PCR和Western-blot技术检测GS对核增殖抗原PCNA、bcl-2和baxmRNA和蛋白质表达的影响。结果与对照组相比,在15~120μmol/L浓度范围内,GS能够抑制HT-29细胞增殖活力;降低S细胞分布比例,将细胞周期滞留在G2/M期;显著性诱导HT-29细胞凋亡(>30μmol/L,P<0.05),并呈现出良好的剂量-效应关系。检测GS对PCNA、bcl-2和bax表达的影响显示,GS能够抑制PCNA和bcl-2mRNA和蛋白质表达,对bax的表达则表现出促进作用。结论GS通过诱导HT-29细胞凋亡而抑制其增殖活力,这些效应与PCNA、bcl-2和bax的表达变化有关。这些资料可为结肠癌的早期预防提供膳食干预新途径,并为临床新药的开发提供新方案。     

石蒜碱诱导结肠癌HT-29 细胞凋亡机制研究

目的 探讨石蒜碱诱导结肠癌HT-29 细胞凋亡及机制。方法 不同浓度的石蒜碱处理体外结肠癌HT-29 细胞,CCK-8法检测细胞活力,流式细胞仪和Hoechst 33258染色法检测细胞凋亡;Real time PCR实验检测Caspase-3、Bcl-2和Bax的mRNA水平;Western blot方法检测Caspase-3、Bcl-2和Bax蛋白的水平。结果 48h时石蒜碱对HT-29 细胞IC50为8.878μmol/L;与对照组比较,1.25μmol/L、2.5μmol/L、5μmol/L石蒜碱能诱导细胞凋亡(P<0.05),上调Caspase-3、Bax的mRNA和Caspase-3、Bax蛋白水平,下调Bcl-2的mRNA和Bcl-2蛋白水平,差异均有统计学意义(P<0.05)。结论 石蒜碱能通过调节Caspase-3、Bcl-2、Bax的表达水平,抑制结肠癌HT-29 细胞生长,诱导细胞凋亡,具有明显的体内抗肿瘤作用。     

Soybean saponins inhibit cell proliferation by suppressing PKC activation and induce differentiation of HT-29 human colon adenocarcinoma cells

Soybeans are major dietary sources of saponins, which have been suggested as possible anticarcinogens. This study was performed to determine the effect of soybean saponins on cell proliferation, differentiation, and apoptosis in human colon cancer cells. HT-29 cells were incubated in various concentrations of saponins for 24, 48, and 72 hours. Cell growth and whole cell protein kinase C (PKC) activity were determined. Alkaline phosphatase activity and carcinoembryonic antigen level were measured as markers for cell differentiation. Apoptotic cells were quantified. Study results indicated that soybean saponin treatment decreased cell growth in a concentration-dependent manner, and pre-treatment of the cells with saponins significantly suppressed the 12-O-tetradecanoyl phorbol 13-acetate-stimulated PKC activity. Cells treated with 300 and 600 ppm of saponins significantly increased alkaline phosphatase activity by 146% and 242% of the control, respectively. Also, 4-10 times more carcinoembryonic antigen was produced in cells treated with saponins. However, at all the concentrations used, saponins did not induce apoptosis, although there were slight decreases in apoptotic activity in cells treated with 240 and 600 ppm of soybean saponins. These results suggest that crude soybean saponin extract effectively suppresses PKC activation and induces differentiation, which possibly mediate the growth inhibition of tumor cells. Further experiments, including preclinical efficacy studies, are required to fully evaluate soybean saponins for their chemopreventive properties.     

Activation of caspase-8 contributes to 3,3'-Diindolylmethane-induced apoptosis in colon cancer cells

3,3'-Diindolylmethane (DIM) is the major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol, which is a promising anticancer agent present in cruciferous vegetables and has itself been reported to have anticarcinogenic properties. This study examined DIM-mediated regulation of apoptosis in the HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer cell lines. DIM (0-30 micromol/L) substantially decreased the number of viable cells and induced apoptosis of HCT116 and HT-29 cells in a concentration-dependent manner. Western-blot analyses of total cell lysates revealed that DIM increased the activation of caspase-3, -7, -8, and -9 and enhanced poly(ADP-ribose) polymerase cleavage in both HCT116 and HT-29 cells. In addition, DIM increased the translocation of cytochrome c and Smac/Diablo from the mitochondria to the cytoplasm. In concert with the caspase-8 activation by DIM, increased levels of Fas and truncated Bid were observed. DIM did not affect the protein levels of p53, Bcl-2, Bax, or Fas ligand (FasL) in HCT116 cells. In HT-29 cells, however, DIM decreased Bcl-2 levels, although the protein levels of Bax or FasL were not affected. The caspase-8 inhibitor Z-IETD-FMK attenuated the DIM-induced apoptosis, indicating that increased activation of this enzyme contributed to the increase in p53-independent apoptosis that was observed in colon cancer cells. We have demonstrated that DIM induces apoptosis in colon cancer cells, providing insights into the mechanisms underlying its antitumorigenic activities.     

Induction of apoptosis in HT-29 colon cancer cells by phloretin

Phloretin, which is present in apples and pears, has been found to inhibit the growth of several cancer cells and induce apoptosis of B16 melanoma and HL60 human leukemia cells. The present study examined whether and how phloretin induces apoptosis of HT-29 human colon cancer cells. Phloretin (0-100 micromol/L) substantially decreased viable cell number and induced apoptosis of HT-29 cells in a dose-dependent manner. Western blot analysis of total cell lysates revealed that phloretin increased the protein levels of Bax but had no effect on Bcl-2. In addition, phloretin induced cleavage of caspase-8, -9, -7, and -3 and poly(ADP-ribose) polymerase. Furthermore, phloretin increased the levels of cytochrome c and Smac/Diablo in the cytosol. The present results indicate that phloretin inhibits HT-29 cell growth by inducing apoptosis, which may be mediated through changes in mitochondrial membrane permeability and activation of the caspase pathways.     

Andrographolide Exhibits Anticancer Potential Against Human Colon Cancer Cells by Inducing Cell Cycle Arrest and Programmed Cell Death via Augmentation of Intracellular Reactive Oxygen Species Level

Andrographolide, a diterpenoid lactone and a major constituent of Andrographis paniculata Nees, exhibits remarkable anticancer activity. However, the effect of andrographolide on colon cancer has not been completely elucidated yet. Thus, we investigated the chemopreventive potential of andrographolide in colon cancer HT-29 cells. The cytotoxic potential of andrographolide on HT-29 cells was determined by MTT assay, trypan blue exclusion assay, colony formation assay, and morphological analysis; and apoptotic property by DAPI and Hoechst staining, FITC-Annexin V assay, DNA fragmentation assay and caspase-3 activity assay. To elucidate andrographolide action, intracellular reactive oxygen species (ROS) level was determined by DCFDA dye; change in mitochondrial potential by Rhodamine123 and Mito Tracker Red CMXRos dye; and cell cycle modulatory property by flow cytometric analysis. Results of the study have shown that andrographolide decreased cell viability of HT-29 cells in a dose- and time-dependent manner. Furthermore, andrographolide induced apoptosis in HT-29 cells which seemed to be linked with augmented intracellular ROS level and disruption of mitochondrial membrane potential. Interestingly, andrographolide caused significant cell cycle arrest in G2/M phase at lower doses, but, in G0/G1 phase at higher doses. In summary, our results indicated that andrographolide exhibited antiproliferative and apoptotic properties against colon cancer HT-29 cells.     

Inhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells

Objectiveγ-Tocotrienol is a major component of the tocotrienol-rich fraction of palm oil, but there is limited evidence that it has antitumor activity. In particular, the effects of γ-tocotrienol on human colon carcinoma cells have not been reported. To investigate the chemopreventive effects of γ-tocotrienol on colon cancer, we examined its capacity to inhibit proliferation and induce apoptosis in HT-29 cells and explored the mechanism underlying these effects.MethodsWe cultured HT-29 cells in the presence of γ-tocotrienol. The effect of γ-tocotrienol on cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mitotic index, and colony formation. The cell-cycle distribution was investigated by flow cytometry. We measured apoptosis by nuclear staining, transmission electron microscopy, and DNA fragmentation. Apoptosis-related proteins and the nuclear factor-κB p65 protein were determined by western blotting and immunofluorescence.Resultsγ-Tocotrienol inhibited cell growth and arrested HT-29 cells in G₀/G₁ phase. The 50% inhibitory concentration was 31.7 μmol/L (48 h). γ-Tocotrienol–induced apoptosis in HT-29 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of caspase-3. Furthermore, we found that γ-tocotrienol reduced the expression level of total nuclear factor-κB p65 protein and inhibited its nuclear translocation.ConclusionThe results indicated that γ-tocotrienol inhibits cell proliferation and induces apoptosis in HT-29 cells in a time- and dose-dependent manner, and that this process is accompanied by cell-cycle arrest at G₀/G₁, an increased Bax/Bcl-2 ratio, and activation of caspase-3. Our data also indicated that nuclear factor-κB p65 protein may be involved in these effects.     

Methanolic extracts of Plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in HT-29 human colon carcinoma cells

Natural marine products have recently become the focus of increased research interest, due to their potential pharmacological activities. Therefore, we have screened 50 varieties of marine seaweed and determined that the methanolic extracts from Plocamium telfairiae (PTE) exhibited a cytotoxic effect against HT-29 human colon carcinoma cells. In this study, we report on the cytotoxic activity and mechanism of PTE-induced apoptosis in HT-29 cells. The treatment of HT-29 cells with various PTE concentrations resulted in the inhibition of growth and the induction of apoptosis in a dose-dependent manner, as determined by the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay, a lactate dehydrogenase release assay, a morphological assay, and a colony formation assay. Interestingly, we also detected apoptotic bodies on Hoechst staining and attempted to determine whether the PTE-induced apoptosis involved the caspase pathway, using a caspase colorimetric assay. The activation of caspases-8, -9, -3, and -7 and the specific proteolytic cleavage of poly(ADP-ribose) polymerase were detected over the course of apoptosis induction. Our results showed that PTE may function as a chemopreventive and/or chemotherapeutic agent in colon carcinoma cells via the reduction of cell viability and the induction of apoptosis.     

Effect of methanolic extract from silkworm droppings on proliferation and caspase activity in HT-29 human colon cancer cells

Colorectal cancer is the third most common cause of cancer-related deaths in the world. Surgical intervention followed by chemotherapy remains the primary approach to treatment since colon cancers remain refractory to most chemotherapeutic agents. Based on that, we established a program to screen natural products for cytotoxic activity, employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay system utilizing HT-29 human colon cancer cells. During the course of our screening, we found that the methanolic extract of silkworm droppings (SDME) has cytotoxic effects on HT-29 cells. In the present study, we investigated the possible mechanisms by which SDME exerts its antiproliferative activity in HT-29 cells. As expected, SDME inhibited growth of HT-29 cells in a dose-dependent manner as assessed by the MTT reduction assay, the lactate dehydrogenase release assay, and the colony formation assay. We also investigated whether the apoptotic effects induced by SDME involve the caspase pathway using the caspase colorimetric assay. Interestingly, caspase-9 and -3, but not caspase-8, were activated in response to SDME treatment. Taken together, these results clearly indicate that the induction of apoptosis by SDME involves a mitochondrial-mediated pathway and strongly suggest that SDME may potentially be a chemotherapeutic agent for human colon cancer.     

Inactivation of akt and NF-kappaB play important roles during indole-3-carbinol-induced apoptosis in breast cancer cells

Despite significant advances in treatment, breast cancer is still the second leading cause of cancer-related deaths in women in the United States. Therefore, significant efforts are being given to develop novel strategies for the prevention of breast cancer in recent years. Our laboratory and others have been studying the effects of a potential chemopreventive agent, indole-3-carbinol (I3C), in breast cancer cells. We have previously shown that I3C induces apoptosis in breast cancer cells and found that the induction of apoptotic processes was partly mediated by dysregulation of anti- and pro-apoptotic molecules. However, the precise molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells has not been fully elucidated. For the present study, we focused our investigation on important cell signaling molecules such as Akt and NF-kappaB during I3C-induced apoptosis in breast cancer cells. We found that I3C induces apoptotic processes in MCF10A-derived cell lines with premalignant (DCIS.com) and malignant (MCF10CA1a) phenotypes but not in nontumorigenic parental MCF10A cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that I3C specifically inhibits Akt kinase activity and abrogates the EGF-induced activation of Akt in breast cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc reporter constructs revealed that Akt gene transfection directly activates NF-kappaB, and this activation was completely abrogated by I3C treatment. In addition, I3C also abrogated the EGF-induced activation of NF-kappaB, which was mediated via the Akt signaling pathway. From these results, we conclude that there is a direct cross-talk between Akt and NF-kappaB pathways and that the inactivation of Akt and NF-kappaB activity plays important roles in mediating I3C-induced apoptosis in breast cancer cells. These results also suggest that I3C may be a potential chemopreventive agent by virtue of its selective apoptosis-inducing ability in premalignant and malignant breast epithelial cells.     

C_2-神经酰胺所致HT-29细胞凋亡中线粒体的变化

目的探讨外源性C2-神经酰胺所致HT-29细胞凋亡中线粒体的变化。方法C2-神经酰胺作用人结肠癌HT-29细胞,采用透射电镜观察细胞线粒体超微结构的变化;采用Mitochondrial Apoptosis Detection Kit和流式细胞仪检测细胞凋亡时线粒体损伤及其膜电位的变化。结果C2-神经酰胺作用HT-29细胞24h,线粒体发生空泡化、变性、嵴肿胀或消失等形态学改变;此时荧光显微镜下可观察到线粒体聚集,发出红色荧光,而正常细胞线粒体发出绿色荧光。C2-神经酰胺作用细胞6h,线粒体膜电位即开始降低,呈时间-剂量-效应关系;结论线粒体参与C2-神经酰胺诱导HT-29细胞凋亡作用。     

Antioxidative effects of glycoprotein isolated from Solanum nigrum L

Glycoprotein from Solanum nigrum L. (SNL glycoprotein) was isolated and tested for antioxidative effects on oxygen free radicals using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the SNL glycoprotein are optimal in acidic pH and up to 60 degrees C. However, it has minimal activities in the presence of EDTA, although such activities are not dependent on M(2+) ions (Ca(2+), Mn(2+), and Mg(2+)) in the presence of EDTA. Interestingly, when SNL glycoprotein was treated with deactivation agents (pronase E and NaIO(4)), the DPPH radical scavenging activity was decreased compared with the SNL glycoprotein treatment alone. The antioxidative effects of SNL glycoprotein on superoxide anion and hydroxyl radical under optimal conditions revealed that SNL glycoprotein has remarkable scavenging effects on both radicals, but exhibited slightly higher scavenging effects on superoxide anion generated by the enzymatic hypoxanthine/xanthine oxidase system than on hydroxyl radicals generated by the Fenton reaction. However, SNL glycoprotein was more effective against hydroxyl radials in cell cultures (NIH/3T3). Consequently, 20 microg/mL SNL glycoprotein has a scavenging ability against superoxide anion corresponding to that of ascorbic acid. On the other hand, its hydroxyl radical scavenging activity corresponds to 0.1 microg/mL catalase. From these results, we suggest that SNL glycoprotein has potent antioxidative potential.     

The mechanism of the action of IFN-gamma and TPA on the modulation of epidermal growth factor receptors of human amnion cells.

We have examined the mechanism of synergistic action occurring between interferon (IFN)-gamma and 12-0-tetradecanoylphorbol-13-acetate (TPA) with respect to the reduction of 125I-epidermal growth factor (125I-EGF) binding to human amnion (WISH) cells [Karasaki Y et al (1989) J Biol Chem 264: 6158-6163]. The cells were treated with protein kinase C (PKC) inhibitors (H7, staurosporine) to investigate the role of PKC in the synergism between IFN-gamma and TPA, since TPA is a strong activator of PKC. The combined effect of IFN-gamma and TPA was blocked by the PKC inhibitor, suggesting that PKC plays an important role in the synergistic action of TPA and IFN-gamma on the inhibition of EGF binding to the cells. The prolonged incubation (24 h) of the cells with TPA resulted in the restoration of EGF binding to the cells. A 24 h treatment of WISH cells with both IFN-gamma and TPA, however, still exhibited greater than 50% inhibition of EGF binding. No PKC activity, however, was observed in the WISH cells treated with both IFN-gamma and TPA for 24 h as well as with TPA alone for 24 h, indicating that IFN-gamma may synergize with the second mediator induced by PKC rather than PKC itself in the reduction of EGF binding to WISH cells. In addition, IFN-gamma showed the synergistic action with calcium ionophores on the reduction of EGF binding to the cells, suggesting that Ca2+ may be one of the second mediators which was induced by TPA and which cooperated with IFN-gamma.     

丹皮酚对人大肠癌HT-29细胞bcl-2和bax基因表达的影响

目的探讨丹皮酚（Pae）对人大肠癌HT-29细胞的增殖抑制、凋亡诱导作用及可能的作用机制,为寻求大肠癌治疗的新策略提供理论依据。方法应用荧光显微镜和透射电镜技术观察不同浓度的Pae对HT-29细胞增殖的抑制及凋亡诱导作用;应用免疫细胞化学技术检测用药前后凋亡相关基因bcl-2、bax表达的变化。结果荧光显微镜及透射电镜观察到Pae作用后HT-29细胞出现典型的细胞凋亡形态;Pae在15.63 mg/L、62.5 mg/L、250 mg/L3种浓度下作用48 h均可诱导HT-29细胞凋亡;免疫细胞化学结果显示Pae作用后HT-29细胞bcl-2蛋白表达显著降低,bax蛋白表达无显著改变。结论Pae可抑制HT-29细胞增殖并诱导其凋亡,并呈现明显的剂量-效应关系和时间-效应关系,其作用机制可能与下调bcl-2/bax的表达比例有关。     

Studies on chemical structure modification and biology of a natural product, gambogic acid (I): Synthesis and biological evaluation of oxidized analogues of gambogic acid

Gambogic acid (GA), a natural product, exhibits high potency in inhibiting cancer cell growth through the effective induction of apoptosis. In order to investigate the structure-activity relationships of GA derivatives, 11 oxidized derivatives of GA were synthesized. Some of them showed strong inhibitory effects on HT-29, Bel-7402, BGC-823, A549, and SKOV 3 cell lines. Moreover, in this paper the cellular growth inhibitor 39-hydroxy-6-methoxy-gambogic acid methyl ester (10) was identified as a HepG2 cell apoptosis inhibitor through Annexin-V/PI double staining assay and the expression of the related apoptotic proteins (Bax and Bcl-2). Compound 10 may serve as a potential lead compound for the development of new anticancer drugs. Further SAR studies of GA derivatives indicated that modification of carbon-carbon double bond at C-32/33 or C-37/38 and of the methyl groups at C-39/C-35 can improve antitumor activity.     

Induction of apoptosis by the aqueous extract of Rubus coreanum in HT-29 human colon cancer cells

OBJECTIVES: The incompletely ripened fruit of Rubus coreanum (IRFRC) has been used in traditional herbal medicine to manage various diseases. To explore the possibility that IRFRC has chemopreventive effects, we examined whether or not extracts of IRFRC inhibits HT-29 cell growth and explored the mechanism for this effect. METHODS: We cultured HT-29 cells in the presence of the aqueous or ethanol extract of IRFRC. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. We measured apoptosis using a DNA fragmentation assay and Annexin V staining. We used western blot analyses to determine the cleavage of caspases and poly (adenosine diphosphate-ribose) polymerase. RESULTS: Aqueous extract of IRFRC substantially inhibited viable HT-29 cell number in a dose-dependent manner, whereas ethanol extract had only a minimal effect. Aqueous extract inhibited DNA synthesis and induced apoptosis of HT-29 cells in a dose-dependent manner. Aqueous extract induced cleavage of caspase-3, -7, and -9 and induced the activity of caspase-3 and cleavage of poly (adenosine diphosphate-ribose) polymerase. CONCLUSIONS: We have shown that aqueous extract of IRFRC inhibits cell proliferation and stimulates apoptosis in HT-29 cells, and that this may be mediated by its ability to activate the caspase-3 pathway. It remains to be determined whether the aqueous extract of IRFRC has chemopreventive activities in animal models.     

姜黄素对氯化铝染毒的NG108 - 15细胞凋亡及PKC - NMDAR通路表达的影响

目的 探讨姜黄素对氯化铝染毒所致NG108 - 15细胞凋亡的影响及其机制,为铝致学习记忆损伤的治疗提供参考。方法 取对数生长期NG108 - 15细胞,随机分为空白对照组、DMSO组（溶剂对照）、姜黄素组（16 μmol/L姜黄素）、铝组（160 mg/L氯化铝染毒 ）、铝+姜黄素组（160 mg/L氯化铝染毒24 h后,给予16 μmol/L姜黄素处理24 h）。收集各组细胞,采用吖啶橙/嗅化乙锭（AO/EB）双荧光染色观察细胞凋亡形态,计数凋亡细胞数并计算凋亡率;流式细胞术检测细胞凋亡率, qRT - PCR检测细胞中PKC、NMDAR1、NMDAR2B 的mRNA表达水平,western blot检测细胞中凋亡相关蛋白（caspase3、Bax）和PKC、NMDAR1、NMDAR2B的蛋白表达水平。多组间均数的比较采用单因素方差分析（one - way ANOVA）,组间两样本均数的比较采用LSD法。结果 与空白对照组相比,铝染毒组的caspase3、Bax蛋白表达水平升高（t = - 5.547、 - 4.948,P＜0.001）,PKC、NMDAR1、NMDAR2B的mRNA（t = 4.926,P = 0.003;t = 6.330,P＜0.001;t = 4.224,P = 0.019）和蛋白（t = 20.638,P＜0.001;t = 4.509,P＜0.001;t = 17.388,P = 0.002）表达水平降低, AO/EB染色和流式细胞术结果均表明细胞凋亡率升高（t = - 5.153、 - 7.390,P＜0.001）;加入姜黄素处理后,与铝染毒组相比,铝+姜黄素组的caspase3、Bax蛋白表达水平降低（t = 2.930,P = 0.006;t = 4.907,P＜0.001）,PKC、NMDAR1、NMDAR2B 的mRNA（t = - 10.337、 - 6.621、 - 6.847,P＜0.001）和蛋白（t = - 30.551、 - 7.451、 - 26.294,P＜0.001）表达水平升高,AO/EB染色和流式细胞术结果均表明细胞凋亡率降低（t = 2.707,P = 0.01;t = 4.632,P＜0.001）。结论 上调PKC - NMDAR信号通路表达,可能是姜黄素减轻氯化铝染毒所致NG108 - 15细胞凋亡的机制之一。